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Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17.2.2010-24.5.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: VELAZ, s.r.o., Kolec u Kladna, Czech Republic (SPF [Specified Pathogen Free] quality guaranteed)
- Age at study initiation: 10 weeks
- Weight at study initiation: (P) Males: cca 309 g; Females: cca 209 g
- Housing: Animals were housed in SPF animal room, 2 rats of the same sex in one plastic cage (40x25x20 cm) containing sterilised clean shavings of soft wood. During mating period: one male and one female in one cage, pregnant females: individually, offspring: with mother.
- Diet (e.g. ad libitum): Complete peleted diet for rats and mice in SPF breeding ST 1 BERGMAN was used (manufacturer: Miroslav Mrkvicka - Výroba krmných smesí, Mlýn Kocanda, Jesenice u Prahy) ad libitum. Diet was sterilised 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 (°C): 22±3°C
- Humidity (%): 30-70 %
- Air changes (per hr): approx. 15×per hour
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The application form (test substance suspension in olive oil) was prepared daily just before administration. The concentrations of suspensions at all dose levels were adjusted to ensure the administration of 1 ml per 100 g of body weight. The vehicle control group was administered by olive oil in the same volume.

VEHICLE
- Concentration in vehicle: concentrations of suspensions at all dose levels were adjusted to ensure the administration of 1 ml per 100 g of body weight
- Amount of vehicle (if gavage): 1 ml per 100 g of body weight
- Lot/batch no. (if required): 4683401 and 4726901
- Purity: pharmaceutical quality
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): alone in cage
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
The treated groups were administered daily for the following period: males and females - 2 weeks prior to the mating period and then during the mating period. Pregnant females were administered during pregnancy and till the 3rd day of lactation. Males were then administered after mating period - totally for 28 days. Non-pregnant females (mated females without parturition) were administered 25 days after the confirmed mating.
Frequency of treatment:
The animals were treated 7 days per week at the same time (8.00 – 10.00 am).
Dose / conc.:
160 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: On the basis of results of the Study No. 106/09/7: Slimes and Sludges, blast furnace and steelmaking - Repeated Dose 28-day Oral Toxicity Study (dose-range finding experiment with 14-day application period) the following dose levels were chosen for current study: 160, 400 and 1000 mg/kg/day.
Parental animals: Observations and examinations:
MORTALITY CONTROL: Yes
daily during the treatment periods for vitality or mortality changes

HEALTH CONDITION CONTROL: Yes
Time schedule: daily during the acclimatization and the experimental part.

CLINICAL OBSERVATIONS: Yes
Clinical Observation of Males and Females daily during the administration period (in their cages) in order to record possible clinical effects after application and all changes in behaviour of animals.

BODY WEIGHT: Yes
Time schedule for examinations: on specified days, all animals were weighed immediately before euthanasia too
Weight increment was computed as an average per group per time interval. Nonpregnant females were not included in calculation of averages in pregnancy and lactation period.
males - weekly
females - weekly in premating period
during pregnancy: 0., 7th, 14th, 20th day
during lactation: 0. or 1st and 4th day

FOOD CONSUMPTION:
In a specified day the remainder of pellets was weighed in each cage, the new food was weighed out and the food consumption for the previous week was computed.
In males average values were calculated for each week of the study (except of mating period). Food consumption for animal/day was calculated from average values of each group.
The same way of calculation of average food consumption was used for females in premating period. In pregnancy and lactation period average individual values (grams/animal/day) were calculated for each week of the study. Average food consumption for each group was calculated from individual values. Nonpregnant and aborted females (females without parturition) were not included in calculation of average food of pregnant females.
males - weekly (except the mating period)
females - weekly during premating period and after mating period
during pregnancy: 0., 7th, 14th, 20th day
during lactation: 0. or 1st and 4th day

EXAMINATION OF VAGINAL SMEARS: Yes
The pregnancy was determined by the presence of spermatozoa in vaginal smear. The vaginal smears were carried out daily in the morning during mating period. The smears were stained and the presence of sperm was evaluated. Day 0 of pregnancy was defined as the day when sperms were found.
Oestrous cyclicity (parental animals):
not recorded
Sperm parameters (parental animals):
Parameters examined in male parental generations:
Sperm motility
Sperm samples were taken from one epididymis and sperm motility was assessed from these samples. The motility of sperm was determined by microscopic examination of the prepared sperm suspension. The result of observation was evaluated subjectively according to following grades: 1 - fast progressive motility, 2 - slow progressive motility, 3 - no progressive motility, 4 - non-motile sperm.

Sperm morphology
Sperm samples were taken from one epididymis and sperm morphology were assessed from these samples. A smear from the sperm suspension was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination. All deviations (e.g. broken tail, abnormal form of tail, double head, amorphous head, abnormal form of neck) were recorded.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, development, pathological and histopathological examination

GROSS EXAMINATION OF DEAD PUPS:
yes, for possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after 28 days
- Maternal animals: All surviving animals on 4th day of lactation period

GROSS NECROPSY
Dead animals were macroscopically examined for any pathological changes with special attention to the organs of the reproductive systems. All macroscopic abnormalities were recorded.
The absolute weights of testes, epididymis, prostate gland and pituitary gland were recorded in males and absolute weight of ovaries, uterus (incl. uterine tube and cervix) and pituitary gland were recorded in females. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.

HISTOPATHOLOGY / ORGAN WEIGHTS
Detailed histological examination was performed on testes (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure). Spermatogenesis and spermatogenic cycle were evaluated according to the publication: Hess, R.A.; Quantitative and qualitative Characteristics of the Stages and Transitions in the Cycle of the Rat Seminiferous Epithelium: Light Microscopic Observation of Perfusion-Fixed and Plastic-Embedded Testes (Biology of Reproduction 43, 525-542, 1990). Pathological changes were evaluated according to the publication: Creasy, D.M.; Evaluation of Testicular Toxicity in Safety Evaluation Studies: The Appropriate Use of Spermatogenic Staging (Toxicologic Pathology 25, 119-131, 1997).
Postmortem examinations (offspring):
GROSS NECROPSY
Dead pups were sexed and externally examined; the stomach was examined for the presence of milk. Pups killed on the 4th day of lactation were sexed and subjected to external examination of the cranium, and to macroscopic examination of the thoracic and abdominal tissues and organs. All macroscopic changes were recorded.
Statistics:
The ANOVA test - 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, biometry of organs, number of pups and number of corpora lutea. Control group with vehicle was compared with three treated groups.
Reproductive indices:
All females (at all groups) were paired so the number of females paired was identical at all groups.
Number of females achieving pregnancy, fertility index, accompanying conception index, duration of mating, average number of corpora lutea, gestation index, pre-implantations, post-implantations were recorded.
Offspring viability indices:
Viability index and post-natal losses were recorded.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Effect were observed, but considered negligible.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Effect were observed, but considered negligible.
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, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see Details on results
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Test substance intake: Effect were observed, but considered negligible.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Percentage of males with impaired motility of sperms was observed in 25 percent of males at the middle dose level and in 33 percent of males at the highest dose level. Percentage portion of morphologically changed sperms was similar the control group in m
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
see Details on results
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no unsheduled deaths during all the study.
In treated males and females of all dose levels no signs of diseases were found out during the check-in, acclimatisation and application period. No treatment-related effects were detected during the health condition control. Only excrements were coloured by the test substance.
No clinical changes were observed in males and females of all dose levels after application of the test substance.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males
The animal body weights at all dose levels were relatively well-balanced with the control group during the whole application period. Only at the middle dose level slightly increased body weight increment was recorded from the 3rd week. No statistically significant differences were detected.
Pregnancy
Average body weight increments of lowest and middle dose levels were relatively well-balanced with the control group. Average body weight and average body weight increment were slightly decreased at the highest dose level since the 7th day of pregnancy. No statistically significant changes were found out during the whole pregnancy.
Lactation
Average body weight at the lowest and highest dose levels was slightly decreased compared to the control.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Sperm motility
Sperm samples were taken from one epididymis and sperm motility was assessed from these samples. The motility of sperm was determined by microscopic examination of the prepared sperm suspension. The result of observation was evaluated subjectively according to following grades: 1 - fast progressive motility, 2 - slow progressive motility, 3 - no progressive motility, 4 - non-motile sperm.
Sperm morphology
Sperm samples were taken from one epididymis and sperm morphology were assessed from these samples. A smear from the sperm suspension was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination. All deviations (e.g. broken tail, abnormal form of tail, double head, amorphous head, abnormal form of neck) were recorded.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
All females (at all groups) were paired so the number of females paired was identical at all groups. Number of females achieving pregnancy, fertility index and accompanying conception index of treated groups were decreased compared to the control, because number of females with live pups born were decreased at all treated groups. Duration of mating of the treated groups was similar to the control group. Average number of corpora lutea was slightly decreased only at the highest dose level (there was lower number of pups) compared to the control. Gestation index of the treated groups was similar to the control group. Pre-implantation losses were increased at the middle and highest dose levels. Post-implantation losses at all dose levels were well-balanced with the control group. Decreased viability index was recorded at the lowest dose level and at the control group, because increase of post-natal losses was recorded in these groups (pups with aerial lungs, with stomach full of milk and cannibalism).

ORGAN WEIGHTS (PARENTAL ANIMALS)
Males
Absolute and relative weights of all observed organs were similar in treated and control males. Only average absolute and relative weights of pituitary gland were slightly increased in dose-related manner. No statistically significant differences were detected.
Females
Average relative and absolute weights of ovaries and pituitary gland were well-balanced with the control group. Average relative and absolute weights of uterus of mothers treated by the test substance were decreased with statistical significance at the highest dose level. Weight of pituitary gland of the highest dose level was slightly decreased compared to the control females.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Males
Examination of the external surface of the body, thoracic and cranial cavity revealed no macroscopic changes in treated and control animals. In abdominal cavity reduced seminal vesicles was recorded in one male of the highest dose level and reduced prostate gland was recorded in one male of the control group and in one male of the highest dose level.
Females
Examination of the external surface of the body revealed no changes. In thoracic cavity no changes were observed. In abdominal cavity autolysed foetus in uterus was recorded in one female at the middle dose level. In stomach hyperaemia of mucosa and haemorrhage in mucosa was sporadically observed at the highest dose level. In vagina dilatation was observed in one female at the control group. Dilatation of uterus was recorded at the control group, the lowest dose level and the middle dose level in nonpaired or nonpregnant females or females with abortion.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Males
Histopathological affections of male reproductive system were detected at all dose levels. Lymphocyte infiltrations (especially interstitial) were described in epididymis of all males (non-pathological finding). In testes atrophy and degeneration of germ epithelium, residual corpora in germ epithelium, segmental separation of basal membrane and vacuolation of cytoplasm of spermatogonium were diagnosed. Functional hyperplasia in prostate gland and lymphocyte infiltration was recorded. Functional hyperplasia in seminal vesicles and atrophy of epithelium in prostate gland were sporadically observed.
Female
In mothers a typical status of the 4th day after parturition was found out in sexual organs: Uterus - focal accumulation of macrophagic elements containing both liposubstances and brown pigment (most probably siderin) were found in mesometrium as signs of previous gravidity; ovarium - follicles and corpora lutea in different phases of development, degeneration of some follicles; uterine cervix - hyperplasia of mucification cells; vagina - atrophy of germinal area, hyperplasia of mucification cells, apoptotic cells, keratinized cells, desquamation of mucification and cornification cells and cell detritus into the lumen.
In nonpregnant and females with abortion a various phase of oestrous cycle was recorded. In reproductive system some affections of uterus and ovaries were detected. In uterus of some females accumulation of ovulatory intraluminal fluid (hydrometra) was found out. It is a spontaneous change during the oestrous cycle. Leucocytes in mucosa of uterus several females were observed. Autolyzed fetus of normal birth size was found in one female of the middle dose level in the right horn of uterus. Histologically this horn was markedly dilated lined with flattened basophilic epithelium with presence of multiple polymorphonuclear cells.
Cysts occurred in ovaries of one female and cysts in pituitary gland of one control females were observed.
Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
reproductive function (sperm measures)
Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
see Details of result
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see Details of result
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
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
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Effect were observed, but considered negligible.
Histopathological findings:
no effects observed
Description (incidence and severity):
Effect were observed, but considered negligible.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
VIABILITY (OFFSPRING)
The total number of live pups and average number of pups per litter (in the day of parturition/1st day and the 4th day after parturition) at all dose levels were decreased compared to the control.
Two pups were found dead (one male - with aerial lungs and one male – cannibalism) at the control group and four pups (two males - with aerial lungs and two males – cannibalism) at the lowest dose level.
Sex ratio (males/females) of pups was relatively well balanced at the lowest and middle dose levels. Number of males was lower at the highest dose level.

BODY WEIGHT (OFFSPRING)
The average body weights of pups at all dose levels were well-balanced with the control group and had an increasing trend during the whole lactation period.

DEVELOPMENT OF PUPS
Two pups were found dead (one male - with aerial lungs and one male – cannibalism) at the control group and four pups (two males - with aerial lungs and two males – cannibalism) at the lowest dose level during lactation period.

GROSS PATHOLOGY (OFFSPRING)
The macroscopic examination was performed in all pups. Stomach without milk was observed in one female at the control group; dark red colour of lungs was sporadically observed at the control and highest dose level. Suspect cavity in the brain of one female of the control group was observed. Marked milk ridge was in two males of the middle dose level. Above-mentioned findings were detected sporadically.

HISTOPATHOLOGY (OFFSPRING)
Dark red colour of lungs was sporadically observed at the control and highest dose level was caused by blood aspiration. The blood aspiration was caused by euthanasia. Suspect cavity in the brain of one female of the control group was most probably slightly dilated brain ventricle.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effect observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
400 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
The negative influence of the test substance treatment expressed mainly at the highest dose level (limit dose): decreased body weight of parental females, impaired sperm quality of parental males and changes of microscopic structure of reproduction organs (testes) in parental males were observed. The test substance administered at the highest dose level had negative influence on reproduction parameters of parental females (number of pregnant, number of females achieving pregnancy, average number of pups, sex ratio, average weight of litter, corpora lutea, implantation).
Food consumption of males and females at all dose levels was slightly decreased.
Clinical status, macroscopic structure of reproductive organs of parental males and females were not markedly affected by treatment of the test substance. Durations of mating and pregnancy, average body weight and postnatal development of pups were unaffected by the test substance treatment.
The NOAEL for REPRODUCTION was established as 400 mg/kg body weight/day.
The NOAEL for DEVELOPMENT of pups was established as 1000 mg/kg body weight/day.
Executive summary:

The test substance, Slimes and Sludges, blast furnace and steelmaking, was tested for reproduction toxicity using the OECD Test Guideline No. 421 Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on July 27th 1995.

Wistar rats of SPF quality were used for testing. The test substance was administered suspended in olive oil using a stomach tube; oral application of rats was made daily. The concentrations of suspension at all dose levels were adjusted to ensure the administered volume of 1 ml per 100 g of body weight. Each group consisted of 12 males and 12 females. On the basis of the results of the Study: Slimes and Sludges, blast furnace and steelmaking - Repeated Dose 28-day Oral Toxicity Study (dose-range finding experiment with 14-day application period) were chosen the dose levels for study - 160, 400 and 1000 mg/kg/day.

The treated groups were administered daily for the following periods:

males and females - 2 weeks prior to the mating period and during the mating period,

pregnant females - during pregnancy and till the 3rd day of lactation,

males - after mating period - totally for 28 days,

nonpregnant females (mated females without parturition) - for 25 days after the confirmed mating.

During the study clinical observation and health status control were performed daily. The body weight and food consumption were measured weekly or in specified time intervals. Vaginal smears were prepared daily during mating period (until the presence of spermatozoa). Reproduction parameters relevant to pups (number of pups, weight of litters, sex or vitality) were also recorded.

The study was finished by gross necropsy of animals. In all males of all groups the sperm parameters: sperm motility and sperm morphology were examined. The selected organs from parental animals were removed for weighing and histopathological examination.

The negative influence of the test substance treatment expressed mainly at the highest dose level (limit dose): decreased body weight of parental females, impaired sperm quality of parental males and changes of microscopic structure of reproduction organs (testes) in parental males were observed. The test substance administered at the highest dose level had negative influence on reproduction parameters of parental females (number of pregnant, number of females achieving pregnancy, average number of pups, sex ratio, average weight of litter, corpora lutea, implantation).

Food consumption of males and females at all dose levels was slightly decreased.

Clinical status, macroscopic structure of reproductive organs of parental males and females were not markedly affected by treatment of the test substance. Durations of mating and pregnancy, average body weight and postnatal development of pups were unaffected by the test substance treatment.

The NOAEL for REPRODUCTION was established as 400 mg/kg body weight/day.        

The NOAEL for DEVELOPMENT of pups was established as 1000 mg/kg body weight/day.   

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
400 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

For the test substance, following values of reproduction toxicity were found:

The NOAEL for REPRODUCTION was established as 400 mg/kg/day body weight/day.           

The NOAEL for DEVELOPMENT of pups was established as 1000 mg/kg/day body weight/day.           

Based on the result of reproductive screening can not be assessed the effect of the test substance on the reproductive ability of male or female. There is not been documented influence on the development of offspring. According to the OECD methodology a negative screening test is not sufficient for determining the hazards of the substance for reproduction.

Exposure to the substance Slimes and Sludges, blast furnace and steelmaking is likely to be predominantly via the dermal route; however the bioavailability of its components is very low following dermal exposure. A data waiving is therefore proposed for this endpoint based on the predictable and low inherent toxicity of the substance and the low level of systemic absorption. The low level of systemic absorption of the substance is proven by the study OECD 428 Skin Absorption: In vitro Method.
The test substance, Slimes and Sludges, blast furnace and steelmaking, was tested for reproduction toxicity using the OECD Test Guideline No. 421 Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on July 27th 1995. The test was carried out under GLP conditions.
Due to low level of systemic absorption of the test substance was waived the further testing.

Effects on developmental toxicity

Link to relevