Disodium sulphide

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1982-1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Very well-documented publication. This study was performed in conformity with GLP regulations and is considered to be of an adequate test design.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2004

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The present study describes the result of a reassessment of the nasal and lung histological specimens obtained from the subchronic CIIT inhalation study (Morgan, JM et al. (1983): A 90-Day Vapour Inhalation Toxicity Study of Hydrogen Sulfide in Sprague-Dawley Rats; summary report; Toxigenics Study 420-0710B). In the sub-chronic inhalation study, Sprague-Dawley rats were exposed whole-body to concentrations of 0, 10, 30, and 80 ppm H2S for 6 h/d, 5d/week for at least 90 days . The goal of this study was to characterize the toxicity of hydrogen sulfide (H2S), including nasal and pulmonary effects.

GLP compliance:

yes

Remarks:

All of the original work relating to this study was done in conformity with U.S. Food and Drug Administration Good Laboratory Practice Regulations (21 CFR 58) that were in force at that time. No data on testing laboratory available.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dihydrogen sulfide

IUPAC Name:

Dihydrogen sulfide

Constituent 2

Reference substance name:

Hydrogen sulphide

EC Number:

231-977-3

EC Name:

Hydrogen sulphide

Cas Number:

7783-06-4

Details on test material:

- Name of test material (as cited in study report): Hydrogen sulfide
- Molecular formula (if other than submission substance): H2S
- Molecular weight (if other than submission substance): 34.08 g/mol
- Smiles notation (if other than submission substance): S
- InChl (if other than submission substance): InChl=1/H2S/h1H2
- Substance type: technical product
- Physical state: gaseous
- Analytical purity: 99.68 % H2S (confirmed by gas chromatography)
- Impurities (identity and concentrations): 0.29 % propylene, 0.03 % propane (confirmed by gas chromatography)
- Origin of test material: Matheson Gas Company (Morrow, GA)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- The test was performed with young Sprague-Dawley rats, Fisher rats and B6C3F1 mice in parallel (see further endpoints studies)
- Source: Charles River Breeding Laboratories, Inc. (Portage, MI)
- Age at study initiation: 6 weeks (at the time of purchase)
- Housing: animals were individually housed in stainless steel inhalation exposure cages
- Diet: Purina Certified Rodent Chow 5002 (St. Louis, MO), ad libitum, except during exposure and on the terminal day of the study
- Water: filtered tap water, ad libitum, except during exposure and on the terminal day of the study
- 4-day quarantine period including body weight measurements (twice) and prestudy ophthalmologic examination


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 to 27 °C
- Humidity (%): 25 to 77 %
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 2-h light-dark cycle


IN-LIFE DATES: From: 1982 To: 1983

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: not applicable

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposures were conducted in 8 m3 stainless steel and glass inhalation chambers.
- Temperature, humidity, pressure in air chamber: temperature of 20 - 26 °C, humidity of 37 - 71 %; measured and recorded approx. hourly with an American Society for Testing and Materials (ASTM) thermometer (Scientific Products, Chicago, IL) and a Certificated Hygrometer Indicator (Abbeon Cal. Inc., Santa Barbara, CA), respectively.
- 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 steamed 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 exposure chamber. The flow of the test material from the flowmeter and the total airflow through the chamber were adjusted to maintain the target concentration within the chamber.
- Air flow rate: monitored continuously throughout the exposure by reading the pressure differential from a minihelic pressure gauge (Dwyer Instruments, Inc., Michigan City, IN) and recording the corresponding airflow from a prepared calibration graph showing airflow vs. differtial pressure.

TEST ATMOSPHERE
- Brief description of analytical method used: A gas chromatograph equipped with a 1.8 m by 0.3 cm Teflon, Chromosil column and a photoionization detector were used to analyse the chamber atmosphere during the experiment as well as to analyse all manually collected samples.
- The test material delivery rate and total airflow were used to estimate the nominal H2S concentration within the chamber.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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 minutes while the control chamber was sampled approximately every 90 min. Chamber samples were also obtained manually each week with precision sample gas syringes. A gas chromatograph (Hewlett-PAckard Model 5710A, Palo Alto, CA) equipped with a 1.8 m by 0.3 cm Teflon, Chromosil column (Supelco, Bellefonte, PA) and a photoionization detector (HNU Systems, Newton, MA) was used to analyse the chamber atmosphere during the experiment as well as to analyse all manually collected samples.

