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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:
25.8.-30.10.2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: Fluidized Bed Combustion (FBC) Fly Ash
- Substance type: inorganic
- Physical state: solid
- Composition of test material, percentage of components: SiO2 - 35.54 %, Al2O3 - 21.56 %, Fe2O3 - 4.21 %, TiO2 - 6.18 %, CaO total - 15.90 %, MgO - 0.57 %, Na2O - 0.38 %, K2O - 0.48 %, MnO - 0.059 %, P2O5 - 0.46 %, SO3 - 5.44 %, CO2 - 0.87 %, Cl - 0.01 %, CaO free - 5.75 %, As, Be, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Se, Tl, V, Zn total < 0.1%
- Lot/batch No.: FUP_ETI_ETH20_2_6_2015
- Expiration date of the lot/batch: 05/2030
- Stability under test conditions: stable
- Appearance: grey solid powder
- Storage condition of test material: in dark, closed container at laboratory temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Únětice, Czech Republic
- 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: at least 5 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 oxides insoluble in the application form (olive oil). A suitable analytical method cannot be found for homogenity and stability testing. Since undissolved particles of the test substance are easily visible in the application form homogeneity was checked by eye (suspension were mixed for 10 minutes by magnetic stirrer – 1000 rpm). Stability of the test substance in the application form cannot be verified but there is no indication that a mixture of rigid oxides would be unstable in its solution (in olive oil) for that short time period (1 hour).

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
- Amount of vehicle (if gavage):
- Lot/batch no. 5726701:
- Purity: pharmaceutical quality
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- 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
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:
25 animals per dose (incliding control group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on previous tests (90-day oral toxicity and reproduction developmental 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 #19
- Organs examined: uterus
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 resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
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 (8th, 11th , 14th , 17th , 20th day of pregnancy)
- mean weight of fetuses (males, females, both sex)
- biometry of uteri (absolute and relative weight )
- corrected body weight (subtraction weight of uterus from surgery body weight of females)
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 fetuses (males, females, both sex)
The two-groups Mann-Whitney test (probability level 0.05) was apllied.
The categorical data (of serious findings - external and internal alteration) was not implemented by the reason of low incidence of these findings at the treated group against the control.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
The reproduction parameters have been changed mostly at the middle and the highest dose level. There were decreased number of implantations, corpora lutea related with an increase of resorptions, preimplantation and postimplantation losses, abortion of two females and presence of two died foetuses. These effects are not sufficiently significant.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: changes in reproduction parameters

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Malformations such as misshapen humerus and misshapen or fused arcus zygomaticus were found out only sporadically. Incidence of these findings was accidental and comparable with control group.
Presence of wavy ribs (undulation along the length of a rib) and misasligned sternebrae were observed mostly at the highest dose level. These findings are not sufficiently significant.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

See Attached background material

Applicant's summary and conclusion

Conclusions:
In the prenatal developmental study the changed reproduction parameters at the middle and markedly at the highest dose level were observed (decreased number of implantations, corpora lutea and related increased preimplantation and postimplantation losses and resorptions including two aborted females). These findings could be caused by the test substance treatment.
The examination of foetuses revealed no changes in body weight and number of foetuses, except the highest dose level, where was reduced the number of foetuses.
No serious macroscopic external and soft tissue changes were observed.
During examination of foetal skeleton delayed ossification of the skeleton at all doses-including control group was observed. Delayed ossification was characterized mostly by incomplete ossification of cranium bones and less frequently by incomplete ossification of vertebrae or unossified sternebrae.
It appears that the delayed ossification of foetuses in this study was not induced by the test substance treatment.
The structural changes such as bipartite ossification of supraoccipital bone, supernumerary ribs, bent ribs or bent humerus were detected without treatment relation in litters across all dose levels. Other structural changes as a misaligned sternebrae and wavy ribs were detected mostly at the highest dose level.
Malformations such as Arcus zygomaticus – misshapen or fused and humerus – misshapen were accidental and probably not related to the treatment.
The occurrence of structural skeletal variations and malformations 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 and comparable with control group.
Presence of wavy ribs (undulation along the length of a rib) hole in basisphenoid and misasligned sternebra were detected mostly at the highest dose level. These findings probably could be related to treatment but most of these findings are reversibile and has no serious adverse effect for newborn pup.
The NOAEL (No Observed Adverse Effect Level) for PRENATAL DEVELOPMENT is 160 mg/kg/day. This NOAEL is based on occurrence of high appearance of postimplantation losses and decreased number of live foetuses. It is not possible excluded the influence on early intrauterine development of foetuses.
The NOAEL (No Observed Adverse Effect Level) for toxicity in PREGNANT FEMALES is 160 mg/kg/day. This NOAEL value is based on occurrence of changes in reproduction parameters where is not possible excluded the influence of toxicity on maternal organism.
Executive summary:

The test substance, Fluidized Bed Combustion (FBC) Fly Ash 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. L142, 2008.

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. 66/09/8: Fluidized Bed Combustion (FBC) Bottom Ash – Repeated Dose 90-day Oral Toxicity (VUOS-CETA Report No. 10-132, 2009) – Annex 1 - Dose-Range Finding Experiment of the study Fluidized Bed Combustion (FBC) Fly Ash – Repeated Dose 90-day Oral Toxicity Study and on the basis of the results of the Study No. 66/09/18: Fluid Bed Combustion (FBC) Bottom Ash – Reproduction Developmental Screening Test (VUOS-CETA Report No. 10-94, 2009).

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.

In prenatal development study the reproduction parameters have been changed mostly at the middle and the highest dose level. There were decreased number of implantations, corpora lutea related with an increase of resorptions, preimplantation and postimplantation losses, abortion of two females and presence of two died foetuses. These findings could be caused by the test substance treatment.

Examination of foetal skeleton indicated delayed development of the skeleton (incomplete ossification of certain bones) at all doses including control group. It appears that the delayed development of foetuses in this study was not induced by the test substance treatment and could be connected with low individual body weight of foetuses.

Other structural changes were recorded in all test groups as well as in control group. Malformations such as misshapen humerus and misshapen or fused arcus zygomaticus were found out only sporadically. Incidence of these findings was accidental and comparable with control group. Presence of wavy ribs (undulation along the length of a rib) and misasligned sternebrae were detected mostly at the highest dose level. These findings probably could be related to treatment.

The occurrence of these structural skeletal variations and malformations 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 and comparable with control group.

The NOAEL (No Observed Adverse Effect Level) for PRENATAL DEVELOPMENT is 160 mg/kg/day.

This NOAEL is based on occurrence of high appearance of postimplantation losses and decreased number of live foetuses. It is not possible excluded the influence on early intrauterine development of foetuses.

The NOAEL (No Observed Adverse Effect Level) for toxicity in PREGNANT FEMALES was established as 160 mg/kg/day.

This NOAEL value is based on occurrence of changes in reproduction parameters where is not possible excluded the influence of toxicity on maternal organism.