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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
THE ANALOGUE APPROACH
Further information in this study record is included in this Green liquor dataset to support the read-across analysis for toxicity to reproduction endpoint

The test material is H2S. The read-across can be performed since hydrogen sulfide (H2S) and sulfides in general are expected to be the toxicologically most hazardous/potent classified group of GL constituents. The analogue approach apply to H2S even if free sulfides in Green liquor are mainly in the form of HS/S2-- anions. 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 pH dependent equilibria between the H2S and S2-/HS- sulfide ions under physiological conditions.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1989

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The pre-mating and post-mating exposure periods of study guideline studies (eg. the OECD 421) were not completely followed.
- Principle of test: Pregnant rats were exposed to 100 ppm of hydrogen sulfide (H2S) alone or in combination with 400 and 800 ppm CS2, 6 h/d during days 6-20 of gestation.
Maternal reproduction and fetal parameters were evaluated on gestational day 21. Treatment with with 100 ppm H2S alone or with 100 or 200 ppm CS2.

In each experiment, a group of control animals was exposed concurrently to filtered room air, in a similar chamber with flow characteristics identical to those of the treatment groups.
GLP compliance:
not specified

Test material

Constituent 1
Reference substance name:
Hydrogen sulphide
EC Number:
231-977-3
EC Name:
Hydrogen sulphide
Cas Number:
7783-06-4
IUPAC Name:
hydrogen sulfide
Test material form:
gas
Specific details on test material used for the study:
Hydrogen sulfide gas by Air Liquide (France).

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
Male (350 g) and primiparous female (180-220 g) Sprague-Dawley (CD) rats obtained from IFFA CREDO Breeding Laboratories.

ENVIRONMENTAL CONDITIONS
The rats were maintained under controlled environmental conditions of temperature
- Temperature (°C): 23 +/- 1
- Humidity (%): 50 +/- 5
- Photoperiod (hrs dark / hrs light):12-h day-night cycle.
Tap water and food (UAR Alimentation Villemoisson) were freely available except during the exposure periods..

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Remarks:
10% H2S gas in nitrogen
Details on exposure:
Exposure atmosphere
H2S (or CS2/H2S mixture) was introduced into 200-l stainless steel inhalation chambers designed to sustain dynamic and adjustable air flows (10-20 m3/h). The chambers were maintained at negative pressure of no more than 3 mm H2O. Input air was filtered, and conditioned to 23 + 2°C and 50 +/- 5 % relative humidity. 10% H2S in nitrogen was delivered from a compressed-gas cylinder via a pressure-regulating valve.The air-flow was regulated by a,mass flow-meter.


Analytical verification of doses or concentrations:
yes
Remarks:
periodical sampling
Details on analytical verification of doses or concentrations:
Atmosphere sampling and analysis H2S concentrations were controlled by periodically pumping a measured volume of test atmosphere through a glass tube packed with silica gel impregnated with cadmium acetate. For H2S analysis, silica gel samples were desorbed with water.
N,N-dimethyl-p-phenylenediamine in sulfuric acid and ferric chloride were added to the cadmium sulfide solution. The adsorbance of the methylene blue solution formed was read at 670 nm. Variations of H2S in the exposure chambers were no more than 5% from the nominal concentrations.
Details on mating procedure:
Male (350 g) and primiparous female (180-220 g) Sprague-Dawley (CD) rats were placed together overnight. The morning on which vaginal smears were found to be sperm-positive was considered to be day 0 of gestation. The females were randomly assigned to experimental groups.
No. of animals per sex per dose:
20-23
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: H2S dose
In the experiment, groups of 20-23 bred rats were exposed to 100 ppm H2S, either individually or in combination with 400 or 800 ppm
CS2.

Exposure levels in air in the preliminary range finding study for H2S were 50, 100 and 150 ppm.

The H2S concentration 100 ppm in the full experiment was selected as the threshold level for maternal and fetal toxicity.
Preliminary experiments (summarized in Table) showed that prenatal exposure to 100 or 150 ppm H2S resulted in a significant (PThe means of implantation sites were significantly increased at 100 and 150 ppm H2S (P < 0.01 and P< 0.05, respectively), but were not considered to be higher than 14.11, which is historical mean of the test laboratory.
Exposure to 50 ppm H2S had no significant effect on any of the parameters explored. Because of the maternal weight loss observed at the concentration of 150 ppm H2S, the concentration of 100 ppm was selected for the full study and studying further the developmental toxicity interaction between CS2 and H2S.

Examinations

Maternal examinations:
Mated females were weighed prior to exposure on days 0 and 6 of gestation and prior to sacrifice on day 21 of gestation. On day 21 of gestation, the females were sacrificed by an intrapulmonary injection of T61-Hoechst. The abdominal wall was opened and the uterus removed and weighed. The uterine horns were then opened and the number of implantation and resorption sites and of live and dead fetuses was noted.
Fetal examinations:
Live fetuses were weighed individually, sexed, and examined for external malformations and cleft palate. Half of the viable fetuses from each litter was fixed in Bouin’s solution and examined for soft tissue anomalies. The remaining viable fetuses were fixed in 70 % alcohol, eviscerated, stored in 1% KOH, stained with alizarin red-S, and then examined for skeletal anomalies.
Statistics:
The litter was considered the experimental unit for analysis of data regarding embryotoxicity including teratogenicity. Incidence of pregnancy and fetal sex ratio were analysed by Yate’s corrected chi-square test. Maternal body weights, maternal absolute weight gains, implantation and resorption sites, dead and live fetuses, and fetal body weights were presented as means f SD, and were compared using Student’s t-test. The Mann-Witney U-test was used to evaluate the incidence of fetal anomalies. The level of significance chosen for all cases was P< 0.05.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No external anomalies were seen in any of the treated groups. The means of implantation sites were significantly increased at 100 and 150 ppm H2S (P < 0.01 and P< 0.05, respectively), but were not considered to be higher than 14.11, which is laboratory historical mean. Exposure to 50 ppm H2S had no significant effect on any of the parameters.
Dermal irritation (if dermal study):
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Preliminary experiments summarized in Table 1 showed that prenatal exposure to 100 or 150 ppm H2S resulted in a significant (P
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No external and soft tissue anomalies were observed in agnathia, enlarged brain ventricles,interventricular septal defect or ectopic testes
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The means of implantation sites were significantly increased at 100 and 150 ppm H2S (P < 0.01 and P< 0.05, respectively), but were not considered to be higher than 14.11, which is lab. historical mean.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOEC
Effect level:
ca. 50 ppm (analytical)
Based on:
test mat.
Basis for effect level:
body weight and weight gain

