Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The whole GL water mixture has not been tested on fertility endpoint. None of the constituents (common inorganic Ca, Na and K salts and sulfur species) have observed effects on human or test animal fertility or development at exposure levels relevant in normal manufacturing and use conditions of the substance (exposures are well below the derived DNELs ).

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
Green liquor (GL) is a complex mixture of inorganic salts in highly alkaline water solution (ca. 80% water). The composition of Green liquor is well known even if it is registered as an UVCB. All anionic and cationic groups present in green liquor is known, even if the speciation and ratio between the ionic constituents may vary in some extent. The variety in ratio/speciation makes the composition of the mixture complex. However, since all groups of the GL constituents are known and in order to avoid unnecessary animal testing, the non-testing read-across method can be performed by applying available hazard/study data from the mono-constituent pure inorganic salts (ie. those salts known to be dissolved in GL).
The referred Key study is part of effects on fertility read-across data evaluation from the green liquor constituent hydrogen sulfide. 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.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Beyond the 421 Guideline study, additional goal of the investigation was to determine whether repeated exposure to H2S during the perinatal period of development resulted in neurotoxicity in the offspring.
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 (n 12 rats/sex/concentration) 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) screening test guideline for reproductive and developmental toxicity.

Another goal of the present investigation was to determine whether repeated exposure to H2S during the perinatal period of development resulted 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
Remarks:
(40 CFR 792).
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Eight-week-old male and female CD rats (80 rats per sex) were obtained from Charles River Laboratories, Inc. (Raleigh, NC). 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). The remaining animals were acclimated for approximately 2 weeks in a HEPA-filtered, mass air-displacement room maintained at 18.5–21.5 °C and 40–70% relative humidity. During the acclimation period, rats were individually housed in suspended stainless steel cages with an automatic watering system. 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. Rats selected for the study were then singly housed (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 ™ cellulose-fiber chip bedding (Shepard Specialty Papers, Kalamazoo, MI). 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. The dams with litters were exposed on PND 5–18 using individual glass exposure cylinders [26] that contained approximately 60 g of ALPHA-Dri bedding.
A 1-h experiment using 10 ppm H2S through the exposure cylinder and bedding in the absence of animals confirmed that adsorption of H2S to the bedding material did not occur.

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. Deionized, filtered tap water was otherwise available ad lib. Following each daily exposure, all animals were transferred back to their home cages for residence and access to feed overnight. Rats were fed NIH-07 rodent chow (Zeigler Bros., Gardners, PA) ad lib, except during the 6-h exposure periods. Fluorescent lighting was kept on a 12-h light–dark cycle (lights on 0700–1900 h).
Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
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 aluminum end plates with neoprene gaskets and an aluminum 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 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 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. Chamber and cylinder exposure atmospheres were measured with a calibrated gas chromatograph (Hewlett Pac ard Model 6890, Hewlett Packard Co., Palo Alto, CA) equipped with a flame photometric detector and a GS-Q (30-m × 53µm) column (Alltech, Deerfield, IL).
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 designated as GD 0.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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 Corporation, Andover, MA).
- Analytical method: Chamber and cylinder exposure atmospheres were measured with a calibrated gas chromatograph equipped with a flame photometric detector and a GS-Q (30-m X 0.53-µm) column
- Samples taken from breathing zone: yes
Duration of treatment / exposure:
Ten-week-old parental (F0) rats were exposed to H2S vapor for a 2-week prebreed exposure period followed by a 2-week mating period.
Presumed pregnant F0 rats were exposed to H2S from GD 0 (gestation day)until GD 19. No exposures occurred through the remainder of
gestation and during the period of parturition (GD 20 through PND 4). The date of parturition was designated as PND 0 (postnatal day).
The dams and their pups were then concurrently exposed to H2S starting on PND 5 with daily exposures continuing through PND 18.
No further H2S exposures to the dam or pups occurred beyond PND 18.

Each litter was weaned on PND 21. Adult (F0) females without positive evidence of insemination were exposed to H2S until 23–24 days
after the end of the breeding period.

