EPA/635/R-03/005 www.ep a.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, DCDISCLAIMER
This document has been reviewed in accordance with U.S. Environmental Protection Agency policy and approved for publication. Mention of trade nam e s or comme rcial products does not constitute endorsem e nt or recomme ndation for use. Note: This document m ay undergo revisions in the f u ture. The m o st up-to-date version will be m a de available electronically via the IRIS Hom e 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 ................................................1 0 4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS, CLINICAL CONTROLS ................................1 0 4.2. PRECHRONIC AND CHRONIC STUDIES IN ANIMALS .................1 8 4.2.1. Oral ......................................................1 8 4.2.2. Inhalation .................................................2 1 4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION ..............................2 7 4.4. OTHER STUDIES ..................................................3 2 4.4.1. Respiratory Toxicity .........................................3 2 4.4.2. Neurotoxicity

.............................................. 3 6 4.4.3. Cardiovascular Toxicity

...................................... 39 4.4.4. Ocular Toxicity .............................................4 0 4.4.5. Genotoxicity

............................................... 4 0 4.5. EVALUATION OF MODE OF ACTION FOR NONCANCER EFFECTS .........................................4 1 4.5.1. Mode of Action of Noncancer Effects ...........................4 1 4.5.2. Oral Exposure ..............................................4 3 4.6. WE IGHT-OF-EVIDENCE EVALUATION AND CANCER CHARACTERIZATION—SYNTHESIS OF HUMAN, ANIMAL, AND OTHER SUPPORTING EVIDENCE, CONCLUSIONS ABOUT HUMAN CARCINOGENICITY, AND LIKELY MODE OF ACTION

.............. 4 3 4.7. SUSCEPTIBLE POPULATIONS ......................................4 4 4.7.1. Possible Childhood Susceptibility ..............................4 4 4.7.2. Possible Gender Differences ...................................4 5

5. DOSE-RESPONSE ASSESSMENTS ..........................................4 5

iii-iv5.1. ORAL REFERENCE DOSE ..........................................4 5 5.2. INHALATION REFERENCE DOSE

................................... 4 5 5.2.1. Choice of Principal Study and Critical Effect with Rationale and Jus ti f i ca ti on

............................................... 4 5 5.2.2. Methods of Analysis—including Models (PBPK, BMD, etc.)

......... 4 7 5.2.3. RfC Derivation—including Applicati on of Uncertainty Factors (UFs) and Mod i f y i ng F ac t o r s ( M F s)

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

............................................ 49

6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND DOSE RESPONSE

..................................... 5 0 6.1. HAZARD IDENTIFICATION

........................................ 5 0 6.2. DOSE RESPONSE

................................................. 5 1

7. RE F E REN C ES

............................................................ 5 3

APPENDIX A: EXTERNAL PEER REVIEW—SUMMARY OF COMMENTS AND DISPOSITION ........................................6 2

ivFOREWORD 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 com p rehensive 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 m ake 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 Cummi ngs, 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 comme nts, 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 Ra diation; Office of Prevention, Pesticides, and Toxic Substances; Office of Solid W a ste and Em ergency Response; Office of W a ter; 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 Wa shington, DC

Robert Beliles National Center for Environmental Assessment U.S. Environmental Protection Agency Wa shington, 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 Wa lnut Creek, CA
Bonnie R. Stern, Ph.D., MPH BR Stern Associates Annandale, VA

Summaries of the external peer reviewers’ comme nts and the disposition of their

recomme ndations 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 assum p tion that thresholds exist for certain toxic effects such as cellular necrosis but m ay not exist for other toxic effects such as som e carcinogenic responses. It is expressed in units of m g /kg-day. In general, the RfD is an estimate (with uncertainty spanning perhaps an order of m a gnitude) 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 expos ure 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 /m 3

. 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/m 3 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); Mem o randum from EPA Adm i nistrator, 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 Num b er and at least one comm on nam e. At a m i nim um, the following databases were searched: HSDB, TSCATS, CCRIS, GENETOX, EMIC, EMICBACK, DART, ETICBACK, TOXLINE, CANCERLINE, MEDLINE, and MEDLINE backfile s. 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 m g /kg-day with an uncertainty factor of 1,000. The RfC for H 2 S was previously listed as 1 x 10 -3 mg /m 3 based on the mouse subchronic inhalation study by CIIT (1983a) using inflamma tion of the nasal

mu cosa as the critical endpoint. The derivation of the RfC used the NOAEL HE C of 1 m g /m 3 and the LOAEL HE C of 2.6 m g /m 3 , as well as an uncertainty factor of 1,000. The cancer assessment for hydrogen sulfide had not yet undergone a com p lete 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. Som e 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: H 2 S 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 Wa ter solubility: 3980 m g /L at 20 

### / C Log K ow Not applicable Dissociation constants: pKa1 = 7.04; pKa2 = 11.96 Conversion factor: 1 ppm = 1.39 m g /m 3 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 m e thanol, 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 H 2 S are not stable; absorbed oxygen causes the formation of elemental sulfur and the solutions becom e turbid rapidly (HSDB, 1998). The primary uses of H 2 S include the production of elemental sulfur and sulfuric acid, the

manufacture of heavy water and other chemicals; in m e tallurgy; 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% m i n.). Occupational exposure to H 2 S 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 m a tter. In residential settings it is m o st comm only the result of decomposition in septic or sewer system s .

3. TOXICOKINETICS RELATIVE TO ASSESSMENTS 3.1. ABSORPTION No studies were identified which quantitatively determined the absorption of H 2 S in humans or animals. However, the inhalation of high concentrations of H 2 S (greater than 2000 ppm or 2780 m g /m 3 ) can be fatal within seconds or m i nutes in both humans and animals, suggesting that it is absorbed rapidly through the lungs. Studies in pigs and rats measuring plasm asulfide levels suggested that H 2 S is absorbed following ingestion. Since H 2 S exists as a

-3-gas, oral exposure is not likely to occur. At physiological pH 7.4 approxim a tely one third of the H 2 S exists in the undissociated form and the rem a inder 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 H 2 S is only moderately water soluble and thus not wellabsorbed by the m o ist tissues of the upper respiratory tract. The percutaneous absorption of H 2 S has been described as slow (Laug and Draize, 1942).

3.2. METABOLISM Hydrogen sulfide is m e tabolized by three pathways: oxidation, m e thylation, and reactions with m e talloproteins. However, the m a jor pathway is oxidation in the liver with m e thylation being a very m i nor pathway. The m a jor oxidation product of H 2 S m e tabolism is sulfate (Beauchamp et al., 1984) although thiosulfate m ay also be isolated from tissues exposed to hydrogen sulfide (Kage et al., 1992). At physiological pH (pH-7.4), H 2 S is approxim a tely 69% ionized (HS
- ). The m e tabolic pathway for H 2 S is depicted in Figure 1. Bartholomew et al. (1980) perfused heparinized blood containing sodium [S 35 ]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 H 2 S (Haggard, 1921). Therefore, adm i nistration of sodium sulfide in th e isolated perfused system would be similar to inhalation exposure to H 2 S. In the isolated perfused liver, approxim a tely 70% sulfide was rapidly m e tabolized to sulfate within 15 m i nutes, and 82% of the detected radioactivity was sulfate after 2 hours perfusion. Wh en unlabeled thiosulfate was added to the liver perfusion system, 54% of the radioactivity was thiosu lfate and 22% was sulfate after 15 m i nutes of perfusion. After 2 hours, these proportions reversed. This would suggest that sulfide is oxidized to thiosulfate with f urther oxidation to sulfate. In the isolated perfused lung, 32% of the adm i nistered radioactivity was lost from the blood as volatile 35 S after 15 m i nutes. However, after 15 m i nutes 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 det ectable 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 thio sulfate as an intermediate m e tabolite. 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 cytochrom e c oxidase inhibition. Wh en glutathione (GSH) was added to the rat liver

m itochondria in vitro , thiosulfate was f urther m e tabolized to sulfate. Kage et al. (1992) exposed adult m a le Japanese white rabbits to 100-200 ppm (139-278 mg /m 3 ) for 60 m i nutes and to 500-1000 ppm (695-1390 m g /m 3 ) until fatal (22 m i nutes, approx.). In the low-level exposure group, blood and urine were collected imme diately after exposure and 1, 2, 4, 6, and 24 hours following exposure. Blood and organs were collected imme diately 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 m u scle. 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 m a jor m e tabolic pathway for H 2 S in humans. Thiosulfate was found in the urine of volunteers exposed to 8, 18, or 30 ppm (11, 25, or 42 mg /m 3 ) H 2 S for 30-45 m i nutes (Kangas and Savolainen, 1987). They also determined thiosulfate levels in m a intenance workers in a pelt processing plant with known exposures to H 2 S, which

were com p ared 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 c ontrol levels suggesting that all of the absorbed sulfide had been oxidized within the 15 hours after exposure. The relationship between sulfide concentration and cytochrom e oxidase in target tissues was explored by exposing m a le CD rats (6/group) to H 2 S at 0, 10, 30, 80, 200, or 400 ppm (0, 14, 42, 111, 278, or 556 m g /m 3 ) once for 3 hr and for 70 days (Dorm an et al., 2002). The only target organ that demonstrated a clear dose response relationship with increased sulfide levels and concomitant decreases in cytochrom e oxidase was the lung examined at the end of the single 3-hour exposure; statistical significance was achieved at 80 ppm (111 m g /m 3 ) and above for increases in sulfide and at 30 ppm (42 m g /m 3 ) and above for decreases in cytochrom e oxidase. Sulfide concentrations in the lung decreased to preexposure levels within 15 m i n of the end of exposure. The response was not seen in the lung after the 70-day repeated exposure. Hindbrain sulfide concentrations and cytochrom e oxidase activities were unaf f ected by single or repeated exposures. Cytochrom e oxidase concentrations were decreased (p 

### # 0.05) in both the nasal respiratory and olfactory epithelium at levels of 30 ppm (42 m g /m 3 ) although sulfide levels did

-5-not appear to be decreased until 400 ppm (556 m g /m 3 ) H 2 S. These results indicate that cytochrom e oxidase is a sensitive biomarker of ef f ect f o r H 2 S 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 m e tabolic pathway for H 2 S. Hydrogen sulfide was reported to be m e thylated to m e thanethiol in vitro by the intestinal m u cosa of Sprague-Dawley rats (Weisiger et al., 1980). Methanethiol can be further m e thylated, although

mu ch m o re slowly, to dim e thylsulfide. Thiol S -methyltransferase was reported to catalyze the two reactions. It is a ubiquitous enzym e , and the investigators reported the highest activity in the colonic and cecal m u cosa (10 -13 mo l/min/mg of protein). High activities were also reported in the liver, lung, and kidney. Levitt et al. (1999) also studied the m e tabolism of H 2 S in the cecal m u cosa of SpragueDawley rats and concluded that oxidation, rather than m e thylation, was the primary m e tabolic route in the cecal m u cosa. Wh en incubated with H 2 S, cecal tissues did not produce m e thanethiol or dim e thylsulfide even when a m e thyl donor, S -adenosylm e thionine, was added to the reaction mi xture. The rates of m e tabolism for cecal and liver homogenates were calculated to be 21 and 2.5 nmol/min/mg of protein, respectively, and the m e tabolic products of the cecal and liver homogenate were reported to be sulfate and thiosulfate, the primary m e tabolite. The oxidation rate is approxim a tely 10,000 times the m e thylation rate reported by W e isiger et al. (1980), which would suggest that m e thylation is not an important pathway for H 2 S detoxification. Another possible pathway of H 2 S m e tabolism is conjugation with GSH. Smith and Abbanat (1966) reported that exogenously-oxidize d, but not reduced, GSH protected m i ce from lethal intraperitoneal (i.p.) injections of sodi um sulfide. They also stated that endogenous oxidized GSH, and other endogenous compounds c ontaining disulfide bridges m i ght provide an important detoxification process following H 2 S exposure. Hydrogen sulfide has also been reported to bind to hem e proteins such as CytOx and

me themoglobin. Wh ile H 2 S interaction with the f o rmer has been implicated in its toxicity, the interaction between m e themoglobin and H 2 S is a possible detoxification pathway. Smith and Gosselin (1964) pretreated rabbits and arm a dillos with sodium nitrite, an inducer of

-6-me themoglobin, and reported increased survival following intracardiac and intravenous (i.v.) injection with lethal doses of sodium sulfide. The investigators also reported that fem a le Charles River CD1 m i ce pretreated with sodium nitrite and p -am i nopropiophenone, also an inducer of

me themoglobin, had increased survival following i.p. injection with sodium sulfide (60 m g /kg). Survival time also significantly increased in m i ce exposed to 722 and 985 ppm (1003 and 1369 mg /m 3 ) H 2 S and pretreated with sodium nitrite and p -am i nopropiophenone.

-7-Figure 1. Metabolic pathways of hydrogen sulfide

.
Source: adapted from Beauchamp et al., 1984.

