Sulfite liquors and Cooking liquors, green

Developmental toxicity / teratogenicity

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

Test material information

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).