Disodium sulphide

Neurotoxicity

Description of key information

Na2S inhibited cytochrome oxidase and carbonic anhydrase prepared from brains of male rats in a concentration-dependent manner. Extensive disruption of respiratory and related mitochondrial functions was determined in homogenates of synaptosomes prepared from brains of CDI mice treated i.p. with Na2S.
Treatment of male Wistar rats with 11.7 mg/kg Na2S i.p. caused a partial inhibition of GABA and dopamine uptake, and it strongly inhibited veratridine-dependent release of these neurotransmitters and reduced veratridine-dependent changes in the trans-membrane potential in synaptosomes isolated from the hemispheres. However, single doses of Na2S (80-200 mg/kg) administered i.p. to rats indicated that very high doses are incapable of producing cerebral necrosis by a direct histotoxic effect.
Inhalation of hydrogen sulfide resulted in a marked, but reversible, decrease in the incorporation of leucine in the cerebral protein and myelin accompanied by a decreased activity of lysosomal acid proteinase after inhalation exposure of adult female mice at 100 ppm H2S for 2 h. 
After repeated inhalation exposure of male rats (5 d, 3h/d) to low levels of H2S, the total power of hippocampal EEG theta activity increased in a concentration-dependent manner in both dentate gyrus and CA1 region that required two weeks for a complete recovery. Neocortical EEG and LIA (Large Amplitude Irregular Activity) were unaffected following exposure to 100 ppm H2S.
Determination of serotonin and norepinephrine levels in developing rat cerebellum and frontal cortex of pups from rats exposed for 7 h/d (GD 5 until PND 21) suggested that alterations of monoamine levels induced by H2S may produce long-lasting neurochemical changes in the CNS.
Short-term exposure of adult male rat to H2S for 3 h/d for 5 d significantly reduced motor activity, water maze performance, and body temperature following exposure to H2S concentrations ≥ 80 ppm, but did not affect regional brain catecholamine concentrations.

Additional information

General:

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.

Read-across concept between sodium sulfide, sodium hydrogensulfide and hydrogen sulfide:

Given that sodium sulfide and sodium hydrogensulfide dissociate in aqueous media, it can safely be assumed that under most physiologically relevant conditions (i. e., neutral pH) sulfide and hydrogen sulfide anions are present at almost equimolar concentrations, thus facilitating unrestricted read-across between both species. Only under extreme conditions such as gastric juice (pH << 2), sulfides will be present predominantly in the form of the non-dissociated hydrogen sulfide. In turn, hydrogen sulfide (H₂S) may be formed from both soluble sulfides, according to the following equilibria:

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

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

Similarly, hydrogen sulfide dissociates in aqueous solution to form two dissociation states involving the hydrogen sulfide anion and the sulfide anion, according to the following equilibrium:

                          H₂S ↔ H⁺ + HS^- ↔ 2 H⁺ + S^2-

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.

Results:

From mechanistic studies with intraperitoneal injection of sodium hydrogensulfide to rats it can be concluded that the lung and not the brain is the primary site of action of hydrogen sulfide, with an afferent neural signal from the lung via the vagus inducing apnea. In addition, substantial changes in neurotransmitter amino acids in the brainstem responsible for neuronal control of breathing were noted. Analyses of effects of sodium hydrogensulfide on brain transmitter systems and monoamine oxidase (MAO) activity after i.p. administration to rats led to the suggestion that inhibition of MAO may be an important contributing factor to the mechanisms underlying loss of respiratory drive after H₂S exposure.

Mechanistic in-vitro and in-vivo studies with Na₂S revealed that sulfide toxicity can be ascribed to the inhibition of cytochrome oxidase as key enzyme of the respiratory chain. Sodium sulfide has also been shown to strongly inhibit neuronal cytochrome oxidase and carbonic anhydrase causing disruption to respiratory and mitochondrial functions in the rat brain in vitro. Inhibitory effects on synaptosomal respiration and transmitter kinetics were shown after i.p. injection into rats with sodium sulfide.

In in-vivo studies with short-term exposures of rats to H₂S, a cumulative effect on hippocampal EEG theta activity was observed at high exposure levels that required two weeks for a complete recovery. Neocortical EEG and Large Amplitude Irregular Activity (LIA) were unaffected. Furthermore, behavioural toxicity was observed in rats only at higher concentrations (≥80 ppm) of H₂S. But, regional brain catecholamine levels or performance on the fixed-interval (FI) schedule were not affected. Perinatal exposure of pregnant rats to hydrogen sulfide resulted in alterations of serotonin and norepinephrine levels in the cerebellum and frontal cortex of the pups.

 

Justification for classification or non-classification

Based on mechanistic in-vitro and in-vivo (i.p.) studies with NaHS and Na₂S, it is not possible to derive a no effect level or low effect level which is relevant for real exposure situations. This holds also true for the study with examination of effects on hippocampal EEG theta activity following short-term inhalation exposure of rats to H₂S, because it cannot be ruled out that these changes are related to a sensory olfactory stimulation, and it is not known whether these changes are related to any adverse clinical effects. However, in short-term behavioural study in rats, some effects on motor activity were observed at high exposure levels. However, study duration was only 5 days, and it is likely that adaptation occurs after longer-term exposure durations, because no behavioural effects were observed in a 90-day inhalation toxicity study with H₂S at the same exposure level. Although it was shown that perinatal exposure of pregnant rats to hydrogen sulfide resulted in alterations of serotonin and norepinephrine levels in the cerebellum and frontal cortex of pups, detailed behavioural tests in offspring of dams exposed by inhalation to H₂S until gestation day 19 and dams and pups from postnatal day 5 to 18 revealed no clinical effects.

Therefore, it can be concluded that neurotoxicological effects of Na₂S and NaHS could be demonstrated in-vitro and following i.p. injection, but subchronic inhalation exposure to H₂S did not result in clinical effects of neurotoxicity in adult rats and their offspring.