-8-3.3. DISTRIBUTION Forensic autopsies of three m e n who were overcome by H 2 S (estimated concentration 767-906 m g /m 3 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 H 2 S by the blood, liver, and kidney was extremely rem arkable m aking the values from these three victim s unreliable. However, sulfide is not norm a lly detected in the brain and lung of control postmortem specim e ns, 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 H 2 S. The investigators also reported that sulfide was detected in the skeletal m u scles of the abdomen and leg.
Distribution of H 2 S in animals is very similar to humans. Kohno et al. (1991) exposed

ma le W i star rats to 75 ppm (104 m g /m 3 ) for 20, 40, or 60 m i n. Following exposure, animals were sacrificed and blood and tissues (brain, lung, heart, liver, spleen, and kidney) were collected within five m i n of sacrifice. After 20 mi nutes of exposure, the concentration of sulfide in the blood, brain, lung, heart, liver, spleen, and kidney was approxim a tely 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 f o llowing H 2 S exposure. Gunina (1959) reported that following exposure to sodium sulfide via i.v. and subcutaneous (s.c.) routes or exposure to H 2 S by inhalation routes in dogs and rats the m a jority 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 m g /m 3 ) H 2 S 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 m g /m 3 ) H 2 S for 30-45 m i nutes and m a intenance workers in a pelt processing plant with known exposures to H 2 S (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 delib erate 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 m aking, whether previously or newly submitted. Som e 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 com p lex scientific and ethical issues posed by EPA’s possi ble 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 H 2 S exposure and subsequent treatm e nt include Milby and Baselt (1999a) and Guidotti (1996). Earlier reviews of the health effects were provided by Glass (1990), Reiffe nstein et al. (1992), and Mehlm an (1994). Exposure to H 2 S has been reported to be an important cause of mo rbidity and m o rtality in the workplace (Snyder et al., 1995) and olfactory dysfunction (Hirsch and Zavala, 1999). These reviews indicate that the typical “rotten-egg odor” of H 2 S is an inadequate warning indicator of exposure since levels in the range of 100-200 ppm (140-280 m g /m 3 ) can lead to loss of smell followed by olfactory paralysis (Reiffenstein et al., 1992). These authors estimate the odor threshold of H 2 S in the range of 3 to 20 ppb (0.004 to 0.03 m g /m 3 ). The case reports cited below involving overexposure have documented a variety of central nervous system (CNS) transitory sym p tom s , such as dizziness, nausea, headache and others m o re long-acting such as abrupt physical collapse or “knockdown,” all of which have been attributed to direct effects of H 2 S on the brain (Milby and Baselt, 1999b). Prolonged uncons ciousness 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 m g /m 3 ). Levels associated with pulmonary edema have been estimated to be in the range of 250-500 ppm (348-695 m g /m 3 ). A rather extensive list of acute sym p tom s of intoxication resulting from an accidental release of H 2 S 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 sem i volatile compounds in addition to hydrogen sulfide (e.g., liquid m anure) 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 H 2 S (Burnett et al., 1977) rendering him unconscious for an indeterminate amount of time. Upon resuscitation, he exhibited m a laise, 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 treatm e nt, his vital signs were norm a l and he was no longer in respiratory distress. He had severe photophobia and blepharospasm s , but no signs of conjunctivitis. He also possessed a cough and som e mo tor weakness of his right arm and leg. A neurologic examination and a chest x-ray revealed no abnorm a lities. After a 3-day stay in the hospital, he was discharged.
Two f a talities were due to m a ssive aspiration of liquid m anure and one f a tality was due to severe pulmonary edema with no aspiration of the m anure (Osbern and Crapo, 1981). Another 41-year-old individual fell unconscious into liquid m anure during a rescue attem p t. After resuscitation, he had difficulty breathi ng and was agitated, but he exhibited no focal neurologic def i cits. His initial chest radiograph showed a f i ve-lobed alveolar infiltrate. After two weeks in the hospital, his chest radiograph showed improvem e nt and his lung function was

norm a l except for a slightly reduced m a xim um mi dexpiratory flow rate. Six months after the accident, he had a norm a l chest radiograph and was asymptomatic. Blood sulfide levels of the two individuals who had fatally aspirated m anure were 5.0 and 3.6 m g /L. The individual who had died by pulmonary edema had a blood sulfide level of 0.8 m g /L. Control blood sam p les exhibited sulfide levels below 0.05 m g /L while blood sam p les from random autopsy cases of badly decomposed subjects did not exceed 0.4 m g /L. No blood sulfide measurements were perform e d on the 41-year-old survivor. Eight days following the accident, air analyzed from the liquid m anure storage tank contained 6,360 ppm me thane, 400 ppm carbon monoxide, 1.5 ppm

amm onia, 2% carbon dioxide, 18% oxygen, and 76 ppm (106 m g /m 3 ) H 2 S. Another case study illustrates the possible long-term sequelae of H 2 S exposure. A 30- year-old m an 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 H 2 S were perform e d. Physical examination and routine laboratory studies revealed no abnorm a lities. However, a chest radiograph detected a mild bilateral interstitial pattern. Bronchoscopy showed a widespread reddish m u cosa. 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 H 2 S suspected as a m a jor component. Several case studies report on the rapid toxicity following exposure to high levels of H 2 S. A 14-year-old boy found a discarded cylinder containing H 2 S in a rubbish dump and imme diately died when he opened the tank (Allyn, 1931). His father also died during a rescue attem p t. Both bodies were deeply cyanosed. A fatality, apparently due to release of H 2 S from a load of refinery waste was described by Sim s on and Sim p son (1971). This report also m e ntioned eyewitness accounts of intense central cyanosis in the victim

. At a poultry feather fertilizer plant, a worker was exposed to H 2 S while attem p ting 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 edem a fluid and numerous pigment-filled macrophages. The diagnosis was pulmonary edema, and the cause of death was H 2 S inhalation. Measurements of H 2 S concentrations at various locations in the fertilizer plant revealed levels as high as 4,000 ppm (5,560 m g /m 3 ) during the cooking and putrefaction of feathers. A 16-year-old boy suffered fatal H 2 S exposure during transport of liquid m anure (Hagley and South, 1983). He was found at the bottom of the m anure tank, pale, unconscious and apneic. There was no evidence that he had aspirated any m anure. The boy began breathing following resuscitation and his color returned to norm a l. His heart rate and blood pressure were norm al though he did not respond to painful stimuli. Over the following hour, he became responsive to stimuli, but then developed extensor spasm s and began to hyperventilate. A chest radiograph and a CT scan of the head were norm a l. 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 dam a ge. Postm o rtem examination revealed that he had cerebral edema. A week later, H 2 S was measured 30 cm below the m anhole of the tank and was found to exceed the upper limit of detection of the monitoring equipment at 150 ppm (208 m g /m 3 ). By contrast, Milby (1962) has reported two cases of severe, but non-fatal H 2 S poisonings. Wh ile disposing of a H 2 S-filled gas cylinder and the subsequent release of its contents, two m en that were exposed to the gas collapsed imme diately and underwent convulsions while unconscious. Upon being rescued from the gas, the m e n required artificial respiration to begin breathing on their own. Both m e n were hospitalized in an unconscious state and were later revived. At the conclusion of their hospital stay, both m e n were without sym p tom s. The author

12-13- states that they “remained well to the present,” but it is unclear how long an interval that represents.
A m a ss exposure to H 2 S took place among workers laying the foundation of a municipal sewage pumping station, leading to the death of a police officer attem p ting to rescue an unconscious worker and persistent neurological se quelae in at least one other individual (Snyder et al., 1995). Com p lications included: decreased ability to comm unicate, slow speech, m arked visual m emo ry impairm e nt 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 erythem a and corneal abrasions. Additional reports associated H 2 S exposure (via sewer gas) with lung function and neurological decrements and, in certa in studies, deaths were reported by W a tt et al. (1997) and Hall and Rumack (1997). Tvedt et al. (1991a) described the follow-up of six H 2 S-exposed patients who lost consciousness and experienced signs of long-termneurological sequelae associated with hypoxic brain dam a ge. Imme diate sym p tom s included cyanosis, pulmonary edema, seizures, and com a. Delayed sym p tom s (5-10 year f o llow-up) ranged f r om worker disability due to neurological sym p tom s to brain dam a ge severe enough to qualify as dem e ntia in one individual. Five patients who had been unconscious in an H 2 S atm o sphere for 5-20 m i nutes showed persistent impairm e nt with m emo ry and m o tor function m o st affected. The two patients with the m o st serious sym p tom s developed pulmonary edema. No measures of H 2 S in air, blood or urine were reported. The investigators concluded that the outcom e of acute exposures can be variable and that neurological sym p tom s may not becom e apparent until a considerable period of time has elapsed. Tvedt et al. (1991b) also described delayed neuropsychiatric sym p tom s in an individual who became unconscious for 15-20 m i nutes during an H 2 S exposure. He regained consciousness after two days, but then deteriorated again and rem a ined com a tose 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 resum e work and evidenced slight m o tor, m emo ry and visual impairm e nts and clinical indications of slight cerebral atrophy. Electroencephalogram and evoked responses were norm a l at this time. Sym p tom s similar to those described by Tvedt et al. (1991b) were also reported by Kilburn (1993) in an oil well tester rendered sem i conscious from exposure to high levels of H 2 S. Follow-up (39 and 49 months) sym p tom s suggested dam a ge to the brain stem, basal ganglia, vestibular apparatus, cortex, and other brai n structures. There was profound impairm e nt of cognitive function, m emo ry, visual perception and coordination, intelligence and neurophysiologic functions. Malingering was ruled out by the reproducibility of perform ance during testing sessions.

13-14- Persistent neurocognitive impairm e nt f r om H 2 S poisoning was also reported by W a sch 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 m emo ry, learning, and perceptual deficits and prolonged abnorm a l event-related potentials (P-300 latency). In one individual, sym p tom s persisted at follow-up three years after exposure. In another study, a 20-month-old child developed intermittent paroxysm a l tonic deviation of the eyes (Gaitonde et al., 1987). After a few months the abnorm a l eye movem e nts resolved, progressive involuntary movem e nts of the entir e 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 norm a l eye movem e nts. Computer tomography revealed bilateral areas of low attenuation in the basal ganglia and som e of the surrounding white m a tter. Virology was negative and there was no evidence of streptococcal infection. The brain scan suggested toxic encephalopathy. Shortly after adm i ssion, the child’s condition improved spontaneously. Ten weeks after adm i ssion, the ataxia had resolved and choreathetoid movem e nts were reduced. A repeat brain scan was norm a l. The child’s illness was attributed to H 2 S exposure. The family lived next to a coal m i ne where a burning tip emitted H 2 S for nearly one year. Three months prior to the child’s adm i ssion, H 2 S emissions were

m onitored for four months, and the m a xim um recorded level in family’s housing schem e was 0.6 ppm (0.8 m g /m 3 ). Local authorities admitted that H 2 S levels m ay have been m any times higher prior to monitoring. The burning tip had been extinguished just prior to the child’s adm i ssion 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 comm unities exposed to industrial sources of H 2 S were assessed by means of a sym p tom

- survey using trained interviewers (Legator et al., 2001). Results of the survey were com p ared to health ef f ects in three ref e rence comm unities not exposed to H 2 S. Sym p tom s were elevated in both exposed comm unities com p ared to the referent populations in: CNS effects, respiratory effect s, 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 comm unities had long-standing involvem e nt with H 2 S exposure issues which m ay have contributed to study bias. Subjects in one of the exposed

comm unities were also assessed by means of several objective psychoneurological tests, the results of which were consistent with results of the sym p tom survey (Borda, 1997; cited by Legator et al., 2001).
A m o rtality study was undertaken among Finnish sulfate mill workers exposed to H 2 S and organic sulfides (Jappinen and Tola, 1990). Wo rkers had been employed for at least one

14-15- year between 1945 and 1961 at three pulp and paper mills owned by the sam e com p any. No exposure data were presented. Deaths from all causes were not increased. However, workers exposed to H 2 S and organic sulfides exhibited an increase in cardiovascular-related deaths

com p ared to national death rates (37 observed com p ared to 24.7 expected). Cardiovascular

mo rtality was higher in workers employed f or 

### $ 5 years com p ared to workers exposed for 1-4 years. The investigators state that increased m o rtality could not be explained by comm on risk factors and that differences in smoking habits did not explain the findings. They suggest that increased m o rtality m ay have been associated with H 2 S and organic sulfide exposure. A cross-sectional study investigated the effects of presumed H 2 S exposure in sewer workers to determine if chronic exposure to the gas was associated with decreased lung function (Richardson, 1995). Sixty-eight sewer workers perform e d spirometric tests and results were

com p ared to 60 nonexposed water treatm e nt workers. Job titles were used to categorize sewer workers according to presumed H 2 S exposure levels. There was a statistically significant decrease in mean FEV 1 /FVC in sewer workers com p ared to water treatm e nt workers. The effect was greater in sewer workers presumed to have higher exposures to H 2 S and longer exposure histories. In nonsmoking subjects, sewer workers were only able to attain 89% of the predicted FEV 1 /FVC value com p ared to 98% in water treatm e nt workers. Although the study author states that chronic low level exposure to H 2 S m ay be associated with decreased lung function, no

m easurements of H 2 S were m a de so any quantitative relationship between “low levels” of exposure and effect are speculative. The pulmonary effects resulting from H 2 S 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 sym p tom s or loss of consciousness. Thirty-four percent of the respondents indicated that they had exposures serious enough to cause sym p tom s, and 8% of the workers stated they had experienced a loss of consciousness due to sour gas exposure. In workers that experienced sym p tom s , no decrease in spirometric values or excess sym p tom s were noted. Wh ile spirometric values were also not affected in workers that lost consciousness, the workers experienced shortn ess of breath during physical activity, wheezing with tightness in the chest, and wheezing attacks. The investigators stated that these sym p tom s are consistent with bronchial hyperresponsiveness. The data also demonstrate the refractiveness of the lung to the effects of H 2 S, with lung function param e ters being unchanged despite unconsciousness from H 2 S exposure. A cross-sectional pulmonary function study was perform e d in m a les that worked in viscose rayon plants (Higashi et al., 1983). A one-workday study was perform e d in which workers were monitored for H 2 S exposure for 8 hr and had a forced expiratory flow-volume test perform e d. Hydrogen sulfide levels were determined using passive diffusion dosimeters worn by the workers (n = 30 in exposed and m a tched control workers). The workers had been

