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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23.08.2017 - 14.12.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
Published in O.J.L. 142, 2008
Deviations:
yes
Remarks:
See Any other information (no impact)
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Adopted on 22nd January 2001
Deviations:
yes
Remarks:
See Any other information (no impact)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Product of Semi-Dry Absorption method of Flue Gas Desulphurization (SDA Product)
- Molecular formula (if other than submission substance): not available
- Molecular weight (if other than submission substance): not available

Composition of test substance
CaSO4*H2O 27.46 %
CaCO3 10.48 %
CaSO3*1/2H2O 34.83 %
CaCl2*2H2O 15.30 %
CaF2 0.62 %
Ca(OH)2 7.78 %
CaO free 5.89 %
Loss by ignition 10.67 %
Sum of toxic metals
(As, Ba, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Sb, Se, Te, Ti, V, Zn)….< 0.1 %

Batch No.: SDA 13.12.16 SKW
Appearance: light grey solid powder

Test animals

Species:
rat
Strain:
Wistar
Remarks:
CRL (SPF quality - guaranteed)
Details on test animals and environmental conditions:
TEST ANIMALS
sexually adult females (males – only for mating)
- Source: Charles River SPF breeding, supplied via VELAZ s.r.o., Czech Republic, RČH CZ 11760500
- Age at study initiation: 11 weeks
- Weight at study initiation: 201.8 - 306.3 g
- Housing: in plastic cages containing sterilised clean shavings of soft wood or sterilized LIGNOCEL in a controlled environment
- Diet (e.g. ad libitum): Complete peleted diet for rats and mice in SPF breeding (manufacturer: Altromin Spezialfutter GmbH&Co.KG, Im Seelenkamp 20, D-3271, Lage, Germany), sterilized before using.
- Water (e.g. ad libitum): drinking water ad libitum; water was sterilised before using
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 30 - 70 %
- Air changes: 15 per hour
- Photoperiod: 12 h light / 12 h dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance consists of various components insoluble in vehicle (olive oil). A suitable analytical method cannot be found for homogeneity and stability testing. Since undissolved particles of the test substance are easily visible in the application form, homogeneity will be checked by eye (suspension will be mixed for 10 minutes by magnetic stirrer). Stability of the test substance in the application form cannot be verified analytically. But the application form will be prepared just before the application and there is no indication that a mixture of rigid components would be unstable in the suspension (in olive oil) for that short time period (1 hour).

The application forms of the test substance (suspensions in olive oil) were prepared daily just before administration. The mixtures were mixed by the magnetic stirrer for 10 minutes and then during administration.

VEHICLE
- Concentration in vehicle: The concentrations of suspensions at all dose levels were adjusted to ensure the administration of 1mL per 100 g of body weight. The vehicle control group was administered by olive oil in the same volume.
- Amount of vehicle (if gavage):
- Lot/batch no.: 8002182001
- Purity: pharmaceutical quality
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Stability of the test substance in the application form cannot be verified analytically. But the application form will be prepared just before the application and there is no indication that a mixture of rigid components would be unstable in the suspension (in olive oil) for that short time period (1 hour).
Details on mating procedure:
Měla Lenka:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 M / 2 F
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy




- Impregnation procedure: [artificial insemination / purchased timed pregnant / cohoused]
- If cohoused:
- M/F ratio per cage: 1 M / 2 F
- Length of cohabitation:
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- Verification of same strain and source of both sexes: [yes / no (explain)]
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy





- Any other deviations from standard protocol:




Duration of treatment / exposure:
5th - 19th day after fertilisation
Frequency of treatment:
7 days per week at the same time (8.00 – 10.00 am)
Duration of test:
20 days
No. of animals per sex per dose:
24 pregnant females for each dose level (total number of animals before mating: 100 females and 25 males)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels for study – 160, 400 and 1000 mg/kg/day – have been chosen with respect to the information given in the Study No. 67/09/18: Semi Dry Absorption (SDA) Product - Reproduction/Developmental Toxicity Screening Test.

