Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2.1.2013 - 8.3.2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Dust, steelmaking
EC Number:
266-005-7
EC Name:
Dust, steelmaking
Cas Number:
65996-72-7
IUPAC Name:
Dust steelmaking
Details on test material:
- Physical state: solid
- Composition of test material, percentage of components: Fe total 57.64% (mainly as oxides), CaO 8.89%, Zn 4.16%, MgO 3.64%, C 0.69%, SiO2 1.56%, Mn 0.57 %, K2O 0.281%, Na2O 0.251%, Al2O3 0.18%
- Lot/batch No.: 21.10.2009
- Expiration date of the lot/batch: unlimited
- Storage condition of test material: stored in PE container at room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Velaz, s.r.o., Koleč u Kladna, Czech Republic (SPF Breeding)
- Age at study initiation: 11 weeks
- Housing: Animals were housed in plastic, individually ventilated cages containing sterilised clean shavings of soft wood. Before mating 2 or 3 rats of the same sex in one cage, during mating period – one male and two females in one cage were housed. During pregnancy 1 female was housed in one cage.
- Diet (e.g. ad libitum): complete peleted diet for rats and mice in SPF breeding (ST 1 BERGMAN)
- Water (e.g. ad libitum): sterilised drinking water (ad libitum).
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 30 - 70 %
- Air changes (per hr): cca 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Remarks:
pharmaceutical quality
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The treated and control females were administered daily by gavage – from the 5th to the 19th day of pregnancy. The animals were treated 7 days per week at the same time (8.00 – 10.00 am).
The concentrations of suspensions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. The application form (test substance suspension in olive oil) was prepared daily just before administration. The vehicle control group was administered by olive oil in the same volume. The application form was mixed during application by magnetic stirrer (10 minutes). The procedure was taken over from analytical report Determination of homogeneity and stability of Dust, steelmaking in vehicle (Annex 1 of Final report, Study No.107/09/7: Dust, steelmaking – Repeated Dose 28-day Oral Toxicity Study, VUOS-CETA Report No. 10-239, 2010).

VEHICLE
- Type of vehicle (if other than water): olive oil
- Concentration in vehicle: according to applied dose (1 mL suspension per 100 g of body weight)
- Amount of vehicle (if gavage): according to applied dose (1 mL suspension per 100 g of body weight)
- Lot/batch no. (if required): 5211201
- Purity: pharmaceutical quality
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male / 2 females
- Length of cohabitation: 2 weeks
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
2 weeks (5th - 19th day of pregnancy)
Frequency of treatment:
daily (8.00 – 10.00 am)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
160 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
400 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
25 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels for study (160, 400 and 1000 mg/kg/day) were chosen on the basis of results of preceding study No.107/09/18: Dust, steelmaking – Reproduction/Developmental Toxicity Screening Test (VUOS-CETA Report No. 10-238, 2010).
- Animal assignment: random

Examinations

Maternal examinations:
GENERAL CLINICAL OBSERVATIONS: Yes
- Time schedule: daily during administration period
- Observation results: No clinical changes indicating dysfunction of organism were found in the treatment and control groups during the whole test.

HEALTH CONDITIONS AND MORTALITY CONTROL: Yes
- Time schedule: daily - during the acclimatization, mating and pregnancy
- Observation results: No significant signs of diseases or toxic effect cause by application of the test substance were found in any groups of animals. No unscheduled deaths were recorded during all the study.

BODY WEIGHT: Yes
- Time schedule for examinations: on the 1st, 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy
- Records: see Table No. 1

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Records: see Table No. 1

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation: 20th day
- Organs examined: uterus
- Records: see Table No. 2

