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Developmental toxicity / teratogenicity

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developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
no data
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 421) plus determination of neurotoxicology in the offspring.
Reason / purpose for cross-reference:
reference to same study

Data source

Reference Type:

Materials and methods

Test guideline
according to guideline
other: OECD 421
GLP compliance:
40 CFR 792
Limit test:

Test material

Constituent 1
Reference substance name:
Dihydrogen sulfide
Dihydrogen sulfide
Constituent 2
Reference substance name:
Hydrogen sulphide
EC Number:
EC Name:
Hydrogen sulphide
Cas Number:
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

Test animals

Details on test animals or test system and environmental conditions:
TEST ANIMALS: virgin male and female rats (80 rats per sex)
- Source: Charles River Laboratories, Inc. (Raleigh, NC)
- Age at study initiation: (P) x 8 wks
- Housing: rats selected for the study were housed individually (except during mating when they were housed 1:1) in polycarbonate cages with stainless steel lids (Laboratory Products, Inc., Rochelle Park, NJ) with Alpha-Dri™ bedding (Shepard Specialty Papers, Kalamazoo, MI). Following each daily exposure, all animals were transferred back to their home cages for residence and access to feed overnight.
- Diet: rats were fed NIH-07 rodent chow (Zeigler Bros., Gardners, PA) ad lib, except during the 6-h exposure periods.
- Water: deionised, filtered tap water available ad lib.
- Acclimation period: approximately 2 weeks in a HEPA-filtered, mass air-displacement room. During the acclimation period, rats were individually housed in suspended stainless steel cages with an automatic watering system.

- Temperature (°C): 18.5–21.5 °C
- Humidity (%): 40–70%
- Photoperiod (hrs dark / hrs light): fluorescent lighting was kept on a 12-h light–dark cycle (lights on 07:00–19:00 h).

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Details on exposure:
- Target concentrations: 0, 10, 30, and 80 ppm H2S
- For each daily exposure, F0 male rats were transferred from their home polycarbonate cages into individual stainless steel inhalation cage units.
- A similar procedure was used for F0 females until GD 19. Dams with litters were exposed on PND 5–18 using individual glass exposure cylinders that contained approximately 60 g of ALPHA-Dri bedding. In addition, approximately 25 g of Transgel © (Charles River Laboratories, Inc. Raleigh, NC) was placed into each exposure cylinder as a water source for the lactating females.
- Gas cylinders containing 5% (50,000 ppm) H2S in nitrogen were purchased from Holox Gases (Cary, NC).

- Adult (F0) male and female rats were exposed in four Hazelton H1000 stainless steel and glass inhalation 1-m3 exposure chambers (Lab Products, Maywood, NJ) contained within permanent 8-m3 Hinners-style stainless steel and glass inhalation exposure chambers.
- The air flow through each 1-m3 chamber was maintained at approximately 200–250 l/min to provide 12–15 air changes per hour during the exposures.
- Hydrogen sulfide was metered through mass flow controllers (MKS Instruments, Andover, MA) and mixed with the chamber air supply to provide the desired target H2S concentrations.