Duration of treatment / exposure:

At least 90 days exposure (actually 91 - 95 days)

Frequency of treatment:

6 h/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
10 ppm (≈14 mg/m3 at 25 °C)
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
30 ppm (≈42 mg/m3 at 25 °C)
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
80 ppm (≈111 mg/m3 at 25 °C)
Basis:
nominal conc.

No. of animals per sex per dose:

10 male and 10 female animals per exposure group

Control animals:

yes

Details on study design:

- Rationale for animal assignment (if not random): random

Positive control:

no data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily, incl. examination for clinical signs and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly from the day of the first exposure examination of the physical conditions

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed twice during the 4-day quarantine and weekly from the day of the first exposure. A final fasted body weight was obtained for each animal just before necropsy

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: yes, weekly from the day of the first exposure
- Mean daily diet consumption calculated as g food/kg body weight/day: No data

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 data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the 4-day quarantine each animal underwent a prestudy ophthalmologic examination; ophthalmologic evaluations were conducted on all test animals by the same board-certified veterinary ophthalmologist at the end of the study (approx. 1 week before necropsy)

HAEMATOLOGY: Yes , from blood samples obtained from anesthetised animals via orbital sinus puncture
- Time schedule for collection of blood: approx. 24 h after the last H2S exposure
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- Parameters checked: erythrocyte count, haemoglobin, hemtocrit, total and differential leukocyte counts, platelet counts, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), methemoglobin, and sulfhaemoglobin. Iin addition, prothrombin times were determined.

CLINICAL CHEMISTRY: Yes, from blood samples obtained from anesthetised animals via abdominal aorta
- Time schedule for collection of blood: approx. 24 h after the last H2S exposure
- Animals fasted: Yes
- Parameters checked: serum chemistry: glutamic pyruvic transaminase, total bilirubin, urea nitrogen, glucose, glutamic oxaloacetic transaminase, total protein, phosphorus sodium, potassium, chloride, calcium, alkaline phosphatase, and gamma glutamyl transpeptidase (whenever possible)

URINALYSIS: Yes, from a subset of animals (that were housed in metabolic cages during an approximately 12-hour fast before necropsy)
- Time schedule for collection of urine: during approximately 12-hour before necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: volume, appearance, occult blood, specific gravity, protein, pH, ketone, and glucose.

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

Approximately 24 h after the last H2S exposure and following an overnight fast, animals were anesthetised with methoxyurane, and blood samples were collected from the orbital sinus and abdominal aorta. Animals were killed by abdominal exsanguination and necropsied. To reduce observer bias, one animal was taken consecutively from each treatment group.

GROSS PATHOLOGY: Yes. The following organs were dissected: brain, kidney, spleen, liver, heart, and ovaries/testes. The animals' noses were dissected free from the head, perfused, trimmed of excess tissue and immersion fixed. The lungs were removed, inflated and immersed in 10 % neutral buffered formalin.

TERMINAL ORGAN WEIGHT: Yes. The following organs were weighed: brain, kidney, spleen, liver, heart, and ovaries/testes.

HISTOPATHOLOGY: Yes .
- Histological reevaluation focused strictly on the respiratory tract, because the original study reported no exposure-related gross or microscopic lesions in any non-respiratory tissues.
- After decalcification, the noses were cut transversely perpendicular to the bridge of the nose at approx. four sites. The lungs were sectioned to include the main stem bronchi, secondary bronchioles, respiratory bronchioles, alveolar ducts, and alveoli. The tissues were processed routinely, paraffin-embedded, sectioned at 5 µm, stained with haematoxylin and eosin, and examined by bright-field light microscopy. The control and the 80 ppm groups were examined first, and if changes were detected, then the other exposure groups were examined.
- Olfactory neuronal loss (ONL) was graded according to its severity and extent (i.e., a visually estimated percentage of the olfactory epithelium affected). The criteria used to grade the severity of the olfactory neuronal lesions were identical to that published by Brennemann et al. (2000a). This subjective grading system consisted of a visual estimation of the average percentage of reduction in the normal thickness of the olfactory neuron layer in the affected portion of the olfactory epithelium (1= mild=26-50 % affected; 2= moderate=51-75 % affected; 3= severe= 76-100 % affected).
- Rhinitis was graded according to its extent only (i.e., a visually estimated percentage of the nasal epithelium affected).
- In the lung, the severity of histological changes was graded subjectively as minimal, mild, moderate, or severe.