PATHOLOGICAL FINDINGS:
- Macroscopic changes were evaluated in all females (including females without foetuses). The dilatations of uterus was detected in two control females.
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
- Records: see Table No. 3
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
- Records: see Table No. 4
Statistics:
The ANOVA software - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis.
This statistical analysis was used for the results of body weight of pregnant females at the end of pregnancy, absolute and relative weight of uterus, number of corpora lutea and implantation, number of foetuses, number of male foetuses, number of female foetuses, weight of foetuses, weight of male foetuses and weight of female foetuses.
Control group with vehicle was compared with three treated groups.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
< 160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Examination of foetal skeleton indicated delayed development of the skeleton at treated dose levels. The increased frequency of presence of incomplete cranial bones (occipital, parietal, interparietal, frontal and their combinations) was detected. The incomplete ossification of parietal bone or parietal bone in combination with interparietal, frontal bone was found in all groups. The increased number of foetuses with decreased number of ossification sites of sternum was recorded also in all groups. These findings (in cranium and sternum) were related with decreased body weights of foetuses. The contrasting increased number of ossification sites of sternum was found only at the highest dose level. Anomaly of ribs – deformation of the false ribs (letter V) was most detected in litters at the highest dose level.
Examination of ribs revealed changes related with treatment not with delayed development. The wavy ribs were observed at all dose levels (not in control). The other findings in ribs -increased number of floating ribs was recorded only in litters at the middle dose level so it did not concern with treatment.
The incomplete ossification of sacral vertebrae (relationship with fetal body weight) was recorded at the all groups (including control group) but in litters at the middle dose level the increase in incidence of this variation (20 %) in comparison with control (11.11 %) was observed.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No toxicity in maternal animals administered by test substance was observed. Delayed development of foetuses (delayed ossification of cranial, sternum skeleton and sacral vertebrae) was detected at all dose levels. These findings were related with decreased individual body weights of some foetuses. The test substance treatment evoked occurrence of variations – wavy ribs in all treated groups so it indicates teratogenic properties of the test substance (it did cause morphologic changes of skeleton) on early prenatal development of organism in uterus.

The NOAEL (No Observed Adverse Effect Level) for toxicity in PREGNANT FEMALES is higher than 1000 mg/kg/day.
The NOAEL (No Observed Adverse Effect Level) for PRENATAL DEVELOPMENT is less than 160 mg/kg/day. This NOAEL value is based on the incidence of variations in the development of ribs.
Executive summary:

The test substance, Dust, steelmaking 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 and OECD Test Guideline No. 414 Prenatal Developmental Toxicity Study, Adopted by the Council on January 22nd 2001.

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 (pharmaceutical quality) 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, were chosen on the basis of results of preceding study No.107/09/18: “Dust, steelmaking – Reproduction/Developmental Toxicity Screening Test”, VUOS-CETA Report No. 10-238, 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 killed, the uterine contents were examined and the foetuses were evaluated for soft tissue and skeletal changes.

No mortality and no signs of toxicity were observed in the dams.

Maternal animals:

After the mating probably pregnant females were randomly assigned to the groups. Two females did not become pregnant after the mating (females without foetuses and implantations). No females aborted (females without foetuses but with implantations). Number of females with implantation sites was sufficient and similar in all groups (according to the request of guideline). The health condition of females was good all the whole study. During examination of reproductive parameters no significant changes were recorded. No toxicity effect of the test substance on maternal animals was observed.

Foetuses:

No death of foetuses was recorded in any litter. Development in uterus was assessed according to the results of weighing, careful necropsy and skeletal examination of foetuses. The number of male foetuses was slightly higher than that of females, and males were also heavier than females. The number and weight of foetuses at all dose levels were in balance with control group. Test substance application did not induce external and/or visceral malformations (permanent structural change that may adversely affect survival). But some variations described further were detected.

Examination of foetal skeleton indicated delayed development of the skeleton at treated dose levels. The increased frequency of presence of incomplete cranial bones (occipital, parietal, interparietal, frontal and their combinations) was detected. The incomplete ossification of parietal bone or parietal bone in combination with interparietal, frontal bone was found in all groups. The increased number of foetuses with decreased number of ossification sites of sternum was recorded also in all groups. These findings (in cranium and sternum) were related with decreased body weights of foetuses. The contrasting increased number of ossification sites of sternum was found only at the highest dose level. Anomaly of ribs – deformation of the false ribs (letter V) was most detected in litters at the highest dose level.

Examination of ribs revealed changes related with treatment not with delayed development. The wavy ribs were observed at all dose levels (not in control). The other findings in ribs -increased number of floating ribs was recorded only in litters at the middle dose level so it did not concern with treatment. 

The incomplete ossification of sacral vertebrae (relationship with fetal body weight) was recorded at the all groups (including control group) but in litters at the middle dose level the increase in incidence of this variation (20 %) in comparison with control (11.11 %) was observed.