Dibismuth trisulphide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Other studies are not required.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-01-16 to 2013-03-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and OECD guideline compliant

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted 27.07.1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species and strain: Hsd.Brl.Han:Wist rat
- Source: Toxi-Coop Zrt. Cserkesz u. 90. H-1103 Budapest Hungary
- Age at study initiation: 71-76 days old
- Weight at study initiation: (P) Males: 296-340 g; Females: 178-215 g;

- Fasting period before study:

- Housing: Before mating: 2 animals of the same sex/ cage; mating: 1 male and 1 female / cage; pregnant females will be housed individually. Males after mating: 2 animals / cage
- Diet: ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum, and tap water from municipal supply, as for human consumption, from 500 mL bottles ad libitum.
- Water: drinking water
- Acclimatization time: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: 8-12 air exchanges/hour by central air-condition system.
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Route of administration:

oral: gavage

Vehicle:

other: Sunflower oil (Helianthii annui oleum raffinatum)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared in the formulation laboratory of the Test Facility daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is not soluble in water therefore sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item
- Concentration in vehicle: The test item was formulated in the vehicle in concentrations of 500, 150 and 50 mg/mL.

Analytical verification of doses or concentrations:

yes

Frequency of treatment:

once per day

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
other: nominal in vehicle

No. of animals per sex per dose:

12 per sex and dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose setting with 1000, 300 and 100 mg/kg bw/day based on findings obtained in a previous 14-Day Oral Gavage Dose Range Finding Study
with Dibismuth Trisulfide in the Rat (Toxi-Coop study no. 559.400.3811) and was in agreement with the Sponsor. The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect.

Positive control:

not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: General clinical observations were made once a day, after treatment at approximately the same time, considering the peak period of anticipated effects after dosing. Pertinent behavioral changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality were recorded including onset, degree and duration of signs.

BODY WEIGHT: Yes
- Time schedule for examinations: All parental animals were weighed with an accuracy of 1 g. Parental males were weighed on the first day of dosing (day 0), at least weekly thereafter and at termination. Parental females were weighed on the first day of dosing (day 0) then weekly, on gestation days 0, 7, 14 and 21 and on post-partal days 0 (within 24 hours after parturition) and 4, as well as on the day of necropsy. Body weight of the female animals were additionally weighed on gestational days 10 and 17 in order to give accurate treatment volumes, but these data will not be evaluated statistically. Body weight data were reported individually for adult animals.

FOOD CONSUMPTION:
The food consumption was determined weekly by reweighing the nonconsumed diet with an accuracy of 1 g during the treatment period except
mating days (pre-mating and post mating for male animals and nonpregnant females, during pre-mating, gestation days 0, 7, 14 and 21, lactation
days 0 and 4 for dams).

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

The investigated organs of reproductive system (testes, epididymides) were histologically normal. The various spermatogenic cells
(the spermatogonia, the spermatocytes, the spermatids and spermatozoa), representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically. Compared to the concurrent control, epididymides
were histologically normal in all animals.

Litter observations:

Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are
significantly smaller than normal pups), and the presence of gross abnormalities. Live pups were counted, sexed and litters were weighed within 24 hours of parturition (on the day when parturition was complete) and on day 4 postpartum with an accuracy of 0.1 g.In addition to the observations on parent animals, any abnormal behavior of the offspring was monitored. All the litters were checked and recorded daily for the number of viable
and dead pups. The pups found dead were subjected to necropsy by a macroscopic examination. On day of birth, a lung flotation test was
performed on all dead pups to separate stillborns from those that died after delivery. The lung flotation test is negative for stillborns (pups that
died intrauterine) but positive for pups that died after delivery.

Postmortem examinations (parental animals):

Pathology
Gross necropsy was performed on each animal one day after the last treatment (day of sacrificing). Animals were anesthetized by Isofluran (details are presented in "Details of Other Materials") and then were exsanguinated. After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality was recorded with details of
the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of
corpora lutea was recorded. The testes, epididymides and brain of all male adult animals were weighed. The uterus with cervix, vagina, testes,
epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, ovaries, pituitary of all adult animals were preserved.
Testes and epididymides were fixed in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.

Postmortem examinations (offspring):

Pathology
Pups were carefully examined for gross (external) abnormalities and euthanized on postnatal day 4.