Statistics:

Parametric data such as body weight and food consumption were analysed using an analysis of variance (ANOVA). Statistically significant differences that were further studied by either Tuey's (equal sample sizes) or Scheffe's (unequal sample sizes) Test of Multiple Comparison. Nonparametric data such as organ weight ratios were analysed 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.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

however, effect not considered as "adverse" with respect to NOAEC derivation

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

however, effect not considered as "adverse" with respect to NOAEC derivation

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

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 specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

however, effect not considered as "adverse" with respect to NOAEC derivation

Gross pathological findings:

no effects observed

Description (incidence and severity):

in any non-respiratory tissues

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

effects only in respiratory tissues

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
- No mortality was observed in rats during the study
- Observed clinical signs were not related to H2S inhalation

BODY WEIGHT AND WEIGHT GAIN
- Female Sprague-Dawley rats exposed to 80 ppm H2S had depressed terminal body weights when compared with air-exposed controls

FOOD CONSUMPTION
- Significantly reduced in rats exposed to 80 ppm H2S during the first week of exposure compared to controls: 148.5±10.1 g and 173.5±12.3 g, respectively for males and 108.3±9.6 g and 118.5±13.2 g, respectively for females

FOOD EFFICIENCY
- no data

WATER CONSUMPTION
- no data

OPHTHALMOSCOPIC EXAMINATION
- No exposure-related eye lesions were observed

HAEMATOLOGY
- 90-day inhalation did not result in toxicologically relevant alterations in haematological indices
- Rats from the 10 ppm group had detectable levels of sulfhaemoglobin: 0.01 ± 0.01 g/dl, and this increase was statistically significant. However, as the levels observed in the rats following high-dose H2S exposure are much lower than those observed in the general human population (Zwart et al, 1986), they thus are unlikely to be toxicologically significant.

CLINICAL CHEMISTRY
- 90-day inhalation did not result in toxicologically relevant alterations in serum chemistries

URINALYSIS
- No treatment-related effects were observed

NEUROBEHAVIOUR
- no data

ORGAN WEIGHTS
- Brain weights from male rats in the 80 ppm exposure group were significantly decreased when compared to air-exposed control animals

GROSS PATHOLOGY
- 90-day inhalation did not result in toxicologically relevant alterations in gross pathology in any non-respiratory tissues

HISTOPATHOLOGY: NON-NEOPLASTIC
- No microscopic lesions in any non-respiratory tissues

NOSE:
- Histological evaluation of the nose showed exposure-related increased incidences of olfactory neuronal loss (ONL) and rhinitis.
- The rats had a high background incidence of rhinitis
- Reevaluation of the nose slides conducted in 2002-2003 established the presence of olfactory neuronal loss resulting in atrophy or thinning of the olfactory epithelium. An increased incidence of this lesions was found in female Sprague-Dawley rats following exposure to 30 ppm H2S. Male Sprague-Dawley rats developed ONL following exposure to 80 ppm H2S only
- At the 80 ppm exposure level, olfactory neuronal loss typically affected most or all of the olfactory epithelium present at nose levels 2 and 3, where it lines the dorsal medial meatus. At nose level 4, this lesion had a lesser extent and affected smaller sites within the olfactory epithelium, which lines the complex ethmoid recess. Sites affected bordered the dorsomedial portionof this cavity and most often included the nasal septum and tips of the ethmoid turbinates.
- The lesions ended to decrease in severity, extent, and sometimes incidence at the 30 ppm exposure level
- other nasal lesions found included abnormalities of the lacrimal duct, lacrimal gland, vomeronasal organs or theeth, sinusitis, or respiratory epithelial hyperthrophy and hyperplasia without inflammation.

LUNG:
- In the lung, exposure to H2S was associated with bronchiolar epithelial hypertrophy and hyperplasia in male and female Sprague-Dawley rats following exposure to ≥30 ppm
- Reevaluation of the lung slides demonstrated an increased incidence of bronchiolar epithelial hypertrophy and hyperplasia in female Sprague-Dawley rats following exposure to 30 or 80ppm H2S, while the males developed this lung lesions following exposure to 80 ppm H2S. The epithelial changes were variably associated with peribronchiolar fibrosis and smooth muscle hypertrophy and often mixed inflammation. The lesions had a variable distribution and severity that ranged from segmental to diffuse and minimal to mild, respectively. There was also a moderate to high background incidence of mixed inflammation in the lung of Sprague-Dawley rats that may prevent detection of treatment related inflammatory changes.