- Rationale for animal assignment: randomly

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the 1st day of pregnancy and then on the 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: uterus (incl. the cervix) was removed and weighed.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Uteri of non-pregnant females were examined to confirm the non-pregnant status
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Sex and individual body weights of foetuses were recorded.
Each foetus was examined for external alterations: symmetry of fore and hind limbs, number of fingers, closing or opening of eye fissures and external auditory canal, symmetry of head, integrity of superior palatum, status of umbilicus and genital papilla were observed.

One half of each litter (one half of female and male foetuses) was examined for soft tissue alterations using careful gross dissection.
Second half of each litter was processed and microscopically examined for skeletal and cartilage alterations according to the internal SOP.
Single staining displayed only ossified skeletal structures was used: the foetuses were fixed in ethanol, macerated in potassium hydroxide solution, stained with Alizarin red and placed in glycerine-based solution.
The skeletal examination was performed using a stereomicroscope and included examination of skull, clavicle, scapula, sternebra and sternum, ribs, vertebrae, pelvic girdle, forelimb/hindlimb.
Statistics:
For statistical evaluation the software Statgraphic® Centurion (version XV, USA) was used. The data from control group were compared with data from treated groups.
The results statistically significant on probability level 0.05 are indicated in the summary tables.

The parametric tests were used for statistical evaluation of:
• body weight of females (5th, 8th, 11th , 14th , 17th , 20th day of pregnancy)
• corrected body weight (subtraction weight of uterus from surgery body weight of females)
• food consumption (per interval)
• mean weight of foetuses (males, females, both sex)
• biometry of uteri (absolute and relative weight )
• preimplantation (IUDE) and postimplantation (IUDL) losses

As the first step the test for normality (Shapiro-Wilk test) was performed. If the data were not normally distributed the transformation of data was performed (Box-Cox transformation). If the data were not normal distributed after transformation,the non-parametric tests (Kruskal-Wallis Test and Mann-Whitney test) for comparison of the medians were performed.
If data were normally distributed after transformation, the Variance check (Levene’s test) to verify standard deviations within each group was used. One-Way ANOVA (probability level 0.05) was used to detect whether there were any significant differences amongst the means and then the post hoc statistical testing (Fisher's least significant difference - LSD test) for only statistical significant differences was performed.

The non-parametric tests were used for statistical evaluation of following parameters:
• number of corpora lutea, number of implantations, number of resorptions
• number of live foetuses (males, females, both sex)
The two-groups Mann-Whitney test (probability level 0.05) was applied.

The categorical data (of serious findings - external and internal alteration) was not implemented by the reason of low or similar incidence of these findings at the treated group against the control.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean food consumption of all treated groups was comparable with control group from the 5th day to the 14th day pregnancy, except 8th day of pregnancy at the lowest dose level.
Statistically significant decrease of food consumption was observed on the 8th day (interval 5th – 8th day) in females of the dose level 160 mg/kg/day and on the 17th and 20th day of pregnancy (interval 14th – 20th day) in females of the dose levels 160 and 400 mg/kg/day.
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The increased absolute uterine weight was observed in females at dose level 1000 mg/kg/day. This increase was noted in relation to higher litter size and without clear dose response and thus did not relate to the test substance treatment. Statistically significant differences of uterus weights were not detected in females of any dose level.
Corrected body weight of all treated females (the necropsy body weight of female minus weight of uterus) was not statistically significantly changed compared to control females.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic examination was performed in all females (including females without foetuses).
The uterus dilatation (probably the change associated with the physiological oestrous cycle) was detected in all groups (2-3-1-1). No finding related with treatment was noted at necropsy in treated females. Only in one female No. 128 was detected dark brown vaginal discharge. This female became pregnant and then all implanted conceptuses in a uterus were totally resorbed.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
The mean number of implantations, corpora lutea and resorptions in treated females was not statistical significantly changed in comparison with the control females.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The numbers of implantations and corpora lutea were slightly higher at the highest dose level.
Preimplantation losses (IUDE) were slightly increased at the middle dose level (10.79 %) in comparison with control group (8.46 %).
Postimplantation losses (IUDL) were statistically insignificantly increased at the lowest (12.19 %) and at the middle (9.06 %) dose levels in comparison with control (5.10 %).
The mean number of implantations, corpora lutea and resorptions in treated females was not statistical significantly changed in comparison with the control females.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The numbers of resorptions were similar at all dose levels and comparable with control group. The mean number of implantations, corpora lutea and resorptions in treated females was not statistical significantly changed in comparison with the control females.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The mean number of implantations, corpora lutea and resorptions in treated females was not statistical significantly changed in comparison with the control females.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Dead foetuses were found out in treated groups as well in control group (1 – 3 – 1 – 0). The three dead foetuses at the lowest dose level came from one litter.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no mortality of females, no changes in health condition status, no pathological findings in the dams, no dose related changes in reproduction parameters

