Slimes and Sludges, blast furnace and steelmaking

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016 - 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

Published in O.J. L. 142 (2008)

Deviations:

yes

Remarks:

Short-term decreased relative humidity from 13.11.2017 (1:00 hours) to 14.11.2017 (9:00 hours). The relative humidity ranged from 27.4 % to 29.6 %.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Iron

EC Number:

231-096-4

EC Name:

Iron

Cas Number:

7439-89-6

Molecular formula:

Fe

IUPAC Name:

iron

Constituent 2

Reference substance name:

Calcium oxide

EC Number:

215-138-9

EC Name:

Calcium oxide

Cas Number:

1305-78-8

Molecular formula:

CaO

IUPAC Name:

oxocalcium

Constituent 3

Reference substance name:

Magnesium oxide

EC Number:

215-171-9

EC Name:

Magnesium oxide

Cas Number:

1309-48-4

Molecular formula:

MgO

IUPAC Name:

magnesium oxide

Constituent 4

Reference substance name:

Zinc

EC Number:

231-175-3

EC Name:

Zinc

Cas Number:

7440-66-6

Molecular formula:

Zn

IUPAC Name:

zinc

Constituent 5

Reference substance name:

Manganese

EC Number:

231-105-1

EC Name:

Manganese

Cas Number:

7439-96-5

Molecular formula:

Mn

IUPAC Name:

manganese(2+)

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River SPF breeding supplied via VELAZ, s.r.o., Czech Republic
- Age at study initiation: 11 weeks
- Weight at study initiation: 231.6 - 310.7 g
- Housing: plastic cages containing sterilized clean shavings of soft wood or sterilized LIGNOCEL; before mating 2 rats of the same sex in one cage, during mating period – one male and two females in one cage were housed. Pregnant females were then placed individually.
- Diet: complete pelleted diet for rats and mice in SPF breeding (Altromin Spezialfutter) was used (manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany)
- Water: drinking water ad libitum
- Acclimation period: 5 days at least

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

The test substance consists of various metals and oxides insoluble in the application form (olive oil). Therefore a suitable analytical method has not been found for homogeneity 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). 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).
The application forms of the test substance (suspensions in olive oil) were prepared daily just before administration. The test substance was pulverized in a mortar before preparation of application form. The mixture was mixed by the stirrer with a glass adapter for 10 minutes and then during administration. 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.
The treated and control groups were administered daily by gavage. Exposition lasted from implantation (the 5th day after fertilization) to one day prior to the day of scheduled euthanasia (the 19th day after fertilization). The animals were treated 7 days per week at the same time (8.00 – 10.00 am). Male rats serve only for mating (they are not administered by the test substance and examined).

Analytical verification of doses or concentrations:

no

Details on mating procedure:

After acclimatisation females were mated with males (1 male and 2 females). Vaginal smears were carried out daily in the morning to control fertilization (first time: 24 hours after
the first removing to male). Presence of sperms was examined. Day 0 of pregnancy was the day on which sperms in vaginal smears were observed. Pregnant females were randomly
distributed to experimental groups.

Duration of treatment / exposure:

Exposition lasted from implantation (the 5th day after fertilization) to one day prior to the day of scheduled euthanasia (the 19th day after fertilization).

Frequency of treatment:

Once a day at the same time (8.00 – 10.00 am).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

160 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

400 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 animals per sex per dose
actually were treated only pregnant females:
control: 16 animals
160 mg/kg/day: 17 animals
400 mg/kg/day: 20 animals
1000 mg/kg/day: 17 animals

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. 106/09/18: Slimes and Sludges, blast furnace and steelmaking - Reproduction/Developmental Toxicity Screening Test.

Examinations

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day in the similar time each day
- Clinical observations: checked in table which is not included

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

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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: reproductive organs only

Ovaries and uterine content:

The 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
- Other: Pathological examination of females (macroscopic evaluation, number of foetuses, uterus evaluation)

Fetal examinations:

Pathological examination of foetuses: mean body weight, external alterations, internal alterations – soft tissues, internal alterations - skeleton

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)
- mean weight of foetuses (males, females, both sex)
- biometry of uteri (absolute and relative weight )
- preimplantation (IUDE) and postimplantation (IUDL) loss
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 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

Description (incidence and severity):

At control and treated females no clinical changes were recorded during the whole study. Only changes related to the colour of the test substance – coloured excrements were recorded (at the dose level 1000 mg/kg/day: from the 2nd week of pregnancy). The short-time piloerection was observed at all females at the middle and at the highest dose levels from the 6th day of pregnancy to 9th day of pregnancy.
In control females and treated females of all dose levels no signs of diseases were recorded during the application period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

One female fom the highest dose level (1000 mg/kg/day) died on 7th day of study because of intubation error. The female was pregnant.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Only females who were found pregnant on the 20th day of gravidity (females with live foetuses) were used for calculation of mean body weights. The statistical evaluation was performed from the 5th to 20th day of pregnancy and differences were found.
The body weight of treated females from 8th up to 20th day of pregnancy was statistically significantly decreased at all dose levels. The decreased body weight of treated females was related to lower body weight increment. The body weight increment decrease with dose dependence.
Values of corrected body weight (the necropsy body weight of female minus weight of uterus) of females were decreased compared to the control group at all dose levels, but the values were not statistically significantly decreased.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Only females which were found to be pregnant 20th day of gestation (females with live foetuses) were used for calculation of mean food consumption. The statistical evaluation was performed from the 5th to 20th day of pregnancy. Mean food consumption in treated groups was significantly decreased compared to control group from 11th day of study (correlation with decreased mean of body increment at all dose levels).

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

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

Description (incidence and severity):

The uterus of all females were weighed, but only females which were found to be pregnant 20th day of gestation were used for calculation of mean weight of uterus.
The absolute weight of uterus was recorded and the relative weight of uterus was computed. The absolute and relative weight of uterus was decreased at the middle dose level but no statistically significant differences in uterus biometry were detected.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Only non-pathological findings revealed related to the colour of the test substance as the coloration of contents of different parts of the gastrointestinal tract (stomach, intestines, appendix) by the test substance (0-2-19-24).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

The test substance had negative effect on the growth of maternal animals. The body weight of treated females from 8th up to 20th day of pregnancy was statistically significantly decreased at all dose levels compared to the control group. The body weight increment was decreased with dose dependence at the all treated females. This correlated with the decrease of food consumption in all treated females during pregnancy.
The corrected body weights of females (without weight of uterus) were decreased compared to the control group at all dose levels, but the values were not statistically significant.
Clinical examinations of treated mothers detected no clinical symptoms of toxicity related to the test substance. The behavior, health condition and clinical status of treated maternal animals were similar compared to controls and no serious changes were found. Only changes related to the colour of the test substance – coloured excrements were recorded (at the dose level 1000 mg/kg/day).
Also pathological examination of females revealed only non-pathological findings related to the colour of the test substance as the coloration of contents of different parts of the gastrointestinal tract (stomach, intestines, appendix) by the test substance (0-2-19-24).

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

No abortion was recorded in any litter.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

For evaluation of IUDE and IUDL only females without foetuses and without implantations were not used.
The numbers of implantations and corpora lutea were reduced at the lowest and the middle dose levels and the numbers of resorptions were decreased at the middle and at the highest dose levels. The mean number of implantations, corpora lutea and resorptions in treated females was not statistical significantly changed in comparison with the control females.
Preimplantation losses (IUDE) were increased at the middle dose level (15.03 %) and slightly increased at the highest dose level (11.88 %) in comparison with control group (10.44 %). Postimplantation losses (IUDL) were statistically insignificantly increased at all dose levels in comparison with control (6.02 % – 10.26 % – 12.97 % – 9.40 %).

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

The numbers of resorptions were decreased at the middle and at the highest dose levels but the change in mean number of resorptions was not statistically significant.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

For evaluation of IUDE and IUDL only females without foetuses and without implantations were not used.
The numbers of implantations and corpora lutea were reduced at the lowest and the middle dose levels and the numbers of resorptions were decreased at the middle and at the highest dose levels. The mean number of implantations, corpora lutea and resorptions in treated females was not statistical significantly changed in comparison with the control females.
Preimplantation losses (IUDE) were increased at the middle dose level (15.03 %) and slightly increased at the highest dose level (11.88 %) in comparison with control group (10.44 %). Postimplantation losses (IUDL) were statistically insignificantly increased at all dose levels in comparison with control (6.02 % – 10.26 % – 12.97 % – 9.40 %).

Dead fetuses:

no effects observed

Description (incidence and severity):

No death of foetuses was recorded in any litter.

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

control: 16 females
160 mg/kg/day: 16 females
400 mg/kg/day: 18 females
1000 mg/kg/day: 16 females

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

< 160 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weight of foetuses was decreased with the dose dependence in all groups compared to the control. Although the variations between individual body weight 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. Reducing of the fetal body weight could be caused by the increasing number of foetuses in treated groups or associated with the decrease in maternal body weight.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The mean body weight of foetuses was decreased with the dose dependence in all groups compared to the control. Although the variations between individual body weight 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. Reducing of the fetal body weight could be caused by the increasing number of foetuses in treated groups or associated with the decrease in maternal body weight.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

The total number of live foetuses in group was increased at all dose levels compared to the control group. The total number of live foetuses in group was statistically significantly increased only at the highest dose level.

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

Sex ratio (mean value) was similar in all groups (7 males : 6 females) except in the highest dose (7 males : 8 females). The number of female foetuses was statistically increased at the highest dose.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

The total number of live foetuses in group was increased at all dose levels compared to the control group. The total number of live foetuses in group was statistically significantly increased only at the highest dose level.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No macroscopic changes of soft tissues and external alteration were found out.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Incomplete ossification of foetal cranium was found out during examination in all groups including control group. With delayed development were affected mostly parietal, interparietal and supraoccipital bones. Total portion of foetuses in litter with these findings of cranium were similar or lower in comparison with control group. This delayed development was not related to the treatment due to a similar percentages in the control group.
Other findings like hole in the supraoccipital bone and bipartite ossification of supraoccipital were observed less frequently. Other changes of foetal cranium were found only sporadically.

Visceral malformations:

no effects observed

Description (incidence and severity):

Pathological examination of soft tissues of foetuses do not reveal any deviation.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no serious structural abnormality of treated foetuses and increased total number of live foetuses

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Conclusions:

In the prenatal developmental study the negative effect of the test substance on the growth of maternal animals was observed at all dose levels. The body weight increment was decreased with dose dependence at the all treated females. There were changed reproduction parameters at all dose levels - increased preimplantation (except the lowest dose level) and postimplantation losses. These findings could be caused by the test substance treatment.
The total number of live foetuses in group was increased at all dose levels compared to the control group. The examination of foetuses revealed decreased of the mean body weight of foetuses in all groups compared to the control group with dose dependence, but statistically significant differences were not detected. But it was questionable whether decrease weight of foetuses in treated groups was due to a higher number of foetuses in the treated groups or was associated with a negative influence of the test substance on growth of maternal animals.
No serious macroscopic external and soft tissue changes were observed. 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 is ¿ 160 mg/kg/day. This NOAEL value is based on the statistically significant decreased body weight of females (related to lower body increment), increased preimplantation and postimplantation losses.
The NOAEL (No Observed Adverse Effect Level) for PRENATAL DEVELOPMENT is 1000 mg/kg/day. This NOAEL is based on no serious structural abnormality of treated foetuses, increased total number of live foetuses.

Executive summary:

The test substance, Slimes and Sludges, blast furnace and 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. L. 142, 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 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 previous study Slimes and Sludges, blast furnace and steelmaking - Reproduction/Developmental Toxicity Screening Test.

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 the prenatal developmental study the negative effect of the test substance on the growth of maternal animals was observed at all dose levels. The body weight increment was decreased with dose dependence at the all treated females. There were changed reproduction parameters at all dose levels - increased preimplantation (except the lowest dose level) and postimplantation losses. These findings could be caused by the test substance treatment.

The total number of live foetuses in group was increased at all dose levels compared to the control group. The examination of foetuses revealed decreased of the mean body weight of foetuses in all groups compared to the control group with dose dependence, but statistically significant differences were not detected. But it was questionable whether decrease weight of foetuses in treated groups was due to a higher number of foetuses in the treated groups or was associated with a negative influence of the test substance on growth of maternal animals.

No serious macroscopic external and soft tissue changes were observed.

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 is ¿ 160 mg/kg/day. This NOAEL value is based on the statistically significant decreased body weight of females (related to lower body increment), increased preimplantation and postimplantation losses.

The NOAEL (No Observed Adverse Effect Level) for PRENATAL DEVELOPMENT is 1000 mg/kg/day. This NOAEL is based on no serious structural abnormality of treated foetuses, increased total number of live foetuses.