15-16- exposed to H 2 S 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 m g /m 3 ) com p ared with < 0.1 ppm (0.1 m g /m 3 ) in m a tched control workers. No significant differences in the results from the pulmonary function tests were observed in exposed com p ared 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 115exposed 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 H 2 S would not cause adverse pulmonary health effects. A survey of 123 m a le viscose rayon workers exposed to H 2 S and/or carbon disulfide (CS 2 ) and 67 nonexposed m e tal, plastic, and starch workers was conducted in which subjects were questioned about eye irritation com p laints (Vanhoorne et al., 1995). Exposed and control workers were employed for at least one year by their employer. Exposure to H 2 S and CS 2 was determined using personal monitoring pumps. Exposure to H 2 S ranged between 0.2 and 8.9 mg /m 3 . Wo rkers exposed to H 2 S also tended to have high exposure to CS 2 . No workers were exposed to H 2 S only. Several types of eye com p laints were reported by workers exposed to CS 2 and H 2 S including: pain, tension, burning, hazy si ght, photophobia, and irritation. Wh ile the percentage of com p laints increased with exposure, the frequency of com p laints was higher in workers exposed to H 2 S and CS 2 com p ared to workers exposed to CS 2 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 prioriof evidence suggests that H 2 S is responsible. Considering all of the evidence, the investigators further suggested that CS 2 enhances the eye irritation properties of H 2 S. A study by Kilburn and W arshaw (1995) included 13 former workers and 22 near-plant residents of a desulfurization unit within a refinery processing “sour’ crude oil. Air monitoring for H 2 S and other chemicals at street level near the subjects’ homes during one week in July 1990 revealed that the H 2 S concentration was 0.01 ppm with periodic peaks of 0.1 ppm along with m e rcaptans (0.002 ppm), ethane (0.5 ppm), propane (0.5 ppm) and unquantified levels of vanadium and thiodiglycolic acid. In additi on, dim e thylsulfide, and vanadium (as vanadium pentoxide) were detected in air. Reduced sulfur gases measured outside the f a cility showed 24- hr averages of up to 21 ppm me rcaptans, 0 to 8.8 ppm H 2 S, carbon oxide sulfide (COS: 6 to 52 ppm) and for total reduced sulfur 6 to 71 ppm

. Questionnaires were com p leted by subjects and controls. Neurobehavioral functions and a profile of mood states were com p ared to 32 controls, m a tched by age and educational level. Neuropsychological functions were also evaluate d, including: two-choice reaction time, balance,

16-17- color discrim i nation, digit symbol, trail-m aking A and B, and imme diate recall. Abnorm a l m e an values of exposed subjects com p ared to controls were determined to be statistically significant for these functions. Mood state scores were found to be m u ch higher in the exposed subjects than the controls. However, m any of the neurophysio logical findings had large standard deviations and consequently were highly-variable. Wh ile 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 H 2 S is a plausible cause of the neurological function deficits, they acknowledged that other effects were attributable to other chemicals that were monitored (e.g., asthm aand dermatitis in workers were attributed to sulfur dioxide and vanadium pentoxide). Given the com p lexity of the exposure environment - in terms of the numbers of chemicals

m onitored, the few numbers of individuals studied and the very limited exposure measurements - it is premature to attribute the def i cits cited with H 2 S exposure.
Neurobehavioral effects of H 2 S were studied in 16 individuals (male and fem a le, ages 21 to 68 years) 2 to 22 years after exposure (Kil burn, 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 m i nutes and the rem a ining six were in the “hours” category. Twelve of the 16 individuals were plaintiffs in lawsuits. Results from a battery of tests were com p ared to those from 353 national referents,

ma tched 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 impairm e nt was apparent in all 16 subjects. Brief high doses were “devastating,” whereas protracted lo w doses of individuals in the chronic category showed effects (p<0.05) on the m o re sensitive neurobehavioral tests, such as balance, visual fields, and choice reaction time. There was no i ndication of bias from the lawsuit and it was considered unlikely that the sym p tom s could be attributed to other causes. Kilburn (1997) concluded that moderate occupational exposure and downwind environmental exposure can cause permanent impairm e nt. This finding was assessed in a subsequent study. Neurobehavioral deficits attributed to low-level environmental exposure of 103 individuals to H 2 S were reported by Kilburn (1999). The neurobehavioral battery included 28 tests. One group of 24 homeowners was exposed to H 2 S 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 abnorm a l balance, color discrim i nation, 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 abnorm a lities

com p ared 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 sam e individuals discussed in the preceding paragra ph 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),

me rcaptans (1.0 - 21.1 ppm), and 24-hr levels of H 2 S at 0 to 8.8 ppm. Referents (357) were selected from four towns (in four states) fr ee 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 dura tion of exposure is predictive of effects, and those exposed to nonlethal levels do not recover com p letely from H 2 S. 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 H 2 S. A five-year retrospective study (Arnold et al., 1985) of 250 workers exposed to H 2 S considered com p ensation claim s submitted from 1979-1983. Fifty-four percent of the workers became unconscious after exposure. Of the clin ical findings, neurologic, respiratory and opthalmologic ailments predominated. The overall fatality rate was 2.8%. In conclusion, H 2 S exposure levels that result in temporary unconsciousness (e.g., 15-30 mi nutes) can cause profound neurophysiological, neurobehavioral, neurocognitive, neurophysical, respiratory, and opthalmologic deficits that are persistent. Wh ile som e of these deficits can occur when the exposed individual rem a ins conscious, it is less clearly established if intermittent or continuous exposure to “low levels” of H 2 S cause similar persistent sym p tom s.

4.2. PRECHRONIC AND CHRONIC STUDIES IN ANIMALS

4.2.1. Oral We tterau et al. (1964) examined the effects of H 2 S 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 H 2 S. The fodder was analyzed for H 2 S content by acidification with hydrochloric acid and iodometric determination prior to animal adm i nistration. The H 2 S content in all of the experim e nts 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 H 2 S-treated alfalfa (2-12% of total feed) and H 2 S-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 H 2 S-treated dried alfalfa (4, 8, and 24% of the diet, respec tively) daily for 105 days. Pigs fed 200 or 400 grams of H 2 S-treated dried alfalfa had decreased weight gains of 4 and 22%, respectively,

com p ared with the control group. No statistical analysis was presented. Food intake decreased

18-19- by 33% in animals fed 400 grams of H 2 S-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 experim e nt, weaned pigs (numbers not provide) were fed diets containing 100, 200, or 400 grams of H 2 S-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 H 2 S in the diet containing 24% dried feed was 0.121%. The investigators concluded that there was no indication th at diets containing a high amount of H 2 S could cause adverse health effects in animals. The diarrhea observed in naive adult pigs m ay have been caused by the sudden change in diet rather than the presence of H 2 S, since increasing the feed containing H 2 S 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 H 2 S, which m ay 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 W e tterau et al. (1964) was used previously on IRIS as the basis for deriving an RfD. However, the low concentration of H 2 S in the feed, estimated at only about 15 m g /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 fem a le Sprague-Dawley rats (20/sex/dose group) were adm i nistered 0, 1, 3.5, or 7 m g /kg-day H 2 S 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 H 2 S gas. Prepared H 2 S solutions were stored in amber vials with Teflon seals and fresh vials were used at each dose adm i nistration. Dosing solutions were prepared weekly and solution stability was determined. Animals were examined f or
mo rtality at the beginning and end of each working day, and clinical signs were monitored imme diately after dose adm i nistration. Food consumption of 10 rats per dose group was determined weekly. Animals were weighed weekly during the treatm e nt period and at necropsy. Blood sam p les were collected from the suborbital sinus of animals prior to study initiation, days 27 to 31 of treatm e nt, and before necropsy. Bl ood and platelet counts were determined, and clinical chemistry was perform e d. At study termination, animals were sacrificed by carbon dioxide asphyxiation. Sacrificed animals and anim als that died during the treatm e nt 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 m i croscopically. In the low- and m i d-dose groups, only lung, kidney, liver, and gross lesions were examined m i croscopically. Mortality was 50% in the high-dose m a les com p ared to 5% in control m a les. No deaths were observed in high-dose f ema les. Mortality in m i d-dose m a les and f ema les was 10 and 5%, respectively, and no deaths were observed in low-dose animals or control f ema les. W eekly m o rtality is reported in Table 1.

Table 1. Cumulative w eekly mortality in male and female Sprague-Da w l ey rats administered H 2 S orally

Dose (mg/kg-day) Males 0
1
3.5

7 Females 0
1
3.5

7 Source: Anderson, 1987 Weeks

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

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 m a le and fem a le animals, with the m a les displaying a higher incidence of these effects (Anderson, 1987). High dose-m a les exhibited abnorm a l posture, convulsions, fist clutching, sedation, unusual vocalization, piloerection, labored breathing, and increased respiratory rates. High-dose fem a les exhibited convulsion, sedation, and labored respiration. Although restlessness was observed in all treated m a les, and increased salivation in all treated f ema les, these clinical signsgenerally occurred sporadically, and the incidence of these findings did not show a clear doseresponse. Therefore, the toxicological significance of restlessness in treated m a les, and salivation in treated f ema les, 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 treatm e nt-related effects in hem a tology or clinical chemistry.

20-21- Ophthalmological findings prior to sacrifice were norm a l. The m o st comm on findings at necropsy were pink and red m o ttled lungs, which occurred in all groups. In high-dose m a les 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 treatm e nt, relative heart weights increased (p < 0.05) in high-dose

ma les, and relative kidney weights increased (p < 0.05) in high-dose fem a les. No gross lesions could be attributed to compound adm i nistration and all m i croscopic findings were considered incidental to compound adm i nistration. 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 m o rtality was 50%, the high dose m a le group. However, the cause of death was not determined. In addition, a m a lady diagnosed as ulcerative dermatitis was comm on across all groups. Good Laboratory Practice would preclude the acceptance of data from such diseased animals for drawing conclusions regarding the effects of H 2 S.

4.2.2. Inhalation The Chemical Industry Institute of Toxicology (CIIT, 1983a) perform e d a 90-day inhalation toxicity study in B6C3F1/CrlBr m i ce using H 2 S. Animals were individually housed and exposed to 0, 10.1, 30.5, or 80 ppm (0, 14, 42, or 111 m g /m 3 ) H 2 S for 6 hr/day, 5 days/week for 90 days (10 m i ce/sex/group). Animals were observed twice daily for m o rtality and clinical signs. Body weight and feed consumption were determined weekly beginning just before the first exposure. A final fasted body weight was de termined 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 wa s perform e d before necropsy in which posture, gait, facial m u scle tone, and pupillary, palpebral, extensor thrust, and crossed-extensor thrust reflexes were assessed. Animals were housed in m e tabolism cages for 12 hr prior to necropsy. Urine sam p les 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 (chem i stry). 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 abnorm a lities. In addition, the following tissues were examined m i croscopically: cerebellum , cerebrum , me dulla, optic nerve, spinal cord, sciatic and anterior tibial nerves, eyes, pituitary, thyroid, parathyroid, salivary glands, heart, lungs,

21-22- spleen, liver, pancreas, adrenals, m e senteric and m andibular lymph nodes, kidneys, bladder, lacrimal glands, ovaries, uterus, oviducts, vagina, cervix, stomach, duodenum, ileum, jejunum , large and small colon, caecum , skeletal m u scle, skin, m ammary glands (both sexes), fem ur, bone

marrow, aorta, ear canal with zym b al gland, nasal turbinates (four levels), trachea, testes, epididymis, esophagus, thymus, prostate, sem i nal vesicle, and gross lesions. Body weights were significantly depressed in both m a les and fem a les in the highexposure groups with sporadic significant decreases in the lower m a le exposure groups (CIIT, 1983a). Decreases in body weights tended to be m o re pronounced in the m a le and fem a le high exposure groups beginning in the 6 thweek of exposure. Feed consumption was significantly reduced in the high-exposure animals. There were no toxicologically- or statistically-significant differences in hem a tology, serum chemistry, urinalysis, ophthalmology, or neurological function except for a 30.5 ppm (42 m g /m 3 ) fem a le and 80 ppm (111 m g /m 3 ) m a le that did not respond to artificial light stimulus.
Two fem a le controls and one 30.5 ppm (42 m g /m 3 ) m a le died prior to study termination. Both control animals exhibited m u ltiple focal black discoloration of the glandular stomach. The 30.5 ppm (42 m g /m 3 ) m a le exhibited a skull fracture with red discolorations and traum a apparently due to a feeding accident. A fem a le and m a le in the 80 ppm (111 m g /m 3 ) 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

sam e black discoloration of the glandular st omach 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 m g /m 3 ) m a le lost the use of anterior appendage with the appearance of paralysis while one 30.5 ppm (42 m g /m 3 ) fem a le and a 80 ppm (111 m g /m 3 ) fem a le were observed to have

a m i ssing front appendage. The investigators stated that it was unknown how the m a le lost its appendage but that the fem a le’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 ma le m i ce exposed to 80 ppm (111 m g /m 3 ) H 2 S were significantly depressed. In addition, the mean absolute kidney weight of 80 ppm (111 m g /m 3 ) fem a le m i ce was also significantly reduced by approxim a tely 20%. However, relative weights were not significantly different and clinical pathology and histology were negative in both sexes. Histologi cal examination of surviving animals revealed the nasal tract as the only site where lesions were considered compound-related. Male (8/9) and fem a le (7/9) m i ce exposed to 80 ppm (111 m g /m 3 ) H 2 S exhibited m i nim a l to mild inf l amma tion of the anterior portion of the nasal m u cosa (section I; section I and II in two m i ce). The lesion was primarily located in the squamous portion of the nasal m u cosa but extended into the respiratory type epithelium (ciliated) in som e animals. In one fem a le, 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 m g /m 3 ) m i ce that were exposed in extremis