OTHER FINDINGS
EXPOSURE ATMOSPHERE
Mean (±SD) H2S atmospheric concentrations were 10.1 ± 0.4 ppm H2S, 30.5 ± 1.1 ppm H2S, and 80.0 ± 1.8 ppm H2S for the 10, 30 and 80 ppm H2S exposure chamber, respectively. H2S was not detected in the control chamber.

Effect levels

1

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

80 ppm

Based on:

test mat.

Remarks:

dihydrogen sulfide

Sex:

male/female

Basis for effect level:

other: 90-day inhalation did not result in toxicologically relevant alterations in haematological indices, serum chemistries, or gross pathology. Therefore, the concentration of 80 ppm H2S can be considered as NOAEC for systemic effects.

2

Dose descriptor:

NOAEC

Remarks:

local effects (for ONL)

Effect level:

10 ppm

Based on:

test mat.

Remarks:

dihydrogen sulfide

Sex:

male/female

Basis for effect level:

other: The results confirm that the rodent nose, and less so the lung, are highly sensitive to H2S-induced toxicity, with 10 ppm H2S representing the NOAEC for ONL (local effects) following sub-chronic inhalation.

3

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

ca. 111 mg/m³ air

Based on:

test mat.

Remarks:

dihydrogen sulfide

Sex:

male/female

Basis for effect level:

other: see 'Remark'

4

Dose descriptor:

NOAEC

Remarks:

local effects (for ONL)

Effect level:

ca. 14 mg/m³ air

Based on:

test mat.

Remarks:

dihydrogen sulfide

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Applicant's summary and conclusion

Conclusions:

90-day inhalation did not result in toxicologically relevant alterations in haematological indices, serum chemistries, or gross pathology in Sprague-Dawley rats. Therefore, the concentration of 80 ppm H2S (ca. 111 mg/m3 air at 25 °C) may be considered as NOAEC for systemic effects.
The main adverse effect caused by hydrogen sulfide was an exposure-related increased incidence of olfactory neuronal loss (ONL). The results showed that the rodent nose, and less so the lung, are highly sensitive to H2S-induced toxicity, with 10 ppm H2S (ca. 14 mg/m3 air at 25 °C) representing the NOAEC for ONL (local effect) following sub-chronic inhalation exposure.
However, because of the gaseous state of hydrogen sulfide, when drawing conclusions with respect to local effects at the olfactory system and respiratory tract, which might be generated by exposure to dust of sodium sulfide, the toxicokinetic behaviour (e.g. total absorption) of H2S and Na2S should be taken into consideration.

Executive summary:

Read across H₂S to Na₂S:

Valid toxicological data on sub-acute or sub-chronic inhalation exposure specifically for sodium sulfide from animal studies are not available. Therefore, because of the lack of appropriate experimental data, read-across from studies with H₂S is proposed based on the following reasoning:

As discussed in the dossier section on toxicokinetics, unrestricted read-across between the substances sodium sulfide, sodium hydrogensulfide and dihydrogen sulfide is considered feasible, in view of the potential systemic toxicity being driven by the sulfide ion as the only relevant species released from any of the sulfide substances under physiological conditions. In this context, it is further considered to be very unlikely that the sodium ions add any toxicological concern.

The soluble compound sodium sulfide (Na₂S) can safely be assumed to be present dissociated in water and relevant biological media (Beauchamp et al., 1984)¹. From sodium sulfide, hydrogen sulfide (H₂S) may be formed according to the following equilibria:

Na₂S + H₂O      →        NaOH + NaHS (2Na⁺+ OH^-+ HS^-)

NaHS + H₂O    →        NaOH + H₂S    (Na⁺+ OH^-+ H₂S)

The toxic effects resulting from the sodium ion is negligible. Hydrogen sulfide dissociates in aqueous solution to form two dissociation states involving the hydrogen sulfide anion and the sulfide anion:

H₂S  ↔  H⁺  +  HS^-  ↔  2 H⁺  +  S^2-

The pKa values for the first and second dissociation steps of H₂S are 7.04 and 11.96, respectively. Therefore, at physiological pH values, hydrogen sulfide in the non-dissociated form (H₂S) and the hydrogen sulfide anion (HS^-) will be present in almost equimolar proportion, whereas only very small amounts of the sulfide anion (S^2-) will be present. In conclusion, under physiological conditions, inorganic sulfides or hydrogensulfides as well as H₂S will dissociate to the respective species relevant to the pH of the physiological medium, irrespective the nature of the “sulfide”, which is why read-across between these substances and H₂S is considered to be feasible without any restrictions.