- For the whole-body exposure of dams and pups, 4.9-liter glass exposure cylinders sealed with two anodized aluminium end plates with neoprene gaskets and an aluminium outer face [CH Technologies (USA), Inc., Westwood, NJ] were used. Once the end plates are in place, the internal volume
of the cylinder is 4.3 litres.
- Airflow through the individual glass exposure cylinders was controlled by an adjustable stainless steel metering valve (Raleigh Valve and Fitting, Raleigh, NC). Airflow through the exposure cylinders was maintained at 2.8– 4.1 l/min during the exposures to provide approximately 35 to 50 air changes per hour. The temperature and humidity in one exposure cylinder per concentration group were measured every 30 min using a thermistor (PreCon, Memphis, TN) and a humidity probe (OMEGA Engineering, Inc., Stamford, CT) located in the outlet end of the glass chamber.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Chamber and cylinder exposure atmospheres were measured with a calibrated gas chromatograph (Hewlett Packard Packard Model 6890, Hewlett Packard Co., Palo Alto, CA) equipped with a flame photometric detector and a GS-Q (30-m x 0.53-µm) column (Alltech, Deerfield, IL). Prior to animals being placed in the 1-m3 chambers, each chamber was checked for uniformity of distribution of the test article H2S by measuring its concentration at nine positions within the chamber.
Details on mating procedure:
- M/F ratio per cage: 1/1 with no change in mating partners
- Each female was placed into the male’s home cage in the afternoon after each daily H2S exposure and then removed the next morning prior to the start of exposure.
- Proof of pregnancy: females were examined daily during the cohabitation period for the presence of sperm or copulation plugs in the vaginal tract. The observation of a copulation plug or sperm in vaginal lavage fluid was considered evidence of successful mating. The day that vaginal sperm or plug was observed was designated as GD 0.
- Presumed pregnant F0 rats were exposed to H2S from GD 0 until GD 19.
- Adult (F0) females without positive evidence of insemination were exposed to H2S until 23–24 days after the end of the breeding period; at that time, they were euthanized, and gross examination of reproductive tracts was performed.
Duration of treatment / exposure:
- Ten-week-old parental (F0) rats were exposed 2 weeks prior to breeding, during a 2-week mating period and then from gestation day (GD) 0 through GD 19
- No exposures occurred through the remainder of gestation and during the period of parturition (GD 20 through PND 4).
- Exposure of dams and their pups (8 rats/litter after culling) resumed between postnatal day (PND) 5 and 18
- No further H2S exposures to the dam or pups occurred beyond PND 18
- Adult male rats were exposed for 70 consecutive days
Frequency of treatment:
6 h/day, 7 days/week
Duration of test:
no data
Doses / concentrationsopen allclose all
Doses / Concentrations:
10.0 ppm (≈14 mg/m3 at 25 °C)
analytical conc.
Doses / Concentrations:
30.1 ppm (≈42 mg/m3 at 25 °C)
analytical conc.
Doses / Concentrations:
79.5 ppm (≈111 mg/m3 at 25 °C)
analytical conc.
No. of animals per sex per dose:
12 rats/sex/group
Control animals:
Details on study design:
- Rationale for animal assignment (if not random): Animals were randomly assigned to the different groups (12 rats/sex/group) at the end of the acclimation period by means of their prestudy body weight.
- The date of parturition was designated as PND 0.
- On PND 4, litters were randomly reduced to four animals per sex whenever possible, and each pup was identified with a foot tattoo
- Surplus pups were euthanized and discarded without further examination.
- Each litter was weaned on PND 21.


Maternal examinations:

- Time schedule: performed on all animals before and after each daily H2S exposure

- Time schedule for examinations:
- Body weights of the F0 female rats were recorded weekly until confirmation of mating.
- Presumed pregnant females were weighed on GD 0, 7, 14, and 20.
- Dams producing litters were weighed individually on PND 0, 4, 7, 14, and 21.

- Food consumption for each animal determined: Yes:
- Food consumption measurements were made weekly for all F0 female rats throughout the prebreed treatment periods.
- During pregnancy, feed consumption of F0 females was recorded for GD 0–7, 7–14, and 14–20.
- Maternal feed consumption was also measured for PND 0–4, 4–7, 7–14,and 14–21.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

- On the day of animal receipt, three rats per sex were randomly chosen and euthanized by CO2 inhalation, and blood was collected for assessment of viral antibody status (Microbiological Associates, Bethesda, MD).

- At the end of the exposure regimen, adult F0 rats were weighed, euthanized with CO2, and exsanguinated.

- A complete necropsy was performed with special emphasis on the reproductive and accessory sex organs.
- The nulliparous adult females (n=11) were necropsied the day after their last day of exposure. The post-parturient adult females (n=37) were necropsied the day of or the day after their pups were weaned.
- The order of necropsy was randomized across dose groups.
- Brain, liver, kidney, adrenal gland, and spleen tissue weights were determined for all F0 rats.
- Ovaries with oviducts, uterus, cervix, and vagina from F0 female rats were collected, weighed, and preserved in 10% neutral buffered formalin.
- Implantation sites were counted in fresh or formalin- fixed, potassium ferricyanide-stained uteri from all adult F0 female rats.
- Tissues were embedded in paraffin, sectioned at 5–6 microns, stained with hematoxylin and eosin, and examined by bright-field light microscopy.