Statistics:

The statistical evaluation of appropriate data (marked †above) were performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-wayanalysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of intergroup differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group
comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible. The frequency of clinical signs, pathology and
histopathology findings were calculated. Results were evaluated in comparison with values of control group (i.e.control value).

Reproductive indices:

The examined parameters of reproductive performance were not affected by the treatment with the test item at 1000, 300 or 100 mg/kg bw/day
doses in male or female animals.

Offspring viability indices:

Parameters listed below were evaluated
- Litter weight on postnatal days 0 and 4,
- Mean body weight gain per litter between postnatal days 0-4
- Number of live births per litter, and number of viable pups per litter on postnatal days 0 and 4
- Survival Index of pups on postnatal day 4
- Sex ratio % (on postnatal days 0 and 4)
There was no test item related effect on pup’s mortality.There were no test item related differences between the control and test item treated groups in the ration or in the litter means of genders on postnatal days 0 or 4. Test item related clinical signs were not detected in the offspring.
The number and percentage of pups with findings (cold, no milk in the stomach, pale) were the highest in the control group. Test item effect on the
body weight development of offspring was not found.
The mean litter weights and mean pup weights were similar in the control and in all test item treated groups on postnatal days 0 and 4.
No test item related macroscopic alterations were found in offspring subjected to gross pathological examination.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Mortality
There was no test item related mortality at any dose level (1000, 300 and 100 mg/kg bw/day).
Clinical observation
The behavior and physical condition of animals were normal during the entire observation period (pre-mating, mating, post-mating, gestation and
lactation periods) in each group.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight and body weight gain The body weight development was undisturbed in the test item treated animals at each dose level (1000, 300 and 100 mg/kg bw/day) during the entire treatment period (pre-mating, mating, post-mating, gestation and lactation periods).
Food consumption
The mean daily food consumption was not affected by the test item in male or female animals at 1000, 300 and 100 mg/kg bw/day during the study (premating and post mating for male animals, during pre-mating for dams and non-pregnant females, during gestation and lactation periods for
dams).

REPRODUCTIVE FUNCTION: (PARENTAL ANIMALS)
Reproduction
There were no test item related differences between the control and test item treated groups in delivery data of dams and in the reproductive
performance of male and female animals.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no test item related changes in brain, testes and epididymides weights.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histopathological examinations of male and female genital organs (ovaries, testes and epididymides) did not reveal any toxic or other test item
related changes at any dose level.

NECROPSY (PARENTAL ANIMALS)
Specific macroscopic alterations related to the test item were not found during the necropsy.

Dose descriptor:

NOEL

Remarks:

for general toxicity

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Dose descriptor:

NOEL

Remarks:

for reproductive performance

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

OFFSPRING
Negative effects of the test item on offspring development (mortality, clinical signs, and body weight and necropsy findings) were not detected
between postnatal days 0 and 4.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Reproductive effects observed:

not specified

Conclusions:

According to OECD guideline 421 a Reproduction/Developmental Toxicity Screening Test with dibismuth trisulfide in the rats was performed. Under the conditions of the present study dibismuth trisulfide did not cause toxic changes and did not influence the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Hsd.Brl.Han: Wistar rats or development of the F1 offspring from conception to day 4 post-partum after repeated dose oral administration at 1000, 300 or 100 mg/kg bw/day. Based on these observations the No Observed Effect Levels (NOEL) were determined as follows:
NOEL for male rats: 1000 mg/kg bw/day
NOEL for female rats: 1000 mg/kg bw/day
NOEL for reproductive performance of the male rats: 1000 mg/kg bw/day
NOEL for reproductive performance of the female rats: 1000 mg/kg bw/day
NOEL for F1 Offspring: 1000 mg/kg bw/day

Executive summary:

According to OECD guideline 421 a Reproduction/Developmental Toxicity Screening Test with dibismuth trisulfide in the rats was performed. Four groups of Hsd.Brl.Han:Wist rats (n=12/sex/group) were administered orally (by gavage) once a day at 0 (vehicle only), 1000, 300 and 100 mg/kg bw/day at concentrations of 500 mg/mL, 150 and 50 mg/mL corresponding to 2 mL/kg bw dose volume. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Concentration of the test item in the dosing formulations was checked twice during the study. Dibismuth trisulfide concentrations in the dosing formulations varied in the range of 84 and 97 % of the nominal values at both analytical occasions, thereby confirming proper dosing. All animals of the parent (P) generation received test item or vehicle prior to mating (14 days) and throughout mating. Test item or vehicle was administered to male animals post mating up to the day before the necropsy (altogether for 43 days). For females, test item was administered through the gestation period and up to lactation days 3-8 i.e. up to the day before the necropsy (altogether for 43 or 49 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offsprings. The dams were allowed to litter, and rear their young up to day 4 postpartum. Litters were weighed and offsprings were observed for possible abnormalities and were euthanized on postnatal day 4. All parental animals were subjected to gross pathology one day after the last treatment. Histopathology examination was performed on reproductive organs (testes, epididymides and ovaries) in the control and high dose groups. The reproductive organs of non-pregnant females and males cohabited with in the low dose group were also processed and evaluated histologically. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with the vehicle (sunflower oil) only.

Under the conditions of the present study Dibismuth trisulfide did not cause toxic changes and did not influence the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Hsd.Brl.Han: Wistar rats or development of the F1 offspring from conception to day 4 post-partum after repeated dose oral administration at 1000, 300 or 100 mg/kg bw/day. Based on these

observations the No Observed Effect Levels (NOEL) were determined as follows:

NOEL for male rats: 1000 mg/kg bw/day

NOEL for female rats: 1000 mg/kg bw/day

NOEL for reproductive performance of the male rats: 1000 mg/kg bw/day

NOEL for reproductive performance of the female rats: 1000 mg/kg bw/day

NOEL for F1 Offspring: 1000 mg/kg bw/day

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is reliable based on its GLP and guideline compliance.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Key study Toxicity to reproduction (according to OECD 421)

The toxicity to reproduction of dibismuth trisulfide was determined according to OECD guideline 421. Four groups of Hsd.Brl.Han:Wist rats were administered orally (by gavage) once a day with 0 (vehicle only), 1000, 300 and 100 mg/kg test item bw/day.

Under the conditions of the present study dibismuth trisulfide did not cause toxic changes and did not influence the reproductive performance (gonad function, mating behaviour, conception, pregnancy, parturition) in parental male and female Hsd.Brl.Han: Wistar rats or development of the F1 offspring from conception to day 4 post-partum after repeated dose oral administration at 1000, 300 or 100 mg/kg bw/day.

Based on these observations the No Observed Effect Levels (NOEL) were determined as follows:

NOEL for male rats: 1000 mg/kg bw/day

NOEL for female rats: 1000 mg/kg bw/day

NOEL for reproductive performance of the male rats: 1000 mg/kg bw/day

NOEL for reproductive performance of the female rats: 1000 mg/kg bw/day

NOEL for F1 Offspring: 1000 mg/kg bw/day

 

 

Waiver

In accordance with column 1 section 8.7.3 of REACH regulation EC (No) 1907/2006 Annex IX, a two-generation reproductive toxicity study is required if the 28- or 90-day study indicates adverse effects on reproductive organs or tissues. In a screening study with dibismuth trisulfide according to OECD 421 no adverse effects on reproductive organs or tissues were observed. Besides, in a 28-Day Repeated Dose Toxicity Study (see 7.5.1 Key.001 Repeated dose toxicity: oral_read across to bismuth) no findings regarding toxicity to reproduction were observed. Due to these results a two-generation study is not required.

Short description of key information:
The toxicity to reproduction of dibismuth trisulfide was determined according to OECD guideline 421. The following results were obtained:
NOEL for male rats: 1000 mg/kg bw/day
NOEL for female rats: 1000 mg/kg bw/day.
NOEL for reproductive performance of the male rats: 1000 mg/kg bw/day
NOEL for reproductive performance of the female rats: 1000 mg/kg bw/day
NOEL for F1 Offspring: 1000 mg/kg bw/day

Based on these results (no effects at all limit dose) the two-generation or a comparable study is not required for the test substance.

Effects on developmental toxicity

Description of key information

A weight of evidence approach was done to advise the endpoint developmental toxicity to avoid new animal testing.
Based on the results in an OECD 421 test (no effects at all to even at the limit dose) and no limits on developmental toxicity in rabbits and rats another bismuth salt being water soluble and more bioavailable as dibismuth trisulfide demonstrate the lack of developmental toxicity of dibismuth trisulfide.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Study published only as an abstract therefore it is not possible to fully assess reliability.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Study published as an abstract only - no information is provided regarding whether any guideline was followed, but the data suggest a similar design as described in OECD 414.