Daily exposure of the F0 males continued until they were exposed for at least 70 consecutive days.
Frequency of treatment:
One daily 6 h exposure, 7 days/week
Dose / conc.:
0 ppm (analytical)
Remarks:
All Doses / Concentrations ppm of H2S. Basis: analytical conc. 0-80 ppm
(0 - 111 mg/m3)
Dose / conc.:
10 ppm (analytical)
Remarks:
14 mg/m3
Dose / conc.:
30 ppm (analytical)
Remarks:
42 mg/m3
Dose / conc.:
80 ppm (analytical)
Remarks:
111 mg/m3
No. of animals per sex per dose:
The study began with 12 male and 12 female rats/group to yield at least eight pregnant females/group. Litters were randomly reduced to four animals per sex whereever possible.
Control animals:
yes
Details on study design:
The pups were tested for motor acitivity, passive avoidance, and acoustice statle, and a functional observation battery was also performed on them..

Motor activity was measured using an automated cage rack photobeam activity system.

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.

Acoustic startle was assessed using a microcomputercontrolled test system. The peak amplitude of each startle response and the interval between the acoustic stimulus and peak amplitude were analysed.

The functional observation battery was performed on the test animals on PND 60 ± 2.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: before and after each daily H2S exposure

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
The body weights of the Fo male rats were determined weekly throughout the study. The body weights of the F0 female rats were recorded in the same manner until confirmation of mating. Presumed pregnant females were weighed on GD 0, 7, 14, and 20. Dams producing litters and their 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.

FOOD CONSUMPTION :
Feed consumption for F0 males was recorded weekly throughout the study except during the period of cohabitation. Feed consumption measurements were made weekly for ail F0 female rats throughout the prehreed treatment periods. During pregnancy, feed consumption of F0 females was recorded for GD 0-7, 7-14, and 14-20. Maternal feed consomption was also measured for PND 0-4, 4-7, 7-14, and 14-21.

WATER CONSUMPTION : No
Oestrous cyclicity (parental animals):
Not recorded.
Sperm parameters (parental animals):
Daily sperm production was calculated for all F0 male rats assuming a 6.1-day spermatid cycle. The results are reported in table 1.


Litter observations:
ASSESSMENT OF DEVELOPMENTAL EFFECTS: All pups were counted, sexed, examined for external anomalitie, and individually weighed on PND0. Each pup was monitored for the appearance of
pinnae detachmend (PND1), surface rightning (PND4), incisor eruption and negative geotaxis (PND7), and eyelid separation (PND12).

BEHAVIORAT TESTING
Motoric activity:
Motoric activity was 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. Spontaneous motor activity was measured during ten 6-min intervals for a total of 60 min using an automated cage rack photobeam activity system.
Passive avoidance:
Passive avoidance with a step-through to darkness para¬digm including one training and one retention trial was used to assess short-term learning and memory [271. Passive avoidance was 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.
Functional observation battery (FOB):
An FOB was performed on one male and one female from each litter on PND 60 ±: 2 using methods described by Moser et al. (1988)..
Acoustic startle:
Acoustic startle was assessed using a microcomputer-controlled automated test system. Acoustic startle was assessed for one male and one female from each litter on PND 21 and 62 ± 3.

Postmortem examinations (parental animals):
SACRIFICE
At the end of the exposure regimen, adult F0 rats were weighed, euthanised with CO, and exsanguinated.
The nulliparous adult females (n = 11) and adult males (n = 48) 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.

GROSS NECROPSY
A complete necropsy was then performed with special emphasis on the reproductive and accessory sex organs.

ORGAN WEIGHT
Brain, liver, kidney, adrenal gland, spleen, ovaries with oviducts, uterus, cervix, vagina, seminal vesicles with coagulating gland, prostate, testis and the head, body, and cauda of the left epididymis.

