Hydrogen sulphide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

2000

Reference 2

Reference Type:

publication

Title:

Olfactory Neuron Loss in Adult Male CD Rats Following Subchronic Inhalation Exposure to Hydrogen Sulfide

Author:

Brenneman, K.A.; R.A. James, E.A. Gross, D.C. Dorman

Year:

2000

Bibliographic source:

Toxicologic Pathology 2000 Vol 28, No 2, 326-333

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 421

Deviations:

no

Principles of method if other than guideline:

The purpose of this study was to evaluate whether repeated 6-h daily exposure of male and female CD rats to H2S atmospheres of 0, 10, 30 or 80 ppm resulted in reproductive or developmental toxicity. This portion of the study was performed, to the extent possible, in compliance with the OECD 421. Another goal was to determine whether repeated exposure to H2S during the perinatal period of development resulted in neurotoxicity in the offspring. Developmental neurotoxicity in pups was assessed by evaluating the ontogeny of a number of developmental milestones, use of a blinded functional observational battery (FOB), an assessment of spontaneous motor activity, and an evaluation of acoustic startle and passive-avoidance behaviors.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

H2S

IUPAC Name:

H2S

Test material form:

other: 5% in nitrogen

Details on test material:

Gas cylinders containing 5% H2S in nitrogen were purchased from Holox Gases (Cary, NC)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River laboratories Inc. (Raleigh, NC)
- Age at study initiation: 8 week
- Housing: polycarbonate cages with stainless steel lids (Laboratory Products, INc. Rochelle Oark, NJ
- Diet: NIH-07 rodent chow, Ziegler Bros., Gardeners, PA ad libitum
- Water: filtered tap water
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.5 - 21.5 °C
- Humidity (%): 40 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: gas

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

The adult (F0) male and female rats were exposed in 4 Hazelton H1000 stainless steel and glass inhalation 1-m³ exposure chambers (Lab Products, Maywood, NJ) contained with in permanent 8-m³ Hinners-style stainless steel and glass inhalation exposure chambers. The air flow through each 1-m³ chamber was maintaines 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 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 2 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 cylinders is 4.3 liters. 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 concentratin group were measured every 30 min using a termistor (PreCon, Memphis, TN) and a humidity probe (ONEGA Engeneering, Inc., Stamford, CT) located in an outlet end of the glass chamber.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber and cylinder exposure atmospheres were measured with calibrated gas chromatograph (Hewlett 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. The generation system was operated by the Andover Infinity control system (Andover Controls Cooperation, Andover, MA).

Details on mating procedure:

The animals were mated (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. 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 designed ad GD 0.

Duration of treatment / exposure:

2 weeks prior to breeding, during 2-week mating period, then from gestation day 0-19. No exposures occurred through the remainder of gestation and during the period of parturition (gestation day (GD) 20 through postnatal day (PND) 4). Dams and pups were concurrently daily exposed starting on PND5 through PND18

Frequency of treatment:

6 hr/day, 7 days/week

Duration of test:

Daily exposure of the F0 Mmales continued until they were exoposed for at least 70 consecutive days.
Adult F0 females without positive evidence of nsemination were exposed to H2S until 23-24 days after the end of the breeding period.
Each litter was weaned on PND 21.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
10, 30. and 80 ppm (0, 14, 42, or 111 mg/m3)
Basis:
other: target conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
10.0 ± 0.6, 30.1 ± 0.8, and 79. 5 ± 2.4 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

12 males and 12 females per group to yield at least 8 pregnant females per group.

Control animals:

yes, sham-exposed

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes , weekly

WATER CONSUMPTION: No
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on post natal day 21
- Organs examined: Brain, liver, kidney, adrenal gland, spleen, ovaries, oviducts, uterus, cervix, vagina, seminal vesicles, coagulating glands, prostrate, left testis, left epididymis and mucosae of the nasal cavity

OTHER:

Fetal examinations:

- External examinations: Yes: all per litter
- Weighing: all per litter
- Histopathologic examination of brains

Statistics:

The unit of comparison was the male, the female, the prgnant female, or the litter, as appropriate. Following an assessement 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) nalysis 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 nonhomogenous. When the ANOVA indicated statistical significance among the 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, nonparametric 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 Kruskal-Wallis test. Incidence data were compared using 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.