. No other histological findings were considered compound-related (CIIT, 1983a).
The critical effect in m i ce was inflamma tion of the nasal m u cosa. This effect was present in m a le (8/9) and fem a le (7/9) animals exposed to 80 ppm (111 m g /m 3 ) H 2 S. The lesions, judged

as m i nim a l to mild in severity, were localized primarily in the squamous portion of the nasal

mu cosa but extended to regions of ciliated respiratory-type epithelium

. Since H 2 S is an irritant gas and other researchers (Brennem an et al., 2000; Dorman et al., 2000; Lopez et al., 1987) have reported nasal inflamma tion following H 2 S exposure, the critical effect in m i ce was considered to be inflamma tion of the nasal m u cosa. The LOAEL for this effect in m i ce was 80 ppm (111 mg /m 3 ) and the NOAEL was 30.5 ppm (42 m g /m 3 ) (CIIT, 1983a).
W ith the following exception, the sam e me thods 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 m a les and fem a les from each group were selected for clinical pathology and histology, while the rem a ining 5 m a les and fem a les from each group were used for special neuropathologic studies. In the neur opathologic 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 lum b ar portions of the spinal cord were dissected and placed in 4% glutaraldehyde. The left sural nerve and large m u scle branch of the tibial nerve were oscim a ted and placed in cedarwood oil for two weeks. Nerve fibers in the oil were teased and individual fibers mounted to glass slides. After staining, specim e ns were examined by light m i croscopy for pathological changes. In Fischer 344 rats, no m o rtality was observed during the 90-day study (CIIT, 1983b). Body weights were significantly reduced from weeks 1 to 13 in m a les and fem a les exposed to 80 ppm (111 m g /m 3 ) H 2 S. Feed consumption was also significantly depressed in m a les and fem a les exposed to 80 ppm (111 m g /m 3 ) H 2 S. No abnorm a lities in ophthalmology, neurological function, serum chemistry, or urinalysis were re ported. Sulfhemoglobin levels were significantly increased in m a les exposed to 80 ppm (111 m g /m 3 ) H 2 S. Although elevated sulfhemoglobin levels were also observed in fem a les exposed to 30.5 or 80 ppm (42 m g /m 3 or 111 m g /m 3 ) H 2 S, the levels were not statistically signif i cant. The investigators state that assigning biological significance to the sulfhemoglobin values should be done with caution because of sensitivity of the m e thod and the size of the groups investigated. Also, there were no H 2 S-related lesions in animals selected for special neuropathologic studi es. 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 m a les exposed to 80 ppm

23-24- (111 m g /m 3 ) H 2 S. Relative, but not absolute, brain weights were significantly increased in these animals. Minim a l m u ltifocal peribronchial lymphocytic infiltrate was observed in the lungs of all rats. Minim a l to mild mononuclear cellular infiltra tion of sections III and IV of the nasal m u cosa and nasolacrimal duct was observed in all rats including the controls. There was also m i nim al to moderate inflamma tion of sections I and II of the nasal m u cosa, but this effect was not dependent on dose with 45, 40, 75, and 55% of animals affected in the 0, 10.1 (14 m g /m 3 ), 30.5 (42 m g /m 3 ) and 80 ppm (111 m g /m 3 ) exposure groups, respectively. None of the histopathologic changes were considered treatm e nt-related by the investigators. The critical effect was depressed body weight gain in fem a le rats. The LOAEL for this effect was 80 ppm (111 m g /m 3 ) and the NOAEL was 30.5 ppm (42 m g /m 3 ).
The sam e me thods described above (CIIT, 1983a,b), including the special neuropathologic studies in CIIT (1983b) were us ed in the following study which used SpragueDawley rats (CIIT, 1983c). In the Sprague-Dawley rats, 15 m a les and 15 fem a les per group, no

mo rtality was observed during the 90-day study. Statistically significant decreases in body weights were observed in m a les exposed to 80 ppm (111 m g /m 3 ) H 2 S during weeks 1 through 3, and in 80 ppm-treated fem a les exposed throughout the 90-day study. Feed consumption was also significantly depressed in m a les and fem a les exposed to 80 ppm (111 m g /m 3 ) H 2 S. No abnorm a lities in ophthalmology, neurological function, hem a tology, serum chemistry, or urinalysis were reported, and there were no H 2 S-related lesions in animals selected f o r the special neuropathologic studies. Gross pathology re vealed a low incidence of lesions that were not compound-related. The black discoloration noted in the glandular stomach of m i ce was also present in 5 treated rats. However, this e ffect was not dose-dependent. Organ weights were statistically similar between control and exposure weights, excluding the absolute brain weight of m a les exposed to 80 ppm (111 m g /m 3 ) H 2 S. 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 H 2 S, e.g., low m o lecular weight and high degree of lipophilicity allowing passage through the blood-brain barrier. Minim a l to mild m u ltifocal peribronchial lymphocytic infiltrate was observed in the lungs of all rats including controls. Minim al to mild

m ononuclear cellular infiltration in sections III and IV of the nasal m u cosa and nasolacrimal duct was observed in all rats, including the controls. Although there was also m i nim a l to moderate inflamma tion in sections I and II of the nasal m u cosa, the effect was not related to dose with 35, 35, 30, and 50% of animals affected in the 0, 10.1 (14 m g /m 3 ), 30.5 (42 m g /m 3 ) and 80 ppm (111 mg /m 3 ) exposure groups, respectively. None of the histopathologic changes were considered treatm e nt-related by the investigators. The critical effect was decreased absolute brain weight in

ma les. The LOAEL for this effect was 80 ppm (111 m g /m 3 ) and the NOAEL was 30.5 ppm (42 mg /m 3 ).

24-25- The following study was actually perform e d 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 /m 3 ) H 2 S 6 hr/day, 5 days/week for 7 weeks. At the start of the week 8, dogs were coexposed to 15 ppm (20.8 m g /m 3 ) H 2 S and 10 ppm carbon disulfide. During week 9, dogs were only exposed to 15 ppm (20.8 m g /m 3 ) H 2 S which was increased to 30 ppm (42 m g /m 3 ) in weeks 10- 12. Blood pressure and heart rate were monitored in the m o rning and afternoon of exposure days. In addition, blood m o rphology (not descri bed by the investigators), and arterial and venous CO 2 and O 2 were determined. The frequency with which the investigators determined these param e ters was described as “time to time.” All measured endpoints were com p ared to control values that were established during a 4-week preexposure period. The dogs exhibited

norm a l health and behavior during the exposure period. Appetites and weights were m a intained satisfactorily, and no blood abnorm a lities were noted. Abnorm a l pulse and blood pressure determinations increased in frequency during the course of the experim e nt (weeks 1-7). The investigators did not state what constituted an abnorm a l blood pressure reading. However, the investigators suggested a cum u lative effect from low exposure. Coexposure with carbon disulfide increased the number of abnorm a l blood pressure readings. Increasing the concentration to 30 ppm (42 m g /m 3 ) H 2 S doubled the number of abnorm a l blood pressure examinations when com p ared to a two-week 15 ppm (21 m g /m 3 ) exposure.
Brennem an et al. (2000) exposed 10-week-old m a le Sprague-Dawley CD rats (12/exposure group) to 0, 10, 30, or 80 ppm (0, 14, 42.7, or 111 m g /m 3 ) H 2 S for 6 hr/day, 7 days/week for 10 weeks. At the end of the 10-week exposure period, animals were euthanized with CO 2 and the noses of the animals were dissected f r ee. 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 m u cosa, and were graded in severity by a subjective scale where 0 = norm a l, 1 = mild, 2 = moderate, 3 = m arked, and 4 =

severe. Table 2 shows that no effects were observed in the control animals or in animals exposed to 10 ppm (14 m g /m 3 ) H 2 S (Brennem an et al., 2000). Lesions, limited to the olfactory m u cosa, were observed in animals exposed to either 30 (427 m g /m 3 ) or 80 ppm (111 m g /m 3 ) H 2 S. These olfactory lesions consisted of mu ltifocal, bilaterally-symme trical olfactory neuron loss and basal cell hyperplasia affecting the lining of the dorsal m e dial meatus and dorsal and m e dial region of the ethmoid recess. The incidence, mean severity, and distribution of the exposure-related lesions increased in a concentration-dependent m anner.

25-26- Table 2. Incidence (Inc) and average severity (Sev) of nasal lesions in male CD rats exposed to H 2 S

Lesion Nasal Cavity Level 0 10 ppm 30 ppm 80 ppm

Inc S ev Inc S ev Inc S ev Inc S ev a 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 3 b — — — — — 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 — — —

aAverage 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 comm on occurrence of basal cell hyperplasia at background levels.

Source: adapted from Brennem an et al. (2000).

Respiratory/transition epithelium largely com p rises level 3 of the nose, the olfactory

mu cosa being only a m i nor component limited to the m o st caudal portion of the section. Olfactory neuron loss was only observed in 80 ppm (111 m g /m 3 ) exposure animals at this level of the nose. Basal cell hyperplasia was not evaluated due to the comm on background occurrence of this lesion at this level. Olfactory m u cosa com p rises a slightly larger portion of level 4 of the nasal cavity. This level had lesion distribution restricted to the olfactory tissue in the dorsal m e dial m e atus and the

mo st rostral projection of the third ethmoturbi nate. Olfactory neuron loss in the 30 ppm (42 mg /m 3 ) exposure animals was mild (grade 1) to moderate (grade 2), but olfactory neuron loss in the 80 ppm (111 m g /m 3 ) 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 m o re severe in the 30 ppm (42 m g /m 3 ) exposure group. Olfactory m u cosa lines m o st 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

mu cosa 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 m a inly affecting the nasal septum, dorsal nasal cavity, and m arginal ethmoturbinate were observed in both exposure groups. The nasal septum was not affected in the 30 ppm (42 m g /m 3 ) exposure group. The sam e pattern and severity of lesions were observed at level 6 in the 80 ppm (111 m g /m 3 ) exposure group. The critical effects in this study are nasal lesions of the olfactory m u cosa, 30 ppm (42 mg /m 3 ) is the LOAEL and 10 ppm (14 m g /m 3 ) the NOAEL (Brennem an et al., 2000). It should be noted that lesions in the nasal tract reported in other studies are not

com p letely in agreement with those reported by Brennem an et al. (2000) where lesions were clearly related to dose and localized in the olfactory epithelium

. In m i ce exposed to similar levels of H 2 S (CIIT, 1983a), inflamma tion of the nasal m u cosa was reported and described as occurring primarily in the squamous nasal m u cosa and extending into the respiratory epithelium , with no specific m e ntion of olfactory m u cosal i nvolvem e nt. Sectioning of the nasal epithelium was through 4- rather than 6-areas examined by Brennem an 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 Brennem an et al. (2000). Reasons for such a discrepancy are unknown.

4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION Dorman et al. (2000) attem p ted to determine if perinatal exposure by inhalation to H 2 S had an adverse effect on reproductive perform ance and pregnancy outcom e s. Virgin m a le and fem a le Sprague-Dawley rats (12/sex/group) we re exposed to 0, 10, 30, or 80 ppm (0, 14, 42, or 111 m g /m 3 ) H 2 S 6 hr/day, 7 days/week for two weeks prior to breeding. Exposure continued during a 2-week m a ting 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 m a ting period was considered GD 0. On postnatal day (PND) 4, litters were randomly reduced to 4 animals per sex when possible. Rem a ining pups were euthanized and discarded without being examined. Dam s and pups were then exposed PND 5-18. Nonpregnant adult fem a les were exposed for an additional 23-24 days following the 2-week breeding period. Adult m a les were exposed to H 2 S for 70 consecutive days. Clinical examinations were perform e d on all animals before and after each exposure (Dorm an et al., 2000). The body weights of the F 0 ma les and f ema les were determined weekly throughout the study, except that fem a le body weights were not determined weekly once evidence of m a ting was present. Presumed pregnant fem a les were weighed on GD 0, 7, 14, and 20, and dam s were weighed on PND 0, 4, 7, 14, and 21. Feed consumption was determined weekly in F 0 ma les and prebreeding fem a les. Feed consumption in presumed pregnant fem a les

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 com p lete necropsy was perform e d 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 F 0 ma le was examined for sperm number, production, m o tility, and m o rphology. No deaths or adverse clinical signs were observed in F 0 ma les and fem a les for any exposure group. There was a statistically significant decrease in feed consumption in m a le rats exposed to 80 ppm (111 m g /m 3 ) H 2 S during the first week of the study. There was a small, but not statistically significant, decrease in body weight (5-6%) observed in F 0 ma les and fem a les exposed to 80 ppm (111 m g /m 3 ) H 2 S 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 F 0 ma les exposed to 10 ppm (14 m g /m 3 ) and 80 ppm (111 m g /m 3 ) but not in the m i d-dose of H 2 S and a decrease in the relative ovary weight of fem a les in the 10 ppm (14 m g /m 3 ) exposure group. There were no statistically significant effects on reproductive perform ance (mating index, fe rtility index, postimplantation loss per litter, and number of late resorptions or stillbirths) in F 0 animals. Also, the number of live pups, litter size, average length of gestation, and average number of implants per fem a le were not affecte d. There were no statistically significant effects on sperm production or m o rphology noted in F 0 ma les; however, a large percentage of abnorm al sperm was observed in one F 0 ma le from both the 30 (42 m g /m 3 ) and 80 ppm (111 m g /m 3 ) exposure groups (29 and 73%, respectively). In com p aring 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 m i neralization, sperm granulomas, and m u ltinucleated giant cells, degenerate sperm forms in the lum e n, aspermia, and oligospermia. Wh ile 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 m g /m 3 ) exposure group. Notable histological findings in fem a les included one each of ovarian cysts and squamous m e taplasia of the endometrium in the 10 ppm (14 m g /m 3 ) and 30 ppm (42 m g /m 3 ) exposure groups. The olfactory neuron loss and basal cell hyperplasia observed in the m a le rats in this study are reported elsewhere (Brennem an 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 a ny 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 m a le or fem a le rats following relatively high H 2 S exposure of 80 ppm (111 m g /m 3 ) under repeated conditions (up to 70 days for m a les).