GLP compliance:

not specified

Limit test:

no

Species:

rabbit

Strain:

other: dutch rabbits

Details on test animals or test system and environmental conditions:

- female Dutch rabbits

Route of administration:

oral: gavage

Vehicle:

other: aqueous aluminium hydroxide

Duration of treatment / exposure:

Female rabbits: Days 8-20 of pregnancy inclusive (day of mating is Day 1 of pregnancy).

Frequency of treatment:

Once daily

Duration of test:

Approximately 23 days

Remarks:

Doses / Concentrations:
50, 100 or 200 mg/kg bw
Basis:

Details on study design:

On Day 30 the rabbits were subjected to autopsy and foetuses examined for external, visceral and skeletal abnormalities.

Fetal examinations:

Rabbit foetuses were examined for external, visceral and skeletal abnormalities.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Marked reduction in maternal body weight occurred in rabbits. No information regarding dose at which toxic effects occurred.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
It was concluded that no adverse effects upon pre- or post-implantation loss, numbers of viable foetuses or foetal development were apparent.

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

no data

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

no data

Basis for effect level:

other: embryotoxicity

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

no data

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

It was concluded that no adverse effects of the test substance bismuth citrate upon pre- or post-implantation loss, numbers of viable foetuses or foetal development were apparent, even tested at systemic toxic doses.

Executive summary:

In this study (Secker, 1993), which has been published as an abstract only, the test substance bismuth citrate was administered to female Dutch rabbits. On Day 30 (day of mating is Day 1 of pregnancy) the rabbits were subjected to autopsy and foetuses examined for external, visceral and skeletal abnormalities. Plasma bismuth levels were determined at the beginning and end of the treatment period. Plasma levels up to 420 ng/g were recorded. Maternal toxicity, manifest as a marked reduction in body weight, was apparent in the rabbit. Numbers of viable foetuses or foetal development was not affected.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Weight of evidence

 

To cover the endpoint developmental toxicity/teratogenicity a weight of evidence approach was chosen.

 

OECD 421 Reproduction/ Developmental Toxicity Screening Test

In a study with dibismuth trisulfide according to OECD 421 no adverse effects neither on reproductive organs or tissues nor on reproduction parameters and the offspring were observed even at the limit dose.

 

Secker,1993: Effects of bismuth citrate on pregnant rats and rabbits

Furthermore a study (Secker, 1993) on effects of bismuth citrate on pregnant rats and rabbits is available. In the study the NOEL (rabbit) for teratogenicity, embryotoxicity and fetotoxicity was determined to be 200 mg/kg bw, no adverse effects were observed at the highest dose tested at which systemic toxicity in dams were noted.

In rats the highest dose tested 1200 mg/kg bw (even exceeding the recommended limit dose) also no effects were noted. Therefore the NOEL (rat) for maternal toxicity, teratogenicity, developmental toxicity, embryotoxicity was determined to be 1200 mg/kg bw.

The substance bismuth citrate and dibismuth trisulfide are both salts. Bismuth citrate is a substance which is more soluble than dibismuth trisulfide and therefore more bioavailable. As a consequence the tests with bismuth citrate represent a worst case scenario.

 

Conclusion

Based on the results noted in a OECD 421 reproduction /developmental toxicity study with dibismuth trisulfide and the study with bismuth citrate on pregnant rats and rabbits a developmental and teratogenic effect can be excluded for the substance dibismuth trisulfide.

Justification for selection of Effect on developmental toxicity: via oral route:
A weight of evidence approach was done to advise the endpoint developmental toxicity to avoid new animal testing.
Based on the results in an OECD 421 test (no effects at all to even at the limit dose) and no limits on developmental toxicity in rabbits and rats another bismuth salt being water soluble and more bioavailable as dibismuth trisulfide demonstrate the lack of developmental toxicity of dibismuth trisulfide.

Toxicity to reproduction: other studies

Additional information

Other studies are not required.

Justification for classification or non-classification

Based on the results for toxicity to reproduction, dibismuth trisulfide is not subject to classification as reproductive or development toxic according to the criteria of EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008.