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEC
Effect level:
100 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
100 ppm (analytical)
Treatment related:
no

Any other information on results incl. tables

Table 1. PRELIMINARY REPRODUCTIVE DATA FROM DAMS AND LITTERS EXPOSED

TO H2S BY INHALATION 6 h/d, ON DAYS 6-20 OF PREGNANCY

 

Air

H2S -50 ppm

H2S -100 ppm

H2S -150 ppm

Number of females treated

8

8

8

9

Number of females pregnant

7

8

8

9

Incidence of pregnancy (%)

87

100

100

100

Body weight gain (g) of dams

on gestational days 6-21 (1)

112 ±17

113±13

97±10

88±8 **

Absolute weight gain (g) of dams on gestational days 6-21 (l)(2)

20 ± 6

20±7

11±7 *

-9±6 **

Mean implantation sites per litter (1)

12.3 ±1.9

13.6±1.9

14.9±1.3 **

14.2±1.3 *

Mean resorption sites per litter (1)

0.7 ± 0.8

0.7 ± 1.5

0.6 ± 0.9

0.4 ± 0.5

Mean dead fetuses per litter (1)

0

0

0

0

Mean live fetuses per litter (1)

11.7 ± 1.2

12.8±2.4

14.2±1.6 **

13.8±1.4 **

Mean fetal body weight (g) (1)

5.76 ± 0.16

5.53±0.18 *

5.35±0.15 **

5.35±0.16 **

External anomalies

0

0

0

0

(I) Group data presented as mean ±SD.

(2) Body weight gain of dams on gestational days 6-21 minus gravid uterine weight (g).

* and **denote significant difference from control value (Air), P<O.05 and P< 0.01, respectively.

Table 2. REPRODUCTIVE DATA FROM DAMS AND LITTERS EXPOSED TO ROOM AIR OR H2S 

BY INHALATION, 6 h/d, ON DAYS 6-20 OF PREGNANCY

 

Air

H2S - 100 ppm

Number of females treated

46

23

Number of females pregnant

40

20

Incidence of pregnancy (%)

87

87

Body weight gain (g) of dams on gestational days 6-21 (1)

130±26

120±26

Absolute weight gain (g) of dams on gestational days 6-21 (l)(2)

25±10

20±12

Mean implantation sites per litter (1)

14.5±3.8

14.6±3.6

Mean resorption sites per litter (1)

0.6±0.8

0.7±0.9

Mean dead fetuses per litter (1)

0

0

Mean live fetuses per litter (1)

13.9±3.8

13.9±4.5

Fetal sex ratio (M:F)

0.90

0.85

Mean fetal body weight (g) (1)

Male

5.5±0.45

5.53±0.50

Female

5.21±0.41

5.16±0.47

(1) Group data presented as mean±SD.

(2) Body weight gain ofdamson gestational days 6-21 minus gravid uterine weight (g).

Table 3. INCIDENCE OF FETAL ALTERATIONS AMONG LITTERS OF RATS EXPOSED TO ROOM AIR OR H2S

BY INHALATION, 6 h/d, ON DAYS 620 OF PREGNANCY

Number of fetuses examined/number of litters examined

Air

H2S - 100 ppm

External examination

558/40

278/20

Soft tissue examination, Males

133/40

64/19

Soft tissue examination, Females

146/40

75/20

Skeletal examination

279/40

139/20

External examination (I) Agnathia

0

0

External examination (1) Club foot

0

0

Oedema of whole body

0

0

Soft tissue examination (1) Enlarged brain ventricles

0

0

Soft tissue examination (1) Interventricular septal defect

0

0

Soft tissue examination (1) Diaphragmatic herrnia

0

0

Soft tissue examination (1) Enlarged renal pelvis

2/2

0

Soft tissue examination (1) Distended ureter

8/5

3/2

Soft tissue examination (1) Ectopic testes

0

0

Skeletal examination (1) Cervical ribs

0

0

Skeletal examination (1) Missing sternebrae

3/3

3/3

Skeletal examination (1) Fused sternebrae

0

0

Skeletal examination (1) Extra 14th ribs

12/10

6/6

Skeletal examination (1) Rudimentary 13th ribs

1/1

0

Skeletal examination (1) Delayed ossification of

thoracic and/or lumbar vertebrae

3/3

5/4

(1) Defects given as number of affected fetuses per number of affected litters,

Applicant's summary and conclusion

Conclusions:
Treatment with 100 ppm hydrogen sulfide caused no maternal toxicity (table 1) or adverse effects on the developing embryo or fetus (table 2).