- Histopathologic examination was performed on reproductive and accessory sex organs from F0 rats in the control and high-exposure groups and all reproductively unsuccessful animals.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes, see maternal examinations
Fetal examinations:
The following parameters were examined in [F1] offspring:

1. Developmental landmarks
- All pups were counted, sexed, examined for external anomalies, and individually weighed on PND 0.
- Each pup was monitored for the appearance of developmental landmarks beginning on PND 1 (pinnae detachment), PND 4 (surface righting), PND 7 (incisor eruption and negative geotaxis), and PND 12 (eyelid separation) using procedures described by Adams and co-workers [Collaborative Behavioral Teratology Study, protocol design and testing procedures, Neurobehav Toxicol Teratol 7 (1985) 579–586.].
- Weight gain: pups were weighed individually on PND 0, 4, 7, 14, and 21; after weaning on PND 21, pup body weights were collected twice weekly throughout the remainder of the study.
- Each female pup was observed for vaginal patency starting on PND 27. The vagina was considered to be patent when no membranous connections were observed on physical examination.
- Each F1 male was observed for preputial separation starting on PND 35.
- The percentage of animals in each litter demonstrating the developmental milestone of interest was calculated for each response.
2. Motor activity
- Measured in the same animal (one male and one female from each litter) before the 6-h H2S exposure on PND 13 and 17 and on PND 21 and 60+/-2
3. Passive avoidance
- Passive avoidance with a step-through to darkness paradigm including one training and one retention trial was used to assess short-term learning and memory.
- Evaluated for one male and female from each litter on PND 22+/-1 and on PND 62+/-3. Naive animals were tested on PND 21, whereas the PND 62+/-3 animals had been previously tested for FOB.
4. Functional observation battery (FOB)
- Performed on one male and one female from each litter on PND 60+/-2 using methods described by Moser et al. [Comparison
of chlordimeform and carbaryl using a functional observational battery, Fundam Appl Toxicol 11 (1988) 189–206].
5. Acoustic startle
- Assessed for one male and one female from each litter on PND 21 and 62+/-3.

Except for animals used for neuropathologic assessments, all weanling animals were euthanized by CO2 asphyxiation.

- Histopathologic examination was performed on brains from rats in control and high-dose groups.
- The remaining F1 rat pups (n = 144) were weighed on PND 63+/-3, euthanized with CO2, and exsanguinated and had a complete necropsy performed. The following organs were weighed: adrenal glands, brain, heart, kidneys, liver, lungs, ovaries with oviducts, spleen, and testes.