HISTOPATHOLOGY / ORGAN WEIGHTS
Ovaries with oviducts, uterus, cervix, vagina, seminal vesicles with coagulating gland, prostate, testis and the head, body, and cauda of the left epididymis, major structures and mucosae (squamous, respiratory, transitional, and olfactory) of the nasal cavity.
Postmortem examinations (offspring):
NEUROPATHOLOGY: Neuropathology in F1 rats was evaluated (one rat/sex/litter) in weanling (PND 23 ± 2) rats tested for passive avoidancc and in adult offspring (PND 61 ± 2) rats tested for motor activity.

The brain, spinal cord, and sciatic nerve with its main branches were exposed, grossly examined, and immersed in perfusion fixative at 4°C 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, center of the cerebrum, center of the midbrain, cer¬ebellum and pons, and medulla oblongata.

GROSS NECROPSY: 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.
Statistics:
The 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 nonhomogenous. 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, 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 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.
Reproductive indices:
The following reproductive indices were reported for H2S exposure:
- mating index (%)
- fertility index (%)
- postimplantation loss per litter(%)
- late resorption or stillbirth (%)
- number of live births
- abnormal sperm (%)
- motile sperm (%)
- Cauda sperm count
- daily sperm production
Clinical signs:
no effects observed
Description (incidence and severity):
There were no deaths and no adverse physical signs observed in F0 male or female rats during the study.
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):
A statistically significant decrease in feed consumption was observed in F0 male rats from the 80-ppm H2S exposure group during the first week of exposure.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease in feed consumption was observed in F0 male rats from the 80-ppm H2S exposure group during the first week of exposure.
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:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
One case each of epididymal sprem granuloma and unilateral necrosis of the cauda in the high exposure group. Ovarian cysts in two rats (10 ppm and 30 exposure).
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No effects on sperm production or morphology. A large % of abnormal sperm was observed in two F0 rats, one from 30 ppm and one from the 80 ppm exposure group.
Reproductive performance:
no effects observed
Description (incidence and severity):
No statistically significant effects on mating index, fertility index, postimplantation loss per litter, or number of late resorptions or still births.
The reproductive and developmental toxicity indeces are reported in Table 1.

The only statistically significant difference from control in either absolute or relative F0 rat organ weights was a decrease in the absolute and
relative weight of the adrenal glands of male F0 rats from the 10 and 80 ppm H2S exposure groups and a decrease in the relative weight of the
ovaries from female rats exposed to 10 ppm H2S. Subchronic exposure of male F0 rats to H2S was associated with mild to marked sensory neuron
loss and basal cell hyperplasia in the olfactory mucosa lining the dorsal medial meatus and the dorsal medial region of the ethmoid recess. Due to
the lack of toxicologically relevant lesions in other body systems, microscopic examination of F0 male rats was limited to the reproductive tracts
in the control and high exposure groups.

In addition, F0 males in the 10- and 30-ppm exposure groups that were not reproductively successful or that had gross observations made in the
reproductive tract were also evaluated histologically. Statistical comparison of the control and high-exposure groups showed no significant
difference from control in the incidences of the histologic diagnoses found. However, there were a few histologic diagnoses with a higher incidence
in the 80-ppm treatment group when compared to control rats. Most of these diagnoses were related to seminiferous tubular degeneration
(including intratubular sperm stasis, tubular mineralization, sperm granulomas, and multinucleated giant cells), and associated changes in the
epididymis (degenerate sperm forms in lumen, aspermia, and oligospermia). One case each of epididymal sperm granulomas and unilateral
necrosis of the cauda were detected only in the high exposure group (n=12 rats).