28-29- W ith regards to developmental effects from H 2 S exposure, there were no statistically significant increases in structural m a lform a tions

. Observed m a lform a tions included kinked tail, agenesis of the tail, anophthalmia, small rear legs and body, frontal bone defects, hypognathia, and skin lesion characterized by detachm e nt of the skin and dermis. However, none of these effects was dose-related. There were no si gnificant differences in pup weight gain or development (pinnae detachm e nt, 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 treatm e nt-related effects on m o tor activity, acoustic startle response, passive avoidance observed, or FOB. Terminal body and organ weights in all exposure groups were com p arable to controls. Although a wide variety of gross observations were noted, they were not considered treatm e nt-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 abnorm a lities. Saillenfait et al. (1989) reported that the exposure of pregnant Sprague-Dawley rats to 0, 70, 140 or 210 m g /m 3 (0, 50, 100, or 150 ppm) H 2 S 6 hr/day during gestational days 6-20 resulted in no signs of m a ternal toxicity or adverse effects on the developing fetus. Wh ile there was a slight but significant decrease in feta l weight in all treated groups, the biological significance of this finding is questionable since the dam s also lost weight and there were greater numbers of live fetuses per litter in those groups. Significant elevations in m a ternal blood glucose levels were observed in Sprague-Dawley rats exposed to 0, 28, 70, or 105 m g /m 3 (0, 20, 50, or 75 ppm) H 2 S, 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 dam s or pups. Pups exhibited decreased serum triglycerides in the 70 m g /m 3 (50 ppm) dose group. In a similarly-designed study using the sam e exposure levels (Hayden et al., 1990b), there was a dose-dependent increase in parturition time of approxim a tely 10, 20, and 40% over m a tched controls at 0, 28, 70, or 105 mg /m 3 (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 dam s. Although no studies have reported effects that prenatal exposure to H 2 S m ay eventually have upon the adult nervous system, early postnatal measurements provide indications of possible neurotoxic effects that m ay occur in adulthood. Hannah et al. (1989) exposed timedpregnant Sprague-Dawley rats (number not reported) to 105 m g /m 3 (75 ppm) H 2 S, 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 gammaamino-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 com p ared to controls. The significance of these amino acid changes to neurobehavioral development is uncertain. In a similarly designed study using the sam e exposure protocol, Hannah and colleagues (Skrajny et al., 1992) exposed groups of 20 pre gnant Sprague-Dawley rats to 0, 28, or 105 m g /m 3 (0, 20, or 75 ppm) H 2 S. 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 m g /m 3 (20 ppm) H 2 S, 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 m g /m 3 (75 ppm) H 2 S. Norepinephrine levels increased (p<0.05) at 105 m g /m 3 (75 ppm) in the cerebellum on PNDs 7, 14, and 21, and in the frontal cortex on PND 21. At 28 m g /m 3 (20 ppm), frontal cortex norepinephrine levels were decreased com p ared to controls on days 14 and 21. In a study designed to evaluate the perinatal effect of H 2 S on developing cerebellar Purkinje cells, Sprague-Dawley dam s were exposed to 28 or 70 m g /m 3 H 2 S (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 H 2 S. Pups born to treated dam s 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 m e thod. One Purkinje cell from the pyramis of each cerebellum was digitized for m o rphometric analysis. Quantitative analysis of dendritic m o rphology is generally regarded as a traditional m o rphometric m e thod 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 m e thod leads to a determination of random versus nonrandom growth. Statistical evaluati on was carried out using the Student’s t -test. Exposure to both 20 and 50 ppm (28 or 70 m g /m 3 ) produced significantly longer segment length than controls over the low and m i ddle branching orders, but not the higher orders. It was stated that segment lengths are typically increased when afferent fi bers are experim e ntally reduced in number. The

m ean 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 com p ared to controls. Wh en considered over the entire dendritic tree, these changes could alter the capacity of Purkinje cells to integrate afferent impulses and m ay be associated with an increased likelihood of an abnorm a l 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 m i ddle orders and a significant decrease in numbers over the higher orders (considered to represent the ‘growth front’). The investigators considered this to be a com p ensatory response when there is restriction on the ‘growth front’. The question of random versus nonrandom growth patterns was evaluated by examining the symme try of the branching. It was found that vertex analysis indicated that the treated cells were undergoing a significant am ount of nonsymme trical or nonrandom growth, suggesting that H 2 S af f ected the branching pattern. The resulting alterations in dendritic arborization appeared to be in response to H 2 S exposure at the 20 and 50 ppm (28 or 70 m g /m 3 ) levels such that the 20 ppm (28 m g /m 3 ) level m ay 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 m ay constitute a chemically-induced change in the growth or organization of structural (or neurochem i cal) elements, they are to be regarded as possible indicators of a neurotoxic effect in accordan ce with guidance in EPA’s neurotoxicity risk assessment guidelines (U.S. EPA, 1998). The question as to whether the alterations are adverse, however, rem a ins 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 sam p ling technique (i.e., one Purkinje cell/pup). Also, the relationship of these alterations to a functional impairm e nt is unclear and cerebellar neurological sequelae are not the m o st obvious sym p tom associated with H 2 S exposure. It is acknowledged, however, that predicting functional impairm e nts 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 dynam i c self-organizing response of the developing brain to injury.
The effect of H 2 S on sperm a togenesis in the rat was assessed by Andrew et al. (1980). A group of 10 14-week-old m a le W i star rats were exposed to 220 ppm (306 m g /m 3 ) H 2 S for 3 hr/day for 7 days, com p ared to a control or nonexposed group. Following the last exposure, each

ma le was caged with two unexposed virgin fem a les for 10 consecutive weeks. Four m a le rats were adm i nistered triethylenemelamine and used as positive controls. The f ema les were sacrificed after 18 days cohabitation. Param e ters evaluated included fertility, corpora lutea, total implants, and dead implants. There were no adverse effects of exposure on these param e ters. Subsequently, the investigators evaluated exposure on prenatal development by exposing pregnant W i star rats to 220 ppm (306 m g /m 3 ) 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 m a lform a tions, and growth retardation. There was no evidence of embryotoxicity or external m a lform a tions.

31-32- 4.4. OTHER STUDIES

4.4.1. Respiratory Toxicity Cytotoxic effects of H 2 S 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 m g /m 3 ) H 2 S 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 perform e d on all animals. Lavage protein, lactate dehydrogenase (LDH), alkaline phosphatase (AP), cellular composition, 

### ( -glutamy l transpeptidase (GGT), and cytopathology were determined. Animals exposed to 400 ppm (556 m g /m 3 ) H 2 S exhibited a significant, but transient increase (800%) in NL protein at 1 hour postexposure that returned to

norm a l at 20 hours postexposure. There was also a transient increase (320%) in LDH from NL fluid in the 400 ppm (556 m g /m 3 ) exposure group. The cellularity in the NL fluid was increased by 139 and 483% in the 10 and 200 ppm (14 and 278 m g /m 3 ) 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 /m 3 ) H 2 S. Differential cell counts indicated that the increase in cellularity was initially due to desquam a tion of nasal epithelial cells (1 hour postexposure) followed by exudation of polymorphonuclear lymphocytes (PMN). Cytopathology revealed that the desquam a ted epithelial cells showed stages of ciliocytophthoria, cytoplasm i c vacuolation, pyknosis, karyorrhexis, cytoplasm i c constriction between the basal region and terminal plate, and separation of ciliated tuf t s. In the 10 ppm (14 m g /m 3 ) and 200 ppm (278 m g /m 3 ) animals, no other effects in NL fluid were observed. Similar to NL fluid, the BAL fluid of rats exposed to 400 ppm (556 m g /m 3 ) had an increase in protein content of approxim a tely 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 m g /m 3 ) H 2 S, 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 m g /m 3 ) exposure group, LDH activity in the BAL fluid was significantly decreased in the 44 hr postexposure group. In animals exposed to 400 ppm (556 m g /m 3 ) H 2 S, 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 m g /m 3 ) H 2 S had AP levels below baseline at 20 and 44 hours postexposure. GGT was elevated (900%) in the 400 ppm (556 m g /m 3 ) 1 hour postexposure animals. Animals exposed to 400 ppm (556 m g /m 3 ) H 2 S had a 42% reduction in nucleated cells in th e BAL fluid at 1 hour postexposure, but which returned to norm a l at 20 hours and increased to 30% over baseline at 44 hours postexposure.

32-33- Pulmonary alveolar macrophages were significantly reduced at 1 hour postexposure in animals exposed to 400 ppm (556 m g /m 3 ) H 2 S, but returned to norm a l by 20 hours postexposure. PMN increased in all postexposure times (highest at 20 hours) in animals exposed to 400 ppm (556 mg /m 3 ) H 2 S. In the 10 ppm (14 m g /m 3 ) and 200 ppm (278 m g /m 3 ) animals, no other effects in BAL were observed. The investigators concluded that vacuolization, ciliocytophthoria (a cellular degenerative change often from accom p anying 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 m g /m 3 ) H 2 S, dramatic changes can rapidly be restored due to the rem arkable repair capacity of the respiratory epithelium

. In addition, the cell counts in rats exposed to 10 ppm (14 m g /m 3 ) 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 dam a ge to the epithelium

. The investigators also suggested that increased LDH and AP observed 1 hour postexposure in BAL fluid is suggestive of dam a ge to the pulmonary epithelium

. However, the investigators stressed that H 2 S appears to be a weaker toxicant to the respiratory epithelium than other pneumotoxicants. The authors stated that the olfactory epithelium appears to be m o re sensitive to the toxic ef f ects of H 2 S 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 m g /m 3 ). The NOAEL is 10 ppm (14 m g /m 3 ). Histologic and ultrastructural alterations in the lungs of rats were reported in a similar study in which m a le Fischer 344 rats were exposed for 4 hours to either 82 or 440 ppm (116 or 613 m g /m 3 ; n = 12 rats per dose level) H 2 S followed by sacrifice at 1, 18, or 42 hours postexposure (Lopez et al., 1988a). Histologic changes were transient and m a inly present in rats exposed to 440 ppm (613 m g /m 3 ) H 2 S. Wh ile som e histologic changes were noted at 82 ppm (116 m g /m 3 ), no pathologic changes were reported at this exposure level. In rats exposed to 440 ppm (613 m g /m 3 ) H 2 S, bronchiolar ciliated cells developed necrosis, but necrotic dam a ge was rapidly repaired through mitosis. Examination of the lung demonstrated the notable edematogenic effect following exposure to 440 ppm (613 m g /m 3 ) H 2 S. Based on findings from the postexposure groups, the investigators suggested a chronology to the edematogenic effect in which fluid first accum u lates around the blood vessels, then the interstitium , and finally in the alveoli. Perivascular edema without involvem e nt of the alveoli in animals exposed to 82 ppm (116 m g /m 3 ) H 2 S suggested that fluid that accum u lated in the interstitium was reabsorbed before it entered the bronchoalveolar spaces. This m ay explain the finding by Lopez et al. (1987) that protein was detected in BAL fluid from animals exposed to 400 ppm (556 m g /m 3 ) H 2 S, but not 200 ppm (278 m g /m 3 ) H 2 S. The investigators also found a lack of structural changes in the

33-34- alveolar endothelium , basem e nt m emb rane, or type I pneumocytes, suggesting that H 2 S exposure to concentrations as high as 440 ppm (613 m g /m 3 ) does not com p rom i se the air-blood barrier. In addition, there was no adverse effect on the m a st cells. If dam a ge to the air-blood barrier and

ma st cell degranulation is not responsible for the observed pulmonary edema following H 2 S exposure, then the investigators suggested that the edema m ay be due to an outflow of liquid from the peribronchovascular connective tissue into the lum e n of small airways via high conductance pathways that fill the alveolar spaces in a retrograde m anner. Lopez et al. (1988b) examined the effect of acute H 2 S exposure on nasal epithelial cells. Male Fischer 344 rats were exposed to 0, 14, 280, or 560 m g /m 3 (0, 10, 200, or 400 ppm) H 2 S 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 m g /m 3 H 2 S. Nasal lesions were present only in the high-dose group and m anifested 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 m o st 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 Brennem an et al. (2000). At 44 hours postexposure, the respiratory

mu cosa was essentially repaired, but the olfactory m u cosa continued to exfoliate. Effects of H 2 S on nasal param e ters were m u ch less severe in the Sprague-Dawley rat study reported by CIIT (1983c). Green et al. (1991) examined the effects of H 2 S from the BAL of rats under the hypothesis that the hem o rrhagic pulmonary edema observed from exposure to high levels of H 2 S is associated with changes in pulmonary surfactant. Male Fischer 344 rats were exposed to 0, 200, or 300 ppm (0, 278, or 417 m g /m 3 ) H 2 S for 4 hours, and at 1 hour postexposure BAL fluid was obtained. Marked abnorm a lities in surfactant activity were demonstrated in the lavagates from rats exposed to the highest concentration only 300 ppm (417 m g /m 3 ) and not in the lavagate from the lower dose 200 ppm (278 m g /m 3 ) 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 H 2 S on lung bacterial defense has been investigated. Rogers and Ferin (1981) exposed m a le Long-Evans rats to 45 ppm (636 m g /m 3 ) H 2 S for 2, 4, or 6 hr followed by bacterial challenge to Staphylococcus epidermidis

. In control animals, m o st of the bacteria was inactivated by the 6-hour postchallenge sacrifice time. Rats exposed to H 2 S for 2 hours responded similarly to controls. However, rats exposed to H 2 S for 4 and 6 hours had 6.5- and 52-fold greater percent bacteria rem a ining, respectively, com p ared to controls. The investigators suggest that an H 2 S-induced absence of bacterial inactivation m ay explain secondary pneumonias in humans subsequent to acute or subacute H 2 S 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

m acrophages lost the phagocytic ability in vitro when exposed to 54 ppm (75 m g /m 3 ) H 2 S for 24 hours. Although the above studies are acute in nature (i.e., m o stly 4 hr) and are not appropriate fo r the establishment of a chronic guideline value, the results m ay of fe r insights into the processes underlying affects observed in the respiratory tract tissues in animals exposed in a

mo re protracted m anner to H 2 S such as by Brennem an et al. (2002). Nasal tissues were examined for histology and cytochrom e oxidase imm unoreactivity in 10-week-old m a le CD rats 24 hours after exposure to 0, 30, 80, 200, or 400 ppm (0, 42, 112, 280, or 560 m g /m 3 ) H 2 S for 3 hr/day for 1 day or for 5 consecutive days (Brennem an 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 H 2 S resulted in regeneration of the respiratory m u cosa and full thickness necrosis of the olfactory

mu cosa localized to the ventral and dorsal meatus, respectively. Repeated exposure to the sam e concentrations caused necrosis of the olfactory m u cosa with early m u cosal regeneration that extended from the dorsal m e dial meatus to the caudal regions of the ethmoid recess. Acute exposure to 400 ppm H 2 S induced severe mitochondrial swelling in sustentacular cells and olfactory neurons, which progressed to olfact ory epithelial necrosis and sloughing. Cytochrom e oxidase imm unoreactive cells were m o re frequently observed in regions of the olfactory m u cosa
comm only affected by H 2 S than in regions that were not. These findings demonstrate that acute exposure to >80 ppm H 2 S resulted in reversible lesions in the respiratory and olfactory m u cosae of the CD rat and, further, that cytochrom e oxidase activity (as judged from cytochrom e oxidase imm unoreactivity) m ay be a susceptibility factor for H 2 S-induced olfactory toxicity in the CD rat.
Transmission electron m i croscopy of cultured rabbit alveolar macrophage (AM) cells exposed in vitro to 54 ppm (75 m g /m 3 ) for 24 hours revealed extensive dam a ge to interior organelles and m emb rane resulting in a decreased percentage of AM capable of phagocytosis (Richardson, 1995). Khan et al. (1991) found evidence that H 2 S 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 /m 3 (0, 50, 200 or 400 ppm) for 4 hours before cell collection. Moulin et al. (2002) correlated nasal pathology in H 2 S-exposed rats with regions of high flux in the rat nasal cavity generated from anatomically accurate computational fluid dynam i cs (CFD). The specific location of lesions from na sal tract sections approxim a ting sections 4 and 5 of a study of Brennem an et al. (2000) were noted and the predominately olfactory-lined portions of the perim e ter of these 2 levels were divi ded into roughly similar size regions. CFD flux of

35-36- H 2 S at these regions and the incidence of nasal pathologies within these regions were then calculated and correlated. The analysis showed that H 2 S-related lesions were observed only at specif i c 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 (approxim ately 50% of the olfactory epithelium affected at 30 ppm [42 m g /m 3 ] and 70% at 80 ppm [111 m g /m 3 ]) whereas the olfactory epithelium of rats exposed to 10 ppm (14 m g /m 3 ) and controls were unaffected. In contrast to the localization of nasal lesions in the olfactory epithelium , CFD sim u lations predicted higher flux (and therefore a higher dose) to those regions lined by respiratory epithelium where no H 2 S-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 (Spearm an’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 ef f ects of H 2 S while respiratory epithelium is resistant. These results therefore lend credence to considering nasal toxicity from H 2 S as relevant to humans having olfactory epithelium that can be impinged upon by inhaled air streams containing H 2 S. The high correlation between predicted H 2 S 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 accomm odate the differences between these species, i.e., the relatively large area of olfactory epithelium in areas of high flux in rats and m i ce (Kimbell et al., 1997) com p ared 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 m o re refined estimates of necessary param e ters such as nasal extraction ef

fi ciency would be required bef o re such a procedure could be applied in a quantitative m anner. 4.4.2. Neurotoxicity Kom b ian et al. (1988) examined the acute effects of H 2 S on amino acid levels in various brain regions of the rat following i.p. adm i nistra tion of 10 or 30 m g /kg doses. Hydrogen sulfide adm i nistration 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 m ay play a role in the neuronal control of breathing. Kom b ian 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 H 2 S through

m echanism s involving respiratory arrest. Hannah et al. (1989; see Section 4.3) also reported depressed levels of these sam e neurotransmitters (save f o r 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 H 2 S at 105 mg /m 3 for 7 hr/day.

36-37- Elovaara et al. (1978) demonstrated a m arked reduction in the incorporation of labeled leucine in cerebral protein and my elin in adult fem a le m i ce exposed to 100 ppm (139 m g /m 3 ) H 2 S for 2 hours. The results indicate decreased br ain protein synthesis. The authors suggested that the changes m ay have resulted from the inhibition of cerebral CytOx activity. The authors also noted that 100 ppm (139 m g /m 3 ) is 5 to 20 times lower than exposure levels associated with human f a talities. Utilizing in vitro whole brain preparations, Roth et al. (1997) showed that the addition of 0.13μM Na 2 S can inhibit CytOx and carbonic anhydrase. Also, KCN, a specific inhibitor of cytochrom e oxidase, altered the rhythmical activity of rat hippocampal neurons in a m anner similar to Na 2 S or H 2 S. The authors speculated that the inhibition of the respiratory enzym e CytOx m ay 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 H 2 S levels on the hippocampus and neocortex of freely moving rats using electroencephalographic activity (EEG) as a m e asurement of neuronal function. The hippocampus was a focus of this study because this exposure of humans to high levels of H 2 S (i.e., approxim a tely 500 ppm or 695 m g /m 3 ) has been associated with m emo ry loss and varying degrees of learning and perceptual deficits (Kilburn, 1993; Tvedt et al., 1991a,b; W a sch 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 m g /m 3 ) H 2 S for 3 hr/day for 5 days. Results, recorded during the last 10 m i nutes of the exposures, indicated that repeated exposure to H 2 S produced a dose-related increase in the amplitude of hippocampal theta activity the total of which was cum u lative with exposure duration. The effect of H 2 S on hippocampal EEG was dose-dependent and highly significant at all concen trations. The authors postulated that elevated catecholamine and/or serotonin levels from th e inhibition of MAO m ay be involved in the sulfide-induced alteration of hippocampal theta activity, since the time f o r reactivation of MAO activity correlated with the time required f o r th e recovery of the total power of hippocampal EEG activity following exposure to H 2 S. The LOAEL for this study is 35 m g /m 3 (25 ppm). The authors also noted that exposure to 100 ppm (139 m g /m 3 ) H 2 S produced no significant effects on rat EEG activity recorded from the neocortex. In an earlier study, Skrajny et al. (1992) examined the effect of H 2 S exposure on serotonin and norepinephrine levels in the deve loping cerebellum and frontal cortex of SpragueDawley rats. Tim e d-pregnant rats were exposed to 20 or 75 ppm (28 or 104 m g /m 3 ) for 7 hr/day from day 5 postcoitus until PND 21. At parturition, treated and control animals were culled to 6 dam s , with a m a xim um 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 dam s was not stated nor was there an indication if pups were selected from randomly selected litters. Exposure to 75

37-38- ppm (104 m g /m 3 ) resulted in a statistically significant (p<0.01) increase in serotonin in the cerebellum from PND 7 to 14, com p ared to controls. At PND 21, the value rem a ined unchanged from PND 14. The increase was even m o re pronounced in the frontal cortex from PND 7 through 21 (p<0.01). The increase at 20 ppm (28 m g /m 3 ) in the frontal cortex was less pronounced than at 75 ppm (104 m g /m 3 ) 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 m o re difficult to interpret as there was no clear pattern of change. The investigators concluded that the effects of exposure on serotonin and norepine phrine, coupled with the laboratory’s prior evidence demonstrating possible m o rphological changes in Purkinje cell development and the known influence of monoam i nes on neural development, suggest a mode of action for H 2 S on the CNS. Higuchi and Fukamachi (1977) examined the effects of inhalation exposure to H 2 S on avoidance behavior in rats. Rats were exposed to 100–500 ppm (139-695 m g /m 3 ) H 2 S. 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 discrim i nated avoidance behavior were assessed. A rapid and significant decrease in discrim i nated avoidance response was observed at concentrations 

### $ 200 ppm (278 m g /m 3 ). At concentrations 

### $ 300ppm (417 m g /m 3 ) , the Sidman-type conditioned avoidance response was also decreased. The behavioral effects were reversed following ve ntilation with clean air or when exposure was terminated.
In an attem p t to demonstrate that the neurological sequelae following exposure to high concentrations of H 2 S is due to neuronal necrosis, Baldelli et al. (1993) adm i nistered sodium sulfide (Na 2 S) to unventilated and anesthetized-ventilated (to com p ensate for the loss of central respiratory drive) m a le W i star rats by i.p. injection. Doses in unventilated rats ranged from 84 to 200 m g /kg, while ventilated rats were adm i nistered 120, 150, or 200 m g /kg. In unventilated animals, all animals that were adm i nistered doses greater than 120 m g /kg (n=11) sulfide died in



### # 10 m i nutes. Doses of 120, 108, 100, 96, and 84 m g /kg were lethal to 7/10, 3/3, 1⁄2, 2/5, and 0/1 animals, respectively. An LD 50 of 94 m g /kg was calculated for 32 unventilated rats by the investigators. Of the 8 surviving unventilated animals, only one 120 m g /kg rat demonstrated histopathological signs of neuronal necrosis of the cerebral cortex. In ventilated animals (5 rats/group), an LD 50 of 190 m g /kg was calculated. None of the 120 or 150 m g /kg group animals died during compound adm i nistration. However, 4/5 of the 200 m g /kg group animals died within m i nutes of compound adm i nistration, and the sole-surviving 200 m g /kg animal and 4/5 of the 150 m g /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 pO 2 or alteration in pH or pCO 2 . Mean arterial blood pressure decreased to 42.8, 34.2, and 16.8 mm Hg for ventilated animals within 2-5 m i nutes of compound

38-39- adm i nistration. Mean arterial blood pressure recovered within 15- 20 m i nutes for the 120 and 150 m g /kg dose groups, but blood pressure did not return to baseline for 50 m i nutes in the lone surviving 200 m g /kg ventilated animal. There were significant deceases in EEG activities that were also dose-dependent. After doses of 

### $ 150 m g /kg, EEG activity decreased to near isoelectric levels. EEG activity returned to norm a l in the two lowest doses and showed som e recovery at the highest dose. Neuronal necrosis of the cerebral cortex, caudate, and brain stem was observed in the surviving 200 m g /kg animals. The investigators concluded that neuronal necrosis is not directly related to H 2 S exposure. Rather, neuronal necrosis is indirectly caused by hypotension induced by H 2 S exposure. The profound hypotension observed in the surviving 200 mg /kg ventilated animal probably resulted in cerebral ischemia. Three reports describe effects of H 2 S on neurological function in rats (Partlo et al., 2001; Struve et al., 2001). Partlo et al. (2001) exposed adult rats to125 ppm (174 m g /m 3 ) H 2 S for 4 hr/day for 5 days/week for 5 weeks. Controls were exposed to a nitrogen/air m i xture. Overt signs of eye irritation, respiratory distress, be havioral dysfunction or impaired consciousness were not observed at any time during the experim e nt. Testing of the animals revealed that this level of exposure did not affect m emo ry reten tion or acquisition, but did impair one measure of perform ance during the reacquisition of a reversed contingency (radial arm ma ze 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 a dult rats to the inhalation of 0, 30, 80, 200, or 400 ppm (0, 43, 111, 278, or 556 m g /m 3 ) H 2 S for 3 hr/day for 5 consecutive days. Wh en exposure levels equaled or exceeded 80 ppm (111 m g /m 3 ), the animals exhibited significant reductions in m o tor activity, body tem p erature, and water maze perform ance (spatial learning); it is to be noted, however, that the latter result m ay 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/m 3 ) H 2 S throughout gestation and postnatally to both dam s and pups (8 rats/litter after culling) PND 5 - 18. No decrement in behavioral perform ance, as judged by testing for m o tor 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 (Dorm an et al., 2000).

4.4.3. Cardiovascular Toxicity In a series of experim e nts, Kosmider et al. (1967) exposed 10 rabbits to 100 m g /m 3 (72 ppm) H 2 S for 1.5 hours and 17 rabbits to 100 m g /m 3 for 0.5 hours for 5 days. Electrocardiograms were taken after exposure and histochem i cal studies of the heart were perform e d. Ten rabbits served as controls for the histochem i cal studies and control

39-40- electrocardiograms were m a de on all animals 10 days prior to exposure. Nine of ten animals exposed for a single 1.5 hour exposure exhibited m a inly 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 bigem i nal 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, m u ltiply exposed animals also exhibited T wave depression. Histochem i cal studies revealed a decrease in ATP phosphohydrolase and NADPH 2 oxidoreductase activity, which suggests a direct e ffect on myocardial cells. A direct effect on ATP phosphohydrolase could alter Na + and K+ transport to the myocardial cell and could result in arrhythmia. Adm i nistration of sodium citrat e, a calcium chelator, attenuated the arrhythmias. Electrocardiogram effects have also been re ported in rats. Kohno et al. (1991) exposed

ma le W i star rats to 75 ppm (105 m g /m 3 ) H 2 S for 20, 40, or 60 m i nutes. Heart rate, blood pressure, and electrocardiogram were monitored during exposure and for one hour postexposure. At 20, 40, and 60 m i nutes, 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 som e 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 m a le Fischer 344 rats to 560 m g /m 3 (403 ppm) H 2 S for 4 hours or 2100 m g /m 3 (1511 ppm) H 2 S for 4 m i n. Ocular lavages were perform e d and cells were examined. There was a significant increase (approxim a tely 2 times) in exfoliated ocular cells associated with both H 2 S exposures. In all animals, the m a jority of recovered cells were corneal epithelial cells. However, a greater percentage of conjuctival epithelial cells was detected in exposed animals. The study demonstrates that H 2 S is an ocular irritant at very high concentrations.

4.4.5. Genotoxicity The m u tagenic potential of H 2 S was investigated using the Ames Salmonella typhimurium mutagenicity assay with and without Aroclor-induced ham s ter and rat liver S9 fractions. Hydrogen sulfide vapor (17-1750 

### : g/plate) was not m utagenic in S. typhimurium strains TA 97, TA 98, or TA 100 with and without m e tabolic activation (Hughes et al., 1984). Although H 2 S is not m utagenic, Berglin and Carlsson (1986) reported that H 2 S gas potentiated the m utagenicity of hydrogen peroxide in S. typhimurium strain TA 102. The investigators

40-41- believed that the increased m u tagenicity was due to H 2 S-m e diated formation of iron sulfide, which converts hydrogen peroxide to hydroxyl radicals m o re 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 H 2 S 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. Som e 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 H 2 S has been investigated. Studies included neurochem i cal, neuroelectrophysiological, and behavioral approaches to address this issue. Baldelli et al. (1993) concluded from studies using H 2 S with or without sim u ltaneous ventilation that the neuronal necrosis seen f o llowing H 2 S exposure is not a direct result of H 2 S, 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, m ay 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 th e brain originates from within the brainstem , while catecholamines and serotonin affect resp iratory rhythm (Warenycia et al., 1989b). To explore this possibility further, experim e nts on monoam i ne oxidase (MAO) enzym e activity and neurotransmitter levels in various regions of the brain have been perform e d 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 H 2 S exposure. On the other hand, som e investigators believe that pulmonary edema m i ght be a primary cause of death following exposure to high concentrations of H 2 S (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 th e afferent endings of the pulmonary vagi, and that high amounts of sulfide tend to paralyze the ventilatory center (Haggard and Henderson, 1922).
Alm e ida and Guidotti (1999), on the basis of rat studies, suggest that the lung m ay be the primary locus of HS ion action that can lead to vagal paralysis. Khan et al. (1990) investigated the action of H 2 S on a m o lecular level in the lung, in which lung mitochondria incubated with H 2 S exhibited a dose-dependent inhibition of cytochrom e oxidase (CytOx) activity. Exposure to

41-42- 50 ppm (70 m g /m 3 ) H 2 S and higher significantly reduced CytOx activity in lung mitochondria in a dose-dependent m anner . Since CytOx is the terminal step in aerobic oxidative m e tabolism , inhibition by H 2 S would stop tissue respiration and lead to the development of hypoxia. Case reports involving individuals in accident al overexposure situations indicate that H 2 S can cause direct neurotoxicity leading to respir atory distress and a variety of secondary adverse effects such as pulmonary edema, ataxia, m o tor weakness, headache, nausea, dyspnea, chest pain and dizziness. In som e 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 m g /m 3 ). Those associated with imme diate physical collapse or “knockdown”have been estimated to be in the range of 500-1,000 ppm (695-1,390 m g /m 3 ). Levels associated with secondary pulmonary edema have been estimated to be in the range of 250-500 ppm (348-695 m g /m 3 ) (Milby and Baselt, 1999a,b; Reiffenstein et al., 1992). Limited epidemiological studies (Richards on, 1995; Hessel et al., 1997; Vanhoorne et al., 1995), supported by findings in laboratory animal s, have reported cardiovascular, pulmonary, and ocular effects attributable to H 2 S. High concentrations of H 2 S, such as in the Breysse (1961) report “as high as 4,000 ppm (5,560 m g /m 3 ) H 2 S,” 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 m g /m 3 ) (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 m u cosa as th e principal target site affected (Dorm an et al., 2000; Brennem an et al., 2000), m o st likely as a consequence of the irritant properties of H 2 S. In these two subchronic studies, nasal lesions of the olfactory m u cosa were observed in animals exposed to 30 or 80 ppm (42 or 111 m g /m 3 ) and consisted of olfactory neuron loss and basal cell hyperplasia. No effects were observed at 10 ppm (14 m g /m 3 ). Male and fem a le B6C3F1/CrlBr mi ce exhibited m i nim a l to mild inflamma tion of the anterior portion of the nasal m u cosa when exposed to 80 ppm (111 m g /m 3 ) H 2 S for 6 hr/day, 5 days/week for 90 days (CIIT, 1983a). Only mi nim a l lesions were found in Sprague-Dawley or Fischer 344 rats (CIIT 1983 b,c). Wh ile strain and species differences m ay explain the discrepancy in response between the SpragueDawley rats and the Fischer-344 rats and B6C3F1 m i ce, it is not known why contrasting effects of H 2 S on nasal lesions were seen by Dorman et al. (2000) vs. CIIT (1983c) since SpragueDawley rats were used in both cases. This discrepancy m ay relate to the use of computational fluid dynam i cs models (e.g., Kim b ell and Subramaniam , 2001) to determine specifically where to look for lesions. This m e thodology was not available at the time of the earlier CIIT (1983a,b,c) studies.
It has been m a de clear that exposure to H 2 S 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 H 2 S near to those at which nasal lesions of Brennem an et al. (2000) are observed.

42-43- Neurotransmitter levels were significantly altered in the brains of postpartum rats pups exposed in utero and postpartum to 28 m g /m 3 (20 ppm) H 2 S (Skrajny et al., 1992). Altered m o rphology of the cerebellar Purkinje cells was noted by Hannah and Roth (1991), also at 28 m g /m 3 H 2 S. Evidence for mechanism s by which neurotoxic effects m ay occur implicates ischemia m e diated by H 2 S inhibition of respiratory chain enzym e activity, in particular cytochrom e oxidase. Khan et al. (1990) reported significant decreases in several respiratory chain enzym e s during exposures at H 2 S above but not at 10 ppm (14 m g /m 3 ). 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 som e instances to discrim i nate the mechanistic transition from being a nonadverse- to an adverseresponse as are the olfactory lesions noted by Brennem an et al. (2000) at a NOAEL of 10 ppm (14 m g /m 3 ).

4.5.2. Oral Exposure In a subchronic oral study in rats, the only one that was found, m o rtality and neuromuscular and behavioral signs have been reported in animals gavaged with 7 m g /kg-day H 2 S in deionized/deoxygenated water for 89 days (Anderson, 1987). Effects included labored breathing, convulsions, and sedation. Mortality was also noted in som e ma les and fem a les exposed to 3.5 m g /kg-day H 2 S for 89 days. Although restlessness was observed in all treated

ma les and increased salivation in all treated f ema les, 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 m a les, and salivation in treated fem a les, is not clear. No histopathological lesions attributable to H 2 S 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 m a les 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 H 2 S were located. Genotoxicity studies also indicate that H 2 S was not m utagenic in S. typhimurium strains TA97, TA98 or TA100 with or without m e tabolic activation (Hughes et al., 1984). Although H 2 S does

43-44- not appear to be m u tagenic, H 2 S gas potentiated the m utagenicity of hydrogen peroxide in S. typhimurium strain TA102 (Berglin and Carlsson, 1986). The investigators reported that the increased m utagenicity was due to H 2 S-m e diated formation of iron sulfide, which converts hydrogen peroxide to hydroxyl radicals m o re 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 m g /m 3 ) H 2 S resulted in abnorm a l growth and

mo rphology of developing cerebellar Purkinje cells and changes in neurotransmitter levels (Skrajny et al., 1992; Hannah et al., 1989). Therefor e pregnant wom e n m i ght be considered as a susceptible subpopulation with the hazard, if any, to the offspring of these wom e n.

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 som e anecdotal evidence (following) that children m ay be m o re susceptible than adults to the adverse effects H 2 S exposure. A case study reported that a 20-month-old child developed intermittent paroxysm a l tonic deviation of the eyes, choreoathetosis and dys tonia with progressive involuntary movem e nts of the entire body following exposure to at least 0.6 ppm (0.8 m g /m 3 ) H 2 S over a period of up to “nearly a year” (Gaitonde et al., 1987). From the text within the report, coexposure to the sam e levels of H 2 S 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 m ay be selectively susceptible to the neurological effects of H 2 S at least at relatively high levels, > 600 ppb. Ascribing relevance of this apparent susceptibility to environmental levels of H 2 S would be, however, conjectural. Based on the findings of the effects of H 2 S 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 m onth 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 H 2 S m ay exist.

4.7.2. Possible Gender Differences Dorman et al. (2000) examined effects on reproductive perform ance from inhalation exposure to H 2 S (see Section 4.3). There were no statistically significant effects on reproductive perform ance (mating index, f e rtility index, postimplantation loss per litter, and number of late

44-45- resorptions or stillbirths) in F 0 fem a le animals. Also, the number of live pups, litter size, average length of gestation, and average number of implants per fem a le were not affected. In

com p aring high-exposure and control m a le 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 wa s found (Anderson, 1987). A number of problem s with that study precluded the derivation of an or al 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 treatm e nt 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 di sease that precludes drawing conclusions from this study regarding the effect of H 2 S, (3) there was a 50% death rate in the high dose m a le group, the only group with significant m o rtality, but the cause of death “could not be determined.” This also inspired a lack of confidence in the data, (4) alopecia was comm on 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 “norm a l” and thus appropriate for the production of reliable data.

5.2. INHALATION REFERENCE DOSE

5.2.1. Choice of Principal Study and Critical Effect w i th 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 H 2 S. Several groups of investigators (Tvedtet al., 1991a,b; Kilburn, 1993, 1997; W a sch et al., 1989) have reported long-term adverse neurological sequelae of H 2 S-induced unconsciousness in humans during occupational, accidental, and chronic exposures including neuropsychological and neurobehavorial decrements, brain dam a ge, and, in one instance, dem e ntia. 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 sam e concentration range at lower levels of H 2 S

45-46- exposure, and both are clearly of relevance to humans. Based on the relative clarity of the

m echanistic basis, the dose-response information at lower levels of H 2 S exposure, and adverse nature of the endpoint for nasal tract lesions as com p ared 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 H 2 S associated with these effects appear to be similar for either endpoint (e.g., the possible indicator of neurotoxic effects reporte d by Hannah and Roth (1991) to occur at 28 m g H 2 S /m 3 and the no-effect level of nasal tract lesi ons reported by Brennem an et al. (2000) at 14 mgH 2 S /m 3 ) is som e indication that consideration f o r the one will also address the other. On the other hand, the sam e judgments m a de here concerning clarity of mechanism or dose-response or adversity could serve as the basis for a reevaluation of the critical endpoint if these m a tters 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. Wh ile such alterations m ay constitute a possible indicator of a neurotoxic effect defined in the U.S. EPA neurotoxicity risk assessment guidelines as "neuroanatomical changes occurring at any le vel of nervous system organization" (U.S. EPA, 1998), their biological significance is not sufficiently understood to clearly ascertain the nature of the adverse outcom e. The effects of certain developmental insults on cerebellar structure and function have been well characterized (Fergus on 1996) and provide a basis with which to gauge the significance of the Hannah and Roth (1991) findi ngs. 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 m i saligned and exhibit aberrant dendritic f o rm

. These animals exhibit tremor, ataxia, altered spontaneous activity and their perform ance on rotorod and cognitive tests (swim

m aze) is impaired. Similar neuroanatom i cal and functional alterations are observed after early life treatm e nt of rats (PND 4 onwards) with antimitotic agents such as m e thylazoxymethanol. Dorman et al., (2002) did not find evidence of the types of neuroanatom i cal alterations or behavioral impairm e nts associated with these models in their dose-response assessment of developmental exposure to hydrogen sulfide. Wh ile it is noteworthy that Dorman et al. (2002) did not did not assess Purkinje cell dendritic m o rphology 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 neuroanatom i cal or behavioral alterations associated with well-characterized models of developmental cerebellar insult. Wh ile 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

46-47- Roth (1991) are nearly the sam e 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 consid ered for derivation of an inhalation RfC: Dorman et al. (2000), CIIT (1983a), and Brenne man et al. (2000). In com p aring high-exposure and control m a le animals exposed subchronically to 80 ppm H 2 S 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 com p ared to the control group. CIIT (1983a) reported that m a le and fem a le m i ce exposed to 80 ppm (111 m g /m g 3 ) H 2 S, 6 hr/day, 5 days/week for 90 days exhibited m i nim a l to mild inflamma tion of the anterior portion of the nasal m u cosa. Only m i nim a l lesions were observed in two species of rats (Sprague-Dawley and Fischer-344) examined by the sam e authors (CIIT, 1983b,c) and in m i ce exposed to lower concentrations of H 2 S. However, in m a le Sprague- Dawley rats exposed to 0, 10, 30, and 80 ppm (0, 14, 42, and 111 m g /m 3 ) H 2 S for 6 hr/day, 7 days/week, for 10 weeks, Brennem an et al. (2000) reported nasal lesions of the olfactory m u cosa in the 30 and 80 ppm (42 and 111 m g /m 3 ) exposure groups. No effects were observed in the control or 10 ppm (14 m g /m 3 ) exposure animals that were considered treatm e nt-related. Nasal lesions consisted of mu ltifocal, bilaterally symme trical olfactory neuron loss and basal cell hyperplasia affecting the lining of the dorsal

me dial meatus and dorsal and m e dial region of the ethmoid recess. The difference in results between Brennem an et al. (2000) and CIIT (1983a) m ay indicate a variability in animal response. The study by Brennem an et al. (2000) was considered to be the m o st appropriate for the derivation of an inhalation RfC for several reasons. First, the critical effect (nasal lesions of the olfactory m u cosa; summarized in Table 2 in Section 4.2.2) has been reported by other investigators (Dorm an 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 H 2 S toxicity by numerous researchers; fourth, the LOAEL (42 m g /m 3 ) and NOAEL (14 m g /m 3 ) 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 Brennem an et al. (2000) (T able 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

47-48- incidence; see Table 2) was not amenable to BMD modeling, thus NOAEL and LOAELs were designated. Converting ppm to m g /m 3 resulted in a NOAEL of 14 m g /m 3 and a LOAEL of 42 mg /m 3 (1 ppm = 1.39 m g /m 3 ). The LOAEL and NOAEL were then adjusted to a weekly continuous exposure such that (N)LOAEL ADJ = (N)LOAEL (mg/m 3 ) x hrs exp/24 hours x day exp/week :

NOAEL ADJ = 14 m g /m 3 x 6/24 hours x 7/7 days/week = 3.5 m g /m 3 LOAEL ADJ= 42 m g /m 3 x 6/24 hours x 7/7 days/week = 10.5 m g /m 3

Thus, a LOAEL ADJof 10.5 m g /m 3 and a NOAEL ADJof 3.5 m g /m 3 were obtained. Conversion of these (adjusted) effect levels to human equivalent concentrations, i.e. HECs, is typically accom p lished through application of procedures specified in the inhalation RfC methodology (U.S. EPA 1994b). Species speci fic param e ters of anatomy, physiology and location of the lesion are utilized to generate a dosimetric adjustm e nt factor (DAF) that is then applied to the effect level of interest, a NOAEL in this instance, to generate a NOAEL HE C For H 2 S, 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 (V E ) was calculated for m a le 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

ma le rats was calculated as f o llows:

lnV E = b 0 + b 1 ln(0.267kg)

The param e ters, b 0 and b 1 , are species specific param e ters listed in Table 4-6 of U.S. EPA (1994). This gave a V E of 0.19 liters/minute for m a le 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 cm 2 , respectively. These values are given on page 4-26 of U.S. EPA (1994b). The RGDR was calculated as follows:

RGDRET = (V E /SA ET ) animal /(V E /SA ET ) human= (0.19/15)/(13.8/200) = 0.184

The animal (N)LOAEL ADJis then m u ltiplied by the RGDRET to yield the (N)LOAEL HE C .

NOAEL HE C = NOAEL ADJx RGDRET = 3.5 m g /m 3 x 0.184 = 0.64 m g /m 3 LOAEL HE C = LOAEL ADJx RGDRET = 10.5 m g /m 3 x 0.184 = 1.9 m g /m 3

48-49- The computational fluid dynam i c (CFD) modeling information provided by Moulin et al. (2002) on the nasal pathology of H 2 S provide useful insight into the results from the current default RfC procedure used in constructing the HEC for H 2 S. From the work of Brennem an et al. (2000) it is clear that the lesions observed in rats from H 2 S 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 sim u lated levels of H 2 S flux (and therefore H 2 S dose) correlated with the incidence of lesions with in the olfactory epithelium , suggesting that the appropriate dose response m e tric for H 2 S 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 adjustm e nt, nor do they use a surface area measure that discrim i nates 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 com p arison of H 2 S exposure (i.e, an HEC) the results m ay be used to provide valuable insight into the limitations of the current procedures. Future work in this area m ay provide inf o rmation to perform a m o re quantitative evaluation of the HEC for H 2 S.

5.2.3. RfC Derivation—including Application of Uncertainty Factors (UFs) and Modifying Factors (MFs) The RfC for H 2 S is derived by dividing the NOAEL HE C 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 (10 1/2 ) was used rather than 10 because of a dosimetric adjustm e nt 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 repr oductive perform ance. 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 m u lti-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 toameliorate concerns that children are a susceptible population at least in the situation of long49-50- term relatively low level exposures. Som e circumstantial data seem to indicate that, under high level exposures, children m ay be m o re 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 NOAEL HE C of 0.64 mg /m 3 , yielding an inhalation RfC of 0.002 m g /m 3

.

RfC = NOAEL HE C / Uncertainty Factors RfC = 0.64 m g /m 3 / (10 x 10 x 3) = 0.002 m g /m 3 = 2E-3 m g /m 3

5.3. CANCER ASSESSMENT No data pertaining to the potential carcinogenicity of H 2 S 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 m anufacture of heavy water and other chemicals, in m e tallurgy, and as an analytical reagent. Although quantitative data are lacking, toxicity studies suggest that H 2 S 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 m e tabolic pathway for H 2 S, with thiosulfate and sulfate as m e tabolites. Metabolism in laboratory animals and in humans appears to be similar. Hydrogen sulfide is excreted in the urine.
Hum an 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 H 2 S (at high concentrations) has profound effects on the respiratory system leading to unconsciousness with attendant neurologic sequelae and,

som e times, death. An increase in cardiovascular-related deaths due, in part, to H 2 S exposure was reported in one occupational study.

50-51- Inhalation studies in adult rodents demonstrate sensitivity of nasal olfactory epithelium to low concentrations of H 2 S. The RfC is based on these lesions. Limited evidence suggests that exposure of humans to low concentrations m ay also cause neurologic sym p tom s although quantitative exposure-response data is lacking. Because of similar access of inhaled H 2 S 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. Wh ereas adverse nasal effects are of relevance and concern in adult human exposure scenarios, inhalation studies of perinatal or neonatal exposure in rats demonstrates abnorm a l cellular development in the brain as well as significant alterations in neurotransmitter levels; the toxicological signif i cance of these f i ndings 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 di sturbances in pigs consuming feed containing hydrogen sulfide was derived in the previous IR IS entry. A review of the RfD (see Section 4.2.1) indicates that the effects on which that va lue were based are not reproducible and probably not related to H 2 S. 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 sam e range or som e what 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) m ay 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 H 2 S, i.e., > 600 ppb. However, the relevance of this apparent susceptibility at environmental levels of H 2 S 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 H 2 S 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 H 2 S in humans can be acute and/or chronic. The weight-of-evidence from the human and animal studies suggest that the effects of H 2 S are dose-dependent. The exposureresponse relationship for acute effects, particul arly CNS and respiratory, can be very steep. Levels in the range of 500 to 1,000 ppm (695 to 1,390 m g /m 3 ) are life-threatening and can cause

51-52- imme diate unconsciousness f o llowed 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

m onitoring data since nearly all cases were those involving acute accidental overexposure. Levels dangerous to human health m ay not be detected by odor since high levels of H 2 S can paralyze the olfactory nerves m aking 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 H 2 S toxicity. Based on the clear nature of their adversity (demonstrable histopathology), information available on exposure-dose-response and the comm onality 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 (Brennem an et al., 2000) m ay be similar to one another such that consideration of one m ay be inclusive of the other. However, m o re extensive and definitive information on the neurologic endpoints could reveal that these should be the m o st relevant endpoint, m o re so than nasal tract lesions. Such information m ay provide sufficient reason to reassess H 2 S.
Som e data exist inf e rring that children/neonates m ay be m o re susceptible than adults to the neurological effects of H 2 S. 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 H 2 S (>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 H 2 S (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 sam e underlying

m echanism , inhibition of mitochondrial cytochrom e oxidase activity. Overall, these observations indicate a possible concern regarding the susceptibility of children exposed to high levels of H 2 S, i.e., > 600 ppb. However, the relevance of this apparent susceptibility at environmental levels of H 2 S 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

52-53- 0.002 m g /m 3 or 1.4 ppb. This value was derived from a subchronic inhalation study in laboratory rats by Brennem an et al. (2000). Th is study reported critical effects, nasal tract lesions in olfactory epithelium , in m a le rats exposed to 30 or 80 ppm (42 or 111 m g /m 3 ) but not at 10 ppm (14 m g /m 3 ), the NOAEL. An overall uncertainty factor of 300 was applied to account fo r interspecies extrapolation f r om 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 respirat ory effects in ambient exposure environments.
The overall confidence in the RfC assessment is m e dium-high; confidence in the principal study is m e dium because it was well-designed and conducted, and examined sensitive endpoints. However, it was subchronic in duration, and examined only m a le rats. Overall confidence in the data base is m e dium-high because the endpoints are supported by other subchronic studies. A chronic inhalation study in rats and m i ce, with additional dose groups and

a m o re extensive com p arative examination of neurologic and nasal tract endpoints from the sam e exposures, would increase the confidence in the data base. Ongoing scientific efforts to provide further refinem e nts to the assessment of H 2 S, particularly in the area of regional dosimetry of the nasal tract, are acknowledged.

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61-62- 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 perform e d by scientists within EPA and a m o re formal external peer review perform e d by scientists in accordance with EPA guidance on peer review (U.S. EPA, 1998, 2000a). Comme nts m a de by the internal reviewers were addressed prior to submitting the documents for external peer review and are not part of this appendix. Public comme nts 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 m a de 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 comme nts

ma de by the external reviewers and EPA’s response to these comme nts 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 som e 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 m o st 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 (Brennem an 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 m o rphological/structural changes in Purkinje cells as the critical effect. A second reviewer

62-63- 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 m u st be given first priority ....the nasal epithelial story in rats ....is not relevant.” This reviewer did not offer a suggestion for addr essing 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 guidelin es 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 impairm e nts from specific structural alterations is difficult due to the selective nature of the observed alterations and the dynam ic 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 accom p lished in this assessment (Sections 4.3.3., 4.4.1, and 4.5.2), both nasal tract lesions and neurologic effects occur in the

sam e concentration range at relatively low levels of H 2 S exposure, and both are clearly of relevance to humans. That the levels of H 2 S 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 m g H 2 S /m 3 and the no-effect level of nasal tract lesions reported by Brennem an et al. at 14 m g H 2 S /m 3 ) is som e indication that consideration f o r the one will also address the other.

Based on the relative clarity of the mechanistic basis, the dose-response information at lower levels of H 2 S exposure, and adverse nature of the endpoint for nasal tract lesions as

com p ared to the neurologic endpoints, as well as the recent information suggesting olfactory tissue to be a sensitive target tissue in rats (and therefore m o st likely in humans), nasal tract lesions are chosen as the critical effect on which to base the noncancer dose-response assessments.

63-64- C. General Question: Have the noncancer assessments been based on the m o st 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 m o re thorough discussion of the neurotoxicity toxicity studies in both the Toxicological Review and IRIS Summary s hould 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 com p arative 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 clarif ication that neurologic changes and nasal tract effects (olfactory epithelium ) occur at about the sam e externally applied concentrations of H 2 S, but that the nasal tract lesions are m o re clearly regarded as adverse than are the m o st sensitive neurologic alterations noted (i.e., the alterations in Purkinje cell arborization). All animal studies were reviewed to see if it w ould be instructive to identify NOAELs and LOAELs for studies other than the principal and supporting studies. The nasal lesions caused by H 2 S are relevant to humans for a variety of reasons, to include (1) a sentinel response alerting individuals to the presence of H 2 S, (2) dam a ge caused by H 2 S to the olfactory nerves causing anosm i a, 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 H 2 S, Moulin et al. (2002) reported modeled sim u lations of H 2 S f l ux to this tissue that was highly correlated with the lesions. These sim u lations imply that human olfactory tissues could also be exposed to inhaled H 2 S, and, as no com p elling reason exists to suspect that human olfactory tissue would react any diffe rently to the irritant properties of H 2 S, 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

64-65- com p rehensiveness 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 som e studies be removed?

Comment: The only reviewer responding to this question suggested that a m o re com p rehensive treatm e nt 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 m o re 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 discu ssed 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 adjustm e nt procedures applied to the effect levels in rats (i.e., to accomm odate the differences in breathing style and anatomy) were m a de 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 m ay not be able to totally address all possibilities regarding neurological ef f ects has been m a de 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 m a terial to the nasal tract tissues, and that interindividual variability to such a direct mechanism would be m i nim a l, the inf o rmation needed to consider this m a tter (and a concomitant reduction of uncertainty) is not available to sufficiently com p el such a reduction at this time. It is also questionable as to whether such a

65-66- direct mechanism would promote effects at lower concentration, even for m u ch longer (i.e., lifetime) exposures, the precept on which the subchronic to chronic uncertainty factor is evoked. However, the sam e response has to be m a de for the interindividual UF, i.e., the information needed to consider this m a tter (and a concomitant reduction of uncertainty) is not available to sufficiently com p el such a reduction at this time.

The results of Moulin et al. (2002) and Br ennem an et al. (2000) indicate that olfactory tissues in rats are sensitive to the ef f ects of H 2 S, mo re so than respiratory epithelium

. Modeling sim u lations (computational fluid dynam i cs) in rats of specific air flows to these tissues indicate olfactory tissues to be specif i cally sensitive to the ef f ects of H 2 S, despite higher regional flow to the respiratory epithelium (Moulin et al., 2002). As discussed above, these sim u lations imply that human olfactory tissues could also be exposed to inhaled H 2 S, and, as no com p elling reason exists to suspect that human olfactory tissue woul d react any differently to the irritant properties of H 2 S (the suspected mechanism of dam a ge), 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 com p rehensiveness of the data base? Do these statements m ake sufficiently apparent all of the underlying assum p tions 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 m e dium

.
Response: The confidence statement was corrected such that the overall confidence in the Rf C reflected that of the data base, i.e., m e dium 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 m o re 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 confirm e d in another species.

66-67- 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 H 2 S-induced brain dam a ge due to cytochrom e oxidase enzym e inhibition causing m o rbidity and m o rtality (Warenycia et al., 1989a,b) versus the peripheral (lung-related) effect of H 2 S that ultimately results in hypoxia due to apnea (Baldelli et al., 1993; Alm e ida and Guidotti, 1999)?

Comment: One reviewer indicated that the cytochrom e 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 H 2 S directly on the brain. There m u st be an exposure to H 2 S lasting f o r a mi nim um of about 5 m i nutes during a period of unconsciousness before hypoxic brain dam a ge occurs. There is no reliable evidence that intermittent exposure to levels of H 2 S below 1 or 2 ppm constitutes a danger to physical health.

Response: This effect progression was m o re clearly expounded upon in the text of the Toxicological Review. The increased emphasis placed in the assessment on the role of cytochrom e oxidase in the progression of toxicity, and the advancement of the concept that such an alteration (i.e., statistically significant inhibition of cytochrom e oxidase activity) at low levels of H 2 S could be considered a sensitive indicator and harbinger of H 2 S toxicity should now be apparent to the reader of this assessment.

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