- Neuropathology in F1 rats was evaluated (one rat/sex/litter) in weanling (PND 23+/-2) rats tested for passive avoidance and in adult offspring (PND 61 62) rats tested for motor activity.
- Rats were given heparin (20,000 units USP/kg body weight, IP) and anesthetized with sodium pentobarbital (150 mg/kg). Animals were perfused in situ with a 0.7% sodium nitrite flush solution in 0.05 M sodium phosphate buffer (pH 7.4) followed by fixation with 1.5% glutaraldehyde and 4% formaldehyde in 0.05 M sodium phosphate buffer (pH 7.4).
- The brain, spinal cord, and sciatic nerve with its main branches were exposed, grossly examined, and immersed in perfusion fixative at 48C for at least 24 h.
- Brains were then removed, weighed, and measured.
- Cross-sections of the brain were collected for neuroanatomic pathology at the following sites: forebrain, caudate nucleus, centre of the cerebrum, centre of the midbrain, cerebellum and pons, and medulla oblongata.
- Tissues were embedded in paraffin, sectioned at 5–6 mm, stained with hematoxylin and eosin, and examined by bright-field light microscopy.
For further details on examination methods, see below "Any other information on materials and methods incl. tables"
he unit of comparison was the male, the female, the pregnant female, or the litter, as appropriate. Following an assessment for homogeneity of variance (Levene’s test), the data for quantitative, continuous variables (e.g., parental and pup body weights, organ weights, feed consumption) were intercompared for the exposure and control groups by tests for two-way fixed effects (dose and sex) analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure for significant ANOVAs (F-tests). The F1 generation data were analyzed jointly for both male and female offspring unless a statistical difference between male and female values within each treatment group was observed. A natural log (ln) transformation of the data was used when the Levene’s test for homogeneity (p < 0.01) indicated the data to be non-homogenous. When the ANOVA indicated statistical significance among experimental groups, the Dunnett’s test was used to delineate which groups differed from the control group. When the assumptions for a parametric ANOVA were not met, non-parametric procedures (Kruskal–Wallis test and Wilcoxon 2-sample rank-sum test) were used. A nested analysis of total motor activity data was performed using a repeated-measures analysis (MANOVA) with exposure as a grouping factor and test period as within subject factors. For developmental landmarks (e.g., vaginal patency and preputial separation), each treatment percentage or mean was compared to the control percentage or mean by the Kruskal–Wallis test. Incidence data were compared using the Fisher’s Exact Test. Categorical FOB data were analyzed using a log-linear model. Statistical analyses were performed using SAS Statistical Software. The significance level for any given statistical test was set at p< 0.05.
Mating index, fertility index, post implantation loss per litter, or number of late resorptions or still births, number of females with live pups, litter size, average lengths of gestation, and the average number of implants per pregnant female.
Historical control data:
no data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
- There were no deaths and no adverse physical signs observed in F0 female rats during the study.
- There were no statistically significant effects on the reproductive performance of F0 rats as assessed by the mating index, fertility index, post implantation loss per litter, or number of late resorptions or still births.
- In addition, the number of females with live pups, litter size, average lengths of gestation, and the average number of implants per pregnant female were also unaffected.
- The only statistically significant difference from control in either absolute or relative F0 rat organ weights was a decrease in the relative weight of the ovaries from female rats exposed to 10 ppm H2S.
- Statistical comparison of the control and high-exposure groups showed no significant difference from control in the incidences of the histologic diagnoses found.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
Effect level:
80 ppm
Based on:
test mat.
dihydrogen sulfide
Basis for effect level:
other: other:
Dose descriptor:
Effect level:
ca. 111 mg/m³ air
Based on:
test mat.
dihydrogen sulfide
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- No statistical significant increase in structural malformations was observed.
- Exposure to H2S did not affect pup growth.
- No statistically significant changes in mean organ weights of male and female F1 exposed animals were observed.
- No relevant gross abnormalities were observed at necropsy in the brain, spinal cord, of peripheral nerves of any pup examined after neuroperfusion.
- Microscopic examination was not performed on tissues from these animals due to a lack of significant statistical correlation between the incidence of gross changes observed and the H2S exposure.
-Exposure to H2S did not affect development as assessed by the onset of pinnae detachment, incisor eruption, negative geotaxis, eyelid separation, vaginal patency, or balano-preputial separation, or performance on any of the behavioural tests.

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Test atmospheres

The actual chamber concentrations (mean+/-SD) were 10.0+/-0.6, 30.1+/-0.8, and 79. 5+/-2.4 ppm for the target concentrations 10, 30, and 80 ppm, respectively. H2S was not detected in the control chamber (limit of detection was 0.6 ppm H2S in air). Overall mean daily chamber temperatures ranged from 22.0 to 23.5°C, and the relative humidity in the 1-m3 inhalation chambers ranged from 41 to 46%.

The authors suggested that H2S exposure is unlikely to result in significant reproductive toxicity or developmental neurotoxicity following exposures at concentrations relevant for most occupational exposures (i.e., ≤ 10 ppm).

Applicant's summary and conclusion

Considering all data of this study, there are no indications of an impairment of fertility or reproductive performance in rats exposed up to the highest concentration of 80 ppm H2S. No teratogenic effects or changes in behavioural tests were observed at those concentrations. Thus, a NOAEC of 80 ppm hydrogen sulfide (ca. 111 mg/m3 at 25 °C) can be derived from this screening study for reproductive toxicity and developmental neurotoxicity in rats.
Executive summary:

Read across H2S to Na2S:

Valid toxicological data on toxicity to reproduction 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+  +  S2-


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 (S2-) 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.