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weight of foetuses was increased with the dose dependence in all groups compared to the control. Although the variations between individual body weights of foetuses at all dose levels against control foetuses were observed, this difference was not statistically significant. Males were heavier than females in all groups (include control). This weight imbalance is common.
The foetal body weight was statistically insignificantly increased at all dose levels.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Although the variations between individual body weights of foetuses at all dose levels against control foetuses were observed, this difference was not statistically significant. Males were heavier than females in all groups (include control). This weight imbalance is common.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The total number of live foetuses in group was increased at the highest dose level compared to the control group but the average total number of foetuses in litter was well balanced with control.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The decreased number of male foetuses in litter at the middle dose and increased number of female foetuses in litter at the highest dose were statistically significant. Sex ratio (mean value) was similar in all groups, except sex ratio at the dose level 400 mg/kg/day (6 male : 8 female).
Changes in postnatal survival:
effects observed, non-treatment-related
Description (incidence and severity):
Dead foetuses were found out in treated groups as well in control group (1 – 3 – 1 – 0). The three dead foetuses at the lowest dose level came from one litter.
Test substance-related foetal mortality was not evident at any dose level.
External malformations:
no effects observed
Description (incidence and severity):
No macroscopic changes of soft tissues and external alteration were found out.
Detailed necropsy of foetuses did not reveal increase of external and visceral variations and malformations at any dose level.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Examination of foetal skeleton indicated mainly delayed development of the skeleton at all doses including control group.
During examination of foetal skeleton delayed ossification of skeleton at all doses including control group was observed. The occurrence of structural skeletal variations could not be considered as teratogenic effect of the test substance on early prenatal development of organism in uterus because incidence of these findings was accidental or comparable with control group.
Visceral malformations:
no effects observed
Description (incidence and severity):
Detailed necropsy of foetuses did not reveal increase of external and visceral variations and malformations at any dose level.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on no altered growth and no serious structural abnormality found in treated foetuses

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
There were no unscheduled deaths of females during the study at any dose level.
No adverse changes of health condition and no clinical symptoms of intoxication were found in females at any dose level after administration of the test substance.
During the control of body weight increments and food consumption in pregnant females at all dose levels: 160, 400 and 1000 mg/kg/day toxicologically significant treatment-related effects were not detected. Evaluation of uterine weights (incl. absolute and relative weight of uterus) did not reveal toxicologically significant treatment-related effects. Macroscopical structure of organs of pregnant females and values of reproduction parameters (number of females with live foetuses, number of live and dead foetuses, early and late resorptions and sex ratio of foetuses), were unaffected by treatment with the test substance.
Test substance-related foetal mortality was not evident at any dose level. The foetal body weight was statistically insignificantly increased at all dose levels. Detailed necropsy of foetuses did not reveal increase of external and visceral variations and malformations at any dose level.
During examination of foetal skeleton delayed ossification of skeleton at all doses including control group was observed. The occurrence of structural skeletal variations could not be considered as teratogenic effect of the test substance on early prenatal development of organism in uterus because incidence of these findings was accidental or comparable with control group.
The NOAEL (No Observed Adverse Effect Level) for toxicity in PREGNANT FEMALES was established as 1000 mg/kg/day. This NOAEL value is based on no mortality of females, no changes in health condition status, no pathological findings in the dams, no dose related changes in reproduction parameters.
The NOAEL (No Observed Adverse Effect Level) for PRENATAL DEVELOPMENT was established as 1000 mg/kg/day. This NOAEL is based on no altered growth and no serious structural abnormality found in treated foetuses.
Executive summary:

Introduction

The test substance, Product of Semi-Dry Absorption method of Flue Gas Desulphurization (SDA Product), was tested for prenatal developmental toxicity using the Method B.31, Prenatal Developmental Toxicity Study, Council Regulation (EC) No. 440/2008, Published in O.J. L. 142, 2008 and OECD Test Guideline No. 414, Prenatal Developmental Toxicity Study, Adopted by the Council on January 22nd 2001.

Study performance

Wistar rat females of SPF quality were used for testing. After acclimatization the females were mated with males. The test substance was then administered to pregnant females – daily from the 5th to the 19th day of pregnancy. The study included four groups of females – 3 treated groups and 1 control group (vehicle only). The test substance was administered suspended in olive oil by stomach tube and the concentrations of suspensions at all dose levels were adjusted to ensure the administered volume of 1 mL per 100 g of body weight. 

The dose levels for study – 160, 400 and 1000 mg/kg/day – have been chosen with respect to the information given in the Study No. 67/09/18: Semi Dry Absorption (SDA) Product - Reproduction/Developmental Toxicity Screening Test (VUOS-CETA Report No. 1053, 2010).

The health condition, clinical status after application, body weight and food consumption of maternal animals were monitored during developmental toxicity study. On the 20th day of pregnancy the maternal animals were euthanized, the uterine contents were examined and the foetuses were assessed for changes on soft tissues and skeleton.

Results

There were no unscheduled deaths of females during the study at any dose level.

No adverse changes of health condition and no clinical symptoms of intoxication were found in females at any dose level after administration of the test substance.

During the control of body weight increments and food consumption in pregnant females at all dose levels: 160, 400 and 1000 mg/kg/day, toxicologically significant treatment-related effects were not detected. Evaluation of uterine weights (incl. absolute and relative weight of uterus) did not reveal toxicologically significant treatment-related effects. Macroscopical structure of organs of pregnant females and values of reproduction parameters (number of females with live foetuses, number of live and dead foetuses, early and late resorptions and sex ratio of foetuses), were unaffected by treatment with the test substance.    

Test substance-related foetal mortality was not evident at any dose level. The foetal body weight was statistically insignificantly increased at all dose levels. Detailed necropsy of foetuses did not reveal increase of external and visceral variations and malformations at any dose level.

During examination of foetal skeleton delayed ossification of skeleton at all doses including control group was observed. The occurrence of structural skeletal variations could not be considered as teratogenic effect of the test substance on early prenatal development of organism in uterus because incidence of these findings was accidental or comparable with control group.

The NOAEL (No Observed Adverse Effect Level) for toxicity in PREGNANT FEMALES was established as 1000 mg/kg/day. This NOAEL value is based on no mortality of females, no changes in health condition status, no pathological findings in the dams, no dose related changes in reproduction parameters.

The NOAEL (No Observed Adverse Effect Level) for PRENATAL DEVELOPMENT was established as 1000 mg/kg/day. This NOAEL is based on no altered growth and no serious structural abnormality found in treated foetuses.