Indices:

Developmental landmarks: pinnae detachment, surface righting, incisor eruption, negative geotaxis, eyelid separation,
Motor activity: spontanous motor activity was measured during ten 6.min intervals for a total of 60 min using an automated cage rack photobeam activity system (San Diego Instruments, San Diego, CA).
Passive avoidance: steo through to darkness paradigm
Functional observation battery

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 male or female rats. There were no statistically significant effects on the reproductive performance of the F0 rats as assessed by the number of females with live pups, litter size, average length of gestation,and the average number of implants per pregnant female.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEC

Effect level:

>= 80 ppm

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEC

Effect level:

>= 80 ppm

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Offspring were evaluated using motor activity, passive avoidance, functional observation battery, acoustic startle response and neuropathology.
No relevant gross abnormalities were observed at necropsy in the brain, spinal cord, or peripheral nerves of any pup. Exposue to H2S did not affect pup growth, development, or performance on any of the behavioral tests.

Effect levels (fetuses)

Dose descriptor:

NOAEC

Effect level:

>= 80 ppm

Basis for effect level:

other: please refer for the basis for effect level to the details under "Details on embryotoxic / teratogenic effects"

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Following exposure to 30 and 80 ppm H2S, a significant increase in nasal lesions limited to the olfactory mucosa of the male animals was observed. The female animals were not examined. The following dose-related effects were observed: olfactory neuron loss and basal cell hyperplasia. The distribution pattern in the nose was multifocal, bilaterally symetrical, and had a characteristic rostrocaudal distribution.

Applicant's summary and conclusion

Conclusions:

The results of this study suggests that H2S is neither a reproductive toxicant nor a behavioral developmental neurotoxicant in the rat.
But following exposure to 30 and 80 ppm H2S, a significant increase in nasal lesions, such as olfactory neuron loss and basal cell hyperplasia, was observed.
The no-observed adverse effect level in this study was 10 ppm.

Executive summary:

Groups of 12 male and female rats were exposed to H2S in concentratins of 0, 10, 30 and 80 ppm in the breathing air 6 hours per day on 7 days per week.

After an acclimatization period of 2 weeks the male animals were exposed for 70 days, the females for 2 weeks followed by a mating period of up to 2 weeks and during gestation until day 19 of gestation. On postnatal day 5 to 18 another period of exposure to H2S followed for the dams and their pups. Each litter was weaned on postnatal day 21 and the F0 females were euthanized. Female rats without positive evidence of insemination were exposed until day 23 -24 after the end of the breeding period and euthanized.

Offspring were evaluated using motor activity (postnatal day 13, 17,21 and 60 +-2), passive avoidance (postnatal day 22 +-1, 62 +- 3), functional observation battery (postnatal day 60 +-2), acoustic startle response (postnatal day 21, 62 +-3) and neuropathology (postnatal day 23 +-2, 61 +-2). The remaining F1 rat pups weere euthanized and had a necropsy performed.

There were no deaths and no adverse physical signs observed in F0 male or female rats. There were no statistically significant effects on the reproductive performance of the F0 rats as assessed by the number of females with live pups, litter size, average length of gestation,and the average number of implants per pregnant female.

No relevant gross abnormalities were observed at necropsy in the brain, spinal cord, or peripheral nerves of any pup. Exposue to H2S did not affect pup growth, development, or performance on any of the behavioral tests.

In the histopathologic examination a significant increase in nasal lesions, such as olfactory neuron loss and basal cell hyperplasia, were observed following exposure to 30 and 80 ppm H2S.

The distribution pattern in the nose was multifocal, bilaterally symetrical, and had a characteristic rostrocaudal distribution. The results of this study suggests that H2S is neither a reproductive toxicant nor a behavioral developmental neurotoxicant in the rat. But following exposure to 30 and 80 ppm H2S, a significant increase in nasal lesions, such as olfactory neuron loss and basal cell hyperplasia, was observed. The no-observed adverse effect level was 10 ppm.