Although not statistically significant, lymphoid interstitial infiltrate in the ventral prostate was observed more frequently in the 80ppm treatment
group (5 of 12) when compared to control animals (3 of 12). Statistical comparison of the control and high-exposure groups showed no
significant difference from control in the incidences of histologic diagnoses found. However, there were a few possibly relevant histologic
lesions that occurred in exposed groups but not in unexposed rats. One rat each in both the 10-ppm and the 30ppm exposure groups had
ovarian cysts. Ovaries of one rat in the 80-ppm group contained only a few corpora lutea, which were regressing, and a relatively large number
of tertiary follicles. This rat, as well as one other in the 30-ppm exposure group, also had squamous metaplasia of the endometrium localized to
the uterus.
Dose descriptor:
NOAEL
Remarks:
nasal olfactory lesions
Effect level:
ca. 10 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
Key result
Dose descriptor:
NOAEC
Effect level:
> 80 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
neuropathology
reproductive function (sperm measures)
reproductive performance
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment-related effects in pups were noted in growth.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related effects in pups were noted in growth or organ/body weight ratios.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment-related effects in pups were noted in development or behavioral tests. The NOAEC for the developmental toxicity was 80 ppm (111 mg/m3).
Developmental immunotoxicity:
no effects observed
The pups (F1 generation): The results of the study suggest that H2S at any studied exposure concentrations is not a behavioral or developmental neurotoxicant in rats.
Key result
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
> 80 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
organ weights and organ / body weight ratios
neuropathology
histopathology: non-neoplastic
developmental neurotoxicity
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
80 ppm (analytical)

 

Table 1. Reproductive and developmental toxicity indices following H2S exposure

 

Endpoint

Exp. group

Mating index

(%)

Fertility index

(%)

Post-implanta-tion loss per litter (%)

Late resorption or stillbirth

Number of

live birthsa

 

Abnormal

sperm (%)

 

Motile

sperm (%)

 

Cauda sperm

countb

 

Daily sperm

productionc

 

0

92 (11/12)

82 (9/11)

2.5

1 (1)

16.3 ± 1.6

0.33 ± 0.44

81.6 ± 6.2

1.02 ± 0.15

17.9 ± 2.2

10

92 (11/12)

100 (11/11)

11.0

3 (3)

14.6 ± 2.6

1.46 ± 1.89

74.9 ± 24.6

0.99 ± 0.17

18.1 ± 1.6

30

75 (9/12)

100 (9/9)

10.2

5 (3)

14.7 ± 1.6

3.33 ± 8.11

75.9 ± 13.5

1.05 ± 0.23

17.3 ± 1.9

80

83 (10/12)

80 (8/10)

7.0

1 (1)

13.9 ± 4.6

7.59 ± 21.71d

76.1 ± 11.1d

0.82 ± 0.40

17.3 ± 5.6

aNumber of live births means and standard deviations.

bCauda sperm count means and standard deviations are expressed as×109.

cDaily sperm production means and standard deviations are expressed as×106.

dThere were no sperm in the sperm motility and morphology sample from one animal (n =11).

Table 2

Structural malformations observed at birth

 

 

Observationa

 

 

 

 

 

 

 

H2S exposure concentration (ppm)

Frontal bone defect

Kinked tail

Agenesis of the tail

Anophthalmia

Small rearlegs and body

Skin and dermis

detachment

Hypognathia

 

10

1 (1)

1 (1)

1 (1)

 

1 (1)

 

 

30

 

1 (1)

 

1 (1)

 

7 (1)

2 (1)

80

 

 

1 (1)

 

 

 

 

 

aData presented are for individual animals. The number of litters affected is in parentheses

Table 3

Acquisition of developmental landmarks in pups exposed to H2S

 

 

Developmental landmarka

 

H2S exposure

Conc. (ppm)

 

Pinnae

detachment

 

Negative

geotaxis

 

Incisor

eruption

 

Eyelid

separation

 

Vaginal

patency

 

Balano-preputial

separation

0

3.4 ± 1.1 (9)

8.0 ± 1.0 (7)

10.9 ± 1.3 (9)

14.6 ± 0.9 (8)

35.2 ± 1.9 (9)

42.3 ± 2.1 (9)

10

3.6 ± 1.2 (11)

8.6 ± 0.5 (5)

11.5 ± 0.5 (11)

15.1 ± 0.5 (11)

33.3 ± 1.2 (11)

43.9 ± 2.3 (11)

30

3.1 ± 0.6 (9)

8.3 ± 0.8 (6)

12.0 ± 1.7 (9)

14.8 ± 0.8 (9)

32.7 ± 2.6 (9)

42.9 ± 2.3 (9)

80

3.3 ± 1.0 (8)

7.8 ± 0.8 (6)

11.3 ± 1.4 (8)

15.0 ± 0.8 (8)

33.5 ± 1.5 (8)

43.1 ± 1.6 (7)

 

a Data presented represent postnatal age at which the entire litter has met criterion (± SD). Data in parentheses represent group size.

 

 

Conclusions:
Neither maternal or male rats exposed to H2S demonstrated evidence of chemical-induced toxicity to reproduction. The low incidence of
developmental effects in pups and the lack of any dose-respose relationship suggest that H2S is not a developmental toxicant either.
Executive summary:

In this study, it was examined whether perinatal exposure by inhalation to hydrogen sulfide (H2S) had an adverse impact on pregnancy outcomes, offspring prenatal and postnatal development, or offspring behavior. Virgin male and female Sprague–Dawley rats (12 rats/sex/concentration) were exposed (0, 10, 30, or 80 ppm H2S; 6 h/day, 7 days/week) for 2 weeks prior to breeding. Exposures continued during a 2-week mating period and then from GD 0 through GD 19. Exposure of dams and their pups (eight rats/litter after culling) resumed between postnatal day (PND) 5 and 18. Adult male rats were exposed for 70 consecutive days. Offspring were evaluated using motor activity (PND 13, 17, 21, and 60±2), passive avoidance (PND 221 and 62±3), functional observation battery (PND 60±2), acoustic startle response (PND 21 and 62±3), and neuropathology (PND 23±2 and 61±2). There were no deaths and no adverse physical signs observed in F0 male or female rats during the study. A statistically significant decrease in feed consumption was observed in F0 male rats from the 80-ppm H2S exposure group during the first week of exposure. 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. Exposure to H2S did not affect pup growth, development, or performance on any of the behavioral tests. The results of our study suggest that H2S is neither a reproductive toxicant nor a behavioral developmental neurotoxicant in the rat at 80 ppm or lower concentrations.

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
THE ANALOGUE APPROACH
Green liquor (GL) is a complex mixture of inorganic salts in highly alkaline water solution (ca. 80% water). The composition of Green liquor is well known even if it is registered as an UVCB. All anionic and cationic groups present in green liquor is known, even if the speciation and ratio between the ionic constituents may vary in some extent. The variety in ratio/speciation makes the composition of the mixture complex.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH

Since all groups of the GL constituents are known and in order to avoid unnecessary animal testing, the non-testing read-across method can be performed by applying available hazard/study data from the mono-constituent pure inorganic salts (ie. those salts known to be dissolved in GL).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Toxicity to reproduction, effects on fertility read-across data evaluation from the following green liquor constituents may be carried out. The SOURCE substances: (1) Disodium sulphide (EC:215-211-5), (2) Hydrogen sulphide (EC:231-977-3), (3) Sodium sulphate (EC:231-820-9), (4) Sodium thiosulphate (EC: 231-867-5), (5) Sodium carbonate (EC:207-838-8), (6) Potassium carbonate (EC:209-529-3), (7) Sodium hydroxide (EC:215-185-5), (8) Potassium hydroxide (EC:215-181-3)(9) Sodium chloride (EC:231-598-3).

3. ANALOGUE APPROACH JUSTIFICATION
The principles of read-across analysis given in REACH Guidance on information requirements and chemical safety assessment, Chapter R.6.2 Guidance on the Grouping of Chemicals (ECHA 2008) has been followed as relevant. In addition, further information in support of the read-across approach has been provided in the attachement(s) and next fields 'Attached justification'. The route of administration in referred source key studies is inhalation and relevant. The inhalation route in human/worker exposure cannot be excluded even if the substance is used in systems of high containment and exposure levels are strictly controlled.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Specific details on test material used for the study:
All the following constituents of Green liquor have been taken into account in the read-across evaluation: Disodium sulphide (EC:215-211-5), Hydrogen sulphide (EC:231-977-3), Sodium sulphate (EC:231-820-9), Sodium thiosulphate (EC: 231-867-5), Sodium carbonate (EC:207-838-8), Potassium carbonate (EC:209-529-3), Sodium hydroxide (EC:215-185-5), Potassium hydroxide (EC:215-181-3), Sodium chloride (EC:231-598-3).
Species:
rat
Sex:
male/female
Route of administration:
other: Several routes tested. Oral, feed and inhalation routes tested for read-across source constituents.
Type of inhalation exposure (if applicable):
whole body
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Reproductive performance:
no effects observed
Key result
Dose descriptor:
NOAEC
Effect level:
> 80 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Test result is for potentially most toxic constituent (sulfide) and route of administration (inhalation).
Key result
Dose descriptor:
NOAEL
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Generation:
F1
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
neuropathology
histopathology: non-neoplastic
developmental neurotoxicity
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
No developmental effects observed in fetuces in any individually tested constituents of Green liquor (if test results are available).
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
80 ppm (analytical)
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
100 other: No toxic effects to reproduction (fertility and development) observed in any of the read-across study results of Green liquor constituents.
Treatment related:
no
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
112 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
The reported study data via inhalation route focus on effects of hydrogen sulfide (H2S). Hydrogen sulfide is regarded as the most relevant constituent of green liquor workers and the general population may be exposed to via inhalation.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Due to the corrosive nature of GL and its poor systemic availability in the body under normal handling, it can be assumed that the substance compound probably will not reach the foetus nor reach male and female reproductive organs at levels of toxicity.

Short- to moderate-terrn exposure for GL constituents does not have effect on fertility according the literature search. No studies were located for hydroxides (= high pH) when evaluated the GL compounds effect on fertility. Short- to moderate-term inhalation exposure for hydrogen sulfide, oxidizied sulfur constituents and carbonates did not have effect on fertility. Measured NOAEL is112 mg/m3for hydrogen sulfide. The NOAEL for sodium sulfate was 7.392 mg/l and NOAEL of disodium disulfite was 942 mg/kg bw/day. Maternal NOAEL for oral Na2CO3has been estimated to be > 179 mg/kg in rabbits, > 245 mg/kg in rats and > 340 mg/kg in mice. NOAEL for parental and maternal toxicity of K+was established as >123 mg/kg/d in mice, and >162 mg/kg/d in rats. No human studies on toxicity of GL components for fertility are available.

There is some evidence of developmental toxicity or teratogenicity for GL compounds. Only one (non standard) study in which sodium hydroxide (2μl of a 1 mM solution) was administered intra amniotically to the foetuses in the right uterine horn of 7 mice on the 13th day of gestation, showed no teratogenic effects but induced a high incidence of fetal lethality. However, the route of administration is irrelevant for further hazard evaluation.

Inhalation exposure for hydrogen sulfide does not cause external malformations for the pups at birth. Developmental NOAEL values for inhaled hydrogen sulfide have been varied from 50 to 220 ppm. However, severe alterations in the neurons architecture and brain neurotransmitters were noticed in the developing pups after hydrogen sulfide exposure from gestation to 21 post-partum period. These alterations during the critical phase of development were noticed possibly later leading to behavioural and structural abnormalities. LOAEL for brain-specific developmental toxicity was 28 H2S/m3(20ppm) for 7 h/day from gestation day 5 through postpartum day 21.

Inhaled sulfates, sulfites, carbonates and cations showed either no evidence of developmental toxicity or teratogenicity. However, decreased fetal body weights and abnormalities in skeletal tissues were noticed in some studies, but generally these are not regarded as indicative of developmental toxicity. The NOAEL for sulfate cannot be evaluated due to validity of experiments mentioned above. NOAELs for sulfide were established at 110 mg/kg bw/day in rats and 123 mg/kg bw/day in rabbits, respectively. Teratogenic NOAEL values were established for oral K2CO3administration as > 290 mg/kg/d in mice, and >180 mg/kg/d in rats. NOAEL values for K+ as >123 mg kg/d in mice, and >162 mg K+/kg/d in rats.

In the light of the literature cited, it seems reasonable to suggest that GL is unlikely to have any relevant potential for toxicity to reproduction or developmental toxicity and no further animal testing is warranted for those endpoints. None of the compounds of GL are either classified for reproductive toxicity in the EU. However, due to the observed neurotoxicity in littermates after respiratory exposure for hydrogen sulfide exposure, the OEL limits for H2S should not be overstepped when working with the GL.

Short description of key information:

Test information is limited. However, the read-across analysis for the GL constituents did not rice any specific concerns regarding reproduction toxicity endpoints and none of the known constituent salt of green liquer has a CLP classification for reproductive and/or developmental toxicity. The substance is corrosive, human exposure is limited and further testing the substance for reproductive effects is not regarded technically and scientifically justifiable.

Effects on developmental toxicity

Description of key information

The whole GL water mixture has not been tested on developmental endpoint. None of the constituents (common inorganic Ca, Na and K salts and sulfur species) have observed effects on human or test animal development at exposure levels relevant in normal manufacturing and use conditions of the substance.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Other information than whole mixture test data is available to make conclusion on no/low potential for developmental toxicity and teratogenicity. All constituent of the mixture are common calcium, sodium and potassium salts (hydroxides, carbonates and sulfides/sulfates). Animal testing of highly alkaline green liquor is not justifiable, since alternative methods are available. Read-across analog approach is a suitable method to fulfill this information requirement.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Reason / purpose for cross-reference:
read-across: supporting information
Reason / purpose for cross-reference:
read-across: supporting information
Specific details on test material used for the study:
All the following constituents of Green liquor have been taken into account in the read-across evaluation: Disodium sulphide (EC:215-211-5), Hydrogen sulphide (EC:231-977-3), Sodium sulphate (EC:231-820-9), Sodium thiosulphate (EC: 231-867-5), Sodium carbonate (EC:207-838-8), Potassium carbonate (EC:209-529-3), Sodium hydroxide (EC:215-185-5), Potassium hydroxide (EC:215-181-3), Sodium chloride (EC:231-598-3).
Species:
other: rat, mouse, rabbit
Route of administration:
other: Both inhalation (H2S) and oral feeding study results (carbonates and oxygenated sulphur constituents) are available for the Green liquor constituents.
Type of inhalation exposure (if applicable):
whole body
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Behaviour (functional findings):
no effects observed
Neuropathological findings:
no effects observed
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
Key result
Dose descriptor:
NOAEL
Remarks:
Several read-across results of Green liquor constituents indicate NOAEL levels > 100 mg/kg/d bw.
Effect level:
> 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
behaviour (functional findings)
changes in number of pregnant
clinical signs
dead fetuses
histopathology: non-neoplastic
maternal abnormalities
mortality
necropsy findings
neuropathology
number of abortions
pre and post implantation loss
total litter losses by resorption
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEC
Remarks:
H2S
Effect level:
> 50 ppm (analytical)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
In inhalation study, 100 ppm or 150 ppm H2S resulted in a significant (P
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
A slight retardation in weight of offspring rat during lactation at dose level 955 mg/kg/ bw/day sodium tiosulphate.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Key result
Dose descriptor:
LOAEC
Remarks:
H2S
Effect level:
>= 100 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Dose descriptor:
LOAEL
Remarks:
constituents of green liquor
Effect level:
955 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
read-across assessment
Sex:
male/female
Basis for effect level:
changes in litter size and weights
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
100 ppm
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

In inhalation study, 100 ppm or 150 ppm H2S resulted in a significant (P<O.01) but slight (-4% of the control value) decrease in fetal body weight.

Conclusions:
The read-across analysis from supporting substance (structural analogue or surrogate): No effects on development observed in maternal animals/fetuces in read-across study analysis of the refered test results of Green liquor constituents. Several valid read-across results of each Green liquor constituents indicate NOAEL levels > 100 mg/kg/d bw (in lowest) threshold studies or significantly higher results (>>100 mg/kg/d bw) in non-treshold developmental toxicity studies.
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.
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
Specific details on test material used for the study:
Hydrogen sulfide gas by Air Liquide (France).
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..
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.
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.
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
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.
Key result
Dose descriptor:
NOEC
Effect level:
ca. 50 ppm (analytical)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
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
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
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
100 ppm (analytical)
Treatment related:
no

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,

Conclusions:
Treatment with 100 ppm hydrogen sulfide caused no maternal toxicity (table 1) or adverse effects on the developing embryo or fetus (table 2).
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
GLP compliance:
no
Limit test:
no
Species:
mouse
Strain:
CD-1
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Mouse: Aqueous solutions of sodium carbonate were administered daily via oral intubation to pregnant mice at doses ranging from 3.4 to 340 mg/kg bw during days 6-15 of gestation.
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Mouse: 25 females were mated with young adult males, observation of the vaginal sperm plug was considered day 0 of gestation.
Control animals:
yes
Maternal examinations:
Body weights were recorded on days 0, 6, 11, 15 and 17 of gestation, observed daily for appearance and behaviour. On day 17 all
dams were subjected to caesarean section under anesthesia, numbers of implantation sites, resorption sites, and live and dead fetuses were
recorded. Body weight of live fetuses were recorded. The urogenital tract of each dam was examined in detail for anatomical normality.
Fetal examinations:
All fetuses were examined grossly for the presence of external congenital abnormalities.
1/3 of fetuses of each litter underwent detailed visceral examinations employing the Wilson technique.
The remaining 2/3 were examined for skeletal defects.
Statistics:
Not reported.
Conclusions:
Developmental toxicity
The study concludes in three species (mouse, rabbit, rat) that regarding sodium carbonate there is no concern with regard to developmental toxicity. The SIAR report concluded that there is no needs to study further.
Toxicity to reproduction
Conclusion of the SIAR (2002) report concluded that a reproduction toxicity test is not available for sodium carbonate. However, the substance will
usually not reach the foetus or the male and female reproductive organs when exposed orally, dermally or by inhalation, as it does not become available systemically. As such, it is considered not useful to perform a toxicity to reproduction study.
Executive summary:

Aqueous solutions of sodium carbonate were administered daily via oral intubation to pregnant mice at doses ranging from 3.4 to 340 mg/kg bw during days 6-15 of gestation. The test substance did not affect implantation nor the survival of dams and foetuses. Soft and skeletal tissue anomalies were noted in the experimental group, but the incidence of these findings did not differ from that of sham-treated controls.

Similar negative results were reported for rats and rabbits for daily doses from 2.45 -245 mg/kg bw and 1.79-179 mg/kg bw, respectively (FDA, 1974).

This study confirms in three species that there is no concern with regard to developmental toxicity, which supports the general consideration that the substance will usually not reach the foetus when exposed to sodium carbonate, as it does not become systemically available.

Additional information from potassium carbonate (Source: European Chemicals Agency, http://echa.europa.eu, REACH registration data)

Read across substance potassium hydrogencarbonate reproduction organs have been evaluated. In these studies the macroscopically and histopathologically evaluated reproductive organs epididymides, testes, ovaries, and uterus were free of treatment related effects, even at lifetime (30 months) treatment with dose levels exceeding the guideline limit dose for one or two generation studies of 1000 mg/kg body weight/day

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
110 mg/m³
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

For the purpose of CLP classification, reproductive toxicity is divided into three main differentiations, which relate to (i) impairment of male and female reproductive functions or capacity (fertility), (ii) the induction of non-heritable harmful effects on the progeny (developmental toxicity), and (iii) effects on or via lactation.

The available data give no indication that Green liquor is toxic for reproduction. Based on the results of the read-across data for green liquor constituents and from reproduction/developmental test and the developmental toxicity/teratogenicity studies, green liquor does not need to be classified for toxic to reproduction according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

As an additional information, none of the known constituents of GL are classified for reproductive toxicity in the EU (year 2019 data, Source: European Chemicals Agency, http://echa.europa.eu/).

Additional information