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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1982–1983. Nasal and lung histologic specimens from the study were reexamined in 2002–2003 by a board-certified veterinary pathologist
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Respiratory tract toxicity of inhaled hydrogen sulfide in Fischer-344 rats, Sprague–Dawley rats, and B6C3F1 mice following subchronic (90-day) exposure
Author:
Dorman DC, Struve MF, Gross EA, and Brenneman KA
Year:
2004
Bibliographic source:
Toxicology and Applied Pharmacology, 198, 29– 39
Reference Type:
study report
Title:
Unnamed
Year:
1983
Reference Type:
secondary source
Title:
TOXICOLOGICAL REVIEW OF HYDROGEN SULFIDE (CAS No. 7783-06-4). In Support of Summary Information on the Integrated Risk Information System (IRIS)
Author:
U.S. Environmental Protection Agency
Year:
2003
Bibliographic source:
EPA/635/R-03/005. www.epa.gov/iris

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Hydrogen sulphide
EC Number:
231-977-3
EC Name:
Hydrogen sulphide
Cas Number:
7783-06-4
Molecular formula:
H2S
IUPAC Name:
hydrogen sulfide
Test material form:
other: gas
Details on test material:
Hydrogen sulfide gas was obtained from Matheson Gas Company (Morrow, GA). Purity of the test material was confirmed using gas chromatography, and it was determined to be composed of 99.68% H2S, 0.29% propylene, and 0.03% propane

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. (Portage, MI)
- Age : 6 weeks old at the time of purchase.
- Weight at study initiation: no data
- Fasting period before study:
- Housing: individually housed in stainless steel inhalation exposure cages
- Diet: ad libitum, Purina Certified Rodent Chow 5002 (St. Louis, MO)
- Water: ad libitum filtered tap water
- Acclimation period: 14-day

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 to 27
- Humidity (%): 25 to 77%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12-h light–dark cycle

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure:
whole body
Vehicle:
clean air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Exposures were conducted in 8 m3 stainless steel and glass inhalation chambers.
- Method of holding animals in test chamber: no data
- Source and rate of air: no data
- Method of conditioning air: no data
- System of generating atmosphere: Exposure atmospheres were generated by metering 3% H2S from a gas cylinder through a regulator (Matheson, East Rutherford, NJ) that maintained the delivery line pressure at 50 psi. The gas then streamed through a flowmeter (Matheson) that measured the flow rate of gas to each chamber. The test chemical was then diluted with filtered dry air and introduced to the top of the 8 m3 exposure chamber. The flow of test material from the flowmeter and the total airflow through the chamber were adjusted to maintain the target concentration within the chamber.
- Temperature, humidity, pressure in air chamber: The temperature and humidity of each test chamber were measured and recorded approximately hourly with an ASTM thermometer and a Certified Hygrometer Indicator, respectively. Chambers were maintained at an average daily temperature between 20 and 26 °C and an average daily humidity between 37% and 71%.
- Air flow rate: Airflow was monitored
- Air change rate: no data
- Method of particle size determination: not appropriate
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: Chamber air samples were automatically collected using a Valco Instruments (Houston, TX) custom-designed, pneumatically operated 10-port sampling system to confirm actual chamber H2S concentrations. Exposure chambers were sampled every 45–60 min while the control chamber was sampled approximately every 90 min. Chamber samples were also obtained manually each week with precision sampling gas syringes. A gas chromatograph (Hewlett-Packard Model 5710A, Palo Alto, CA) equipped with a 1.8mby 0.3 cm Teflon, Chromosil 310 column (Supelco, Bellefonte, PA) and a photoionization detector (HNU Systems, Newton, MA) was used to analyze the chamber atmosphere during the experiment as well as to analyze all manually collected samples. The chromatograph oven temperature was held at 50 jC, the injector port was maintained at 100 jC, and the photoionization detector was
kept at 130 jC. Prior to animals being placed in the 8 m3 chambers, each chamber was checked for uniformity of distribution of the H2S vapor by verifying its concentration at six to nine positions within the chamber.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
see below
Duration of treatment / exposure:
90 days
Frequency of treatment:
6 hr/day, 5 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
10.1, 30.5, or 80 ppm (0, 14, 42, or 111 mg/m3)
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
10; 30 and 80 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes, sham-exposed

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prestudy during the 14-day quarantine period, and approximately 1 week before necropsy
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: approximately 24 h after the last H2S exposure
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters : erythrocyte count, hemoglobin, hematocrit, total and differential leukocyte counts, platelet counts, mean corpuscular volume (MCV),
mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), methemoglobin, and sulfhemoglobin.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: approximately 24 h after the last H2S exposure
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters : serum clinical chemistry evaluations were performed: glutamic pyruvic transaminase, total bilirubin, urea nitrogen, glucose, glutamic oxaloacetic transaminase, total protein, phosphorus, sodium, potassium, chloride, calcium, alkaline phosphatase, and gamma glutamyl transpeptidase

URINALYSIS: Yes
- Time schedule for collection of urine: approximately 12-h fast before necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters: volume,
appearance, occult blood, specific gravity, protein, pH, ketone, and glucose.

NEUROBEHAVIOURAL EXAMINATION: Yes
A neurological exam was performed before necropsy in which posture, gait, facial muscle tone, and pupillary, palpebral, extensor thrust, and crossed-extensor thrust reflexes were assessed.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
ORGAN WEIGHTS: brain, kidney, spleen, liver, heart, and ovaries/testes.
HISTOPATHOLOGY: Yes
cerebellum, cerebrum, medulla, optic nerve, spinal cord, sciatic and anterior tibial nerves, eyes, pituitary, thyroid, parathyroid, salivary glands, heart, lungs, spleen, liver, pancreas, adrenals, mesenteric and mandibular lymph nodes, kidneys, bladder, lacrimal glands, ovaries, uterus, oviducts, vagina, cervix, stomach, duodenum, ileum, jejunum, large and small colon, caecum, skeletal muscle, skin, mammary glands (both sexes), femur, bone marrow, aorta, ear canal with zymbal gland, nasal turbinates (four levels), trachea, testes, epididymis, esophagus, thymus, prostate, seminal vesicle, and gross lesions.
Statistics:
Parametric data such as body weight and food consumption were analyzed using an analysis of variance (ANOVA). Statistically significant differences that were noted were further studied by either Tukey’s (equal sample sizes) or Scheffe’s (unequal sample sizes) Test of Multiple Comparison. Nonparametric data such as organ weight ratios were analyzed using a Kruskal–Wallis ANOVA and a Test of Multiple Comparison. Discontinuous data, such as appropriate indices of histopathological findings, were compared using chi-square or Fisher’s Exact Probability Test with a Bonferroni correction. A probability value of <0.05 was used as the critical level of significance for all statistical tests. Unless otherwise noted, data presented are mean values +/- SD.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Two mice, one female and one male, from the 80 ppm H2S exposure group were euthanized on study days 5 and 6, respectively, after being found prostrate and hypoactive. Two other female mice from the control group and one male mouse from the 30 ppm H2S exposure group also died during the study due to injuries associated with moving the animals or from unknown causes.
Observed clinical signs were not related to H2S inhalation

BODY WEIGHT AND WEIGHT GAIN
Exposure of mice to 80 ppm H2S was also associated with decreased body weight gain (Fig. 1) or terminal body weights (Tables 1) when compared with control animals.

FOOD CONSUMPTION
Exposure of mice to 80 ppm H2S resulted in reduced food consumption during the majority of the exposure period. The total amount of food consumed by male mice during the 13-week study following exposure to either air or 80 ppm H2S were 631.0 ± 91.4 and 465.9 ± 18.2 g, respectively. During the study, female mice exposed to either air or 80 ppm H2S consumed 621.2 ± 64.6 and 435.0 ± 41.3 g of food, respectively.

OPHTHALMOSCOPIC EXAMINATION
No exposure-related eye lesions were observed.

HAEMATOLOGY
Control mice had low levels of sulfhemoglobin (0.03 ± 0.02 g/dl) and exposure to H2S did not result in increased sulfhemoglobin concentrations in this species. No other changes in hematological indices were observed in H2S-exposed mice.

CLINICAL CHEMISTRY
No changes in serum chemistries were observed in H2S-exposed mice.

URINALYSIS
No treatment-related effects were observed on any of the urinalysis parameters measured in the study.

ORGAN WEIGHTS
Brain weights from the male B6C3F1 mice in the 80 ppm exposure group were significantly significantly decreased when compared to air-exposed control animals. Liver, heart, and kidney weights from the male B6C3F1 mice in the 80 ppm exposure group were significantly decreased relative to controls, but when normalized to either brain or body weights, these values were not significantly different (data not shown). Female mice in the 80 ppm H2S exposure group had significantly decreased kidney and ovary weights when compared to control animals, but similar to the male mice, normalized relative values were not significantly different.
However, relative weights were not significantly different and clinical pathology and histology were negative in both sexes.

NEUROBEHAVIOR
There were no toxicologically- or statistically-significant differences in neurological function except for a 30.5 ppm (42 mg/m3) female and 80 ppm (111 mg/m3) male that did not respond to artificial light stimulus.

GROSS PATHOLOGY
No data.

HISTOPATHOLOGY: NON-NEOPLASTIC
The reevaluation of the nasal tissues confirmed the presence of an increased incidence (100%) of rhinitis in male and female B6C3F1 mice following exposure to 80 ppm H2S. This lesion consisted of infiltration of the nasal epithelium or lamina propria by inflammatory cells, predominantly neutrophils. The infiltrate was often accompanied by serocellular exudate, epithelial hyperplasia or hypertrophy, squamous metaplasia, erosion or ulceration, cystic epithelial invaginations, fibrosis, hyperkeratosis, or goblet cell hyperplasia. Rhinitis was most prevalent in the more anterior parts of the nose
(corresponding to nasal Sections 1 and 2) and decreased in extent more caudally. On average, 53–62% of the nasal epithelium in Section 1 was affected following exposure to 80 ppm H2S, whereas 17–32% and 4–6% of the nasal epithelium in Sections 2 and 3, respectively, were affected. No gender difference in either the incidence or extent of the rhinitis lesion was observed in mice. The B6C3F1 mice had no background incidence of rhinitis; however.
The reevaluation of the nose slides conducted in 2002–2003 also established the presence of olfactory neuronal loss (Table 2) resulting in atrophy or thinning of the olfactory epithelium. An increased incidence of this lesion was found in male and female B6C3F1 mice following exposure to 30 and 80 ppm H2S.
Other histologic changes noted during the reevaluation were often similar in character to those noted in the original read and were interpreted as
likely background findings without clinical significance to the species, strain, and age of animal evaluated.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
30.5 ppm
Sex:
male/female
Basis for effect level:
other: = 42 mg/m3
Dose descriptor:
LOAEC
Remarks:
systemic toxicity
Effect level:
80 ppm
Sex:
male/female
Basis for effect level:
other: = 111 mg/m3, decreased body weight gain
Dose descriptor:
NOAEC
Remarks:
nasal irritation
Effect level:
10.1 ppm (analytical)
Sex:
male/female
Basis for effect level:
other: = 14 mg/m3
Dose descriptor:
LOAEC
Remarks:
nasal irritation
Effect level:
30.5 ppm (analytical)
Sex:
male/female
Basis for effect level:
other: =42 mg/m3, , increased incidence of olfactory neuronal loss

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Mean (±SD) terminal body weight and absolute mean (±SD) organ weights for male and female B6C3F1  mice exposed to air, 30, or 80 ppm H2S

 

Male B6C3F1 mice

Female B6C3F1 mice

 

0

30ppm H2S

80ppm H2S

 

0

30ppm H2S

80ppm H2S

Body weight(g)

25.5±1.38

25.6±1.11

24.7±1.45

Body weight(g)

21.7±1.66

22.2±1.26

20.0±1.93*

Organ weight(g)

 

 

 

Organ weight(g)

 

 

 

Brain

0.48±0.01

0.49 ± 0.02

0.46 ± 0.02a

Brain

0.48±0.02

0.49±0.01

0.46±0.03

Heart

0.17±0.03

0.18±0.02

0.15±0.02*

Heart

0.13±0.01

0.13±0.01

0.12±0.01

Kidney

0.53±0.03

0.53±0.03

0.45±0.05*

Kidney

0.34±0.02

0.34±0.02

0.31±0.03*

Liver

1.24±0.13

1.28±0.10

1.06±0.09*

Liver

1.00±0.13

1.04±0.12

0.92±0.12

Spleen

0.05±0.01

0.06±0.01

0.05±0.01

Spleen

0.06±0.01

0.06±0.01

0.06±0.01

Testicle

0.23±0.01

0.22±0.02

0.23±0.01

Ovary

0.02±0.003

0.02±0.002

0.01±0.001*

aP-0.05.

 

 

 

 

 

 

 

*P<0.05.

 

 

 

 

 

 

 

Table2: Incidence of olfactory neuronal loss in B6C3F1  mice following subchronic exposure to air, 10, 30, or80 ppm H2S

 

 

Male

Female

 

0

10

30

80

0

10

30

80

2(Incidence)

0/9

0/7

6/6*

7/7*

0/8

1/9

10/10*

7/7*

2(S/E)

NA

NA

3.0/100%

3.0/100%

NA

2.0/15%

2.7/91%

3.0/100%

3(Incidence)

0/9

0/7

7/7*

8/8*

0/7

0/7

8/9*

6/6*

3(S/E)

NA

NA

2.0/59%

3.0/100%

NA

NA

2.0/39%

3.0/100%

4(Incidence)

0/8

0/9

1/6

9/9*

0/4

0/6

0/6

7/7*

4(S/E)

NA

NA

1.5/2%

2.6/45%

NA

NA

NA

2.4/41%

Averageseverity(S)andextent(E)ofthelesionatthreenasallevels(2–4)havealsobeenprovided. NA:notapplicable(lesionwasnotpresent).

Seetextfordescriptionofseverityandextentscoringsystem.

*P<0.05.

Applicant's summary and conclusion

Conclusions:
The LOAEL for this effect in mice was 30.5 ppm (42 mg/m3) and the NOAEL was 10.1 ppm (14 mg/m3). The LOAEL for the systemic toxicity was 80 ppm (111 mg/m3) based on a decreased body weight gain and the NOAEL was 30.5 ppm (42 mg/m3).
Executive summary:

The Chemical Industry Institute of Toxicology (1983) performed a 90-day inhalation toxicity study in B6C3F1/CrlBr mice using H2S. Animals were individually housed and exposed to 0, 10.1, 30.5, or 80 ppm (0, 14, 42, or 111 mg/m3) H2S for 6 hr/day, 5 days/week for 90 days (10 mice/sex/group). Animals were observed twice daily for mortality and clinical signs. Body weight and feed consumption were determined weekly beginning just before the first exposure. A final fasted body weight was determined for all animals just before necropsy. Each animal also received an ophthalmological exam before the first day of exposure and within seven days of necropsy. A neurological exam was performed before necropsy in which posture, gait, facial muscle tone, and pupillary, palpebral, extensor thrust, and crossed-extensor thrust reflexes were assessed. Animals were housed in metabolism cages for 12 hr prior to necropsy. Urine samples were collected for all animals and volume, appearance, occult blood, specific gravity, protein, pH, ketone, and glucose were determined. On the day of necropsy, animals were anesthetized with ether and blood was drawn from the suborbital sinus (hematology) and abdominal aorta (chemistry). Animals were sacrificed on study day 91, 92, 93, 94, 95, and 99. The brain, kidney, spleen, liver, heart, and ovaries/testes were removed, weighed, and examined for gross and histopathologically observable abnormalities. In addition, the following tissues were examined microscopically: cerebellum, cerebrum, medulla, optic nerve, spinal cord, sciatic and anterior tibial nerves, eyes, pituitary, thyroid, parathyroid, salivary glands, heart, lungs, spleen, liver, pancreas, adrenals, mesenteric and mandibular lymph nodes, kidneys, bladder, lacrimal glands, ovaries, uterus, oviducts, vagina, cervix, stomach, duodenum, ileum, jejunum, large and small colon, caecum, skeletal muscle, skin, mammary glands (both sexes), femur, bone marrow, aorta, ear canal with zymbal gland, nasal turbinates (four levels), trachea, testes, epididymis, esophagus, thymus, prostate, seminal vesicle, and gross lesions.

Body weights were significantly depressed in both males and females in the high-exposure groups with sporadic significant decreases in the lower male exposure groups. Decreases in body weights tended to be more pronounced in the male and female high exposure groups beginning in the 6thweek of exposure. Feed consumption was significantly reduced in the high-exposure animals. There were no toxicologically- or statistically-significant differences in hematology, serum chemistry, urinalysis, ophthalmology, or neurological function except for a 30.5 ppm (42 mg/m3) female and 80 ppm (111 mg/m3) male that did not respond to artificial light stimulus.

Two female controls and one 30.5 ppm (42 mg/m3) male died prior to study termination. Both control animals exhibited multiple focal black discoloration of the glandular stomach. The 30.5 ppm (42 mg/m3) male exhibited a skull fracture with red discolorations and trauma apparently due to a feeding accident. A female and male in the 80 ppm (111 mg/m3) exposure group were sacrificedin extremison study days five and six, respectively. Both sacrificed animals exhibited prostration and/or hypoactivity prior to sacrifice. Both animals also had the same black discoloration of the glandular stomach as those in the control group. A low incidence of alopecia and emaciation was observed in control and exposed animals. In addition, a 10.1 ppm (14 mg/m3) male lost the use of anterior appendage with the appearance of paralysis while one 30.5 ppm (42 mg/m3) female and a 80 ppm (111 mg/m3) female were observed to have a missing front appendage. The investigators stated that it was unknown how the male lost its appendage but that the female’s loss was apparently congenital. The investigators considered none of the above findings to be compound-related. Gross pathology of surviving animals also revealed no gross lesions that were considered compound-related.

The mean absolute weights of the heart, liver, and spleen from male mice exposed to 80 ppm (111 mg/m3) H2S were significantly depressed. In addition, the mean absolute kidney weight of 80 ppm (111 mg/m3) female mice was also significantly reduced by approximately 20%. However, relative weights were not significantly different and clinical pathology and histology were negative in both sexes. Histological examination of surviving animals revealed the nasal tract as the only site where lesions were considered compound-related. Male (8/9) and female (7/9) mice exposed to 80 ppm (111 mg/m3) H2S exhibited minimal to mild inflammation of the anterior portion of the nasal mucosa (section I; section I and II in two mice). The lesion was primarily located in the squamous portion of the nasal mucosa but extended into the respiratory type epithelium (ciliated) in some animals. In one female, the lesion was suppurative and severe involving the entire nasal passage and associated structures. The lesion was also observed in two 80 ppm (111 mg/m3) mice that were exposedin extremis. No other histological findings were considered compound-related.

The critical effect in mice was inflammation of the nasal mucosa. This effect was present in male (8/9) and female (7/9) animals exposed to 80 ppm (111 mg/m3) H2S. The lesions, judged as minimal to mild in severity, were localized primarily in the squamous portion of the nasal mucosa but extended to regions of ciliated respiratory-type epithelium. The reevaluation of the nose slides conducted in 2002–2003 (Dorman et al., 2004) also established the presence of olfactory neuronal loss resulting in atrophy or thinning of the olfactory epithelium. An increased incidence of this lesion was found in male and female B6C3F1 mice following exposure to 30 and 80 ppm H2S. Since H2S is an irritant gas and other researchers (Brenneman et al., 2000; Dorman et al., 2000; Lopez et al., 1987) have reported nasal inflammation following H2S exposure, the critical effect in mice was considered to be olfactory neuronal loss. The LOAEL for this effect in mice was 30.5 ppm (42 mg/m3) and the NOAEL was 10.1 ppm (14 mg/m3). The LOAEL for the systemic toxicity was 80 ppm (111 mg/m3) based on a decreased body weight gain and the NOAEL was 30.5 ppm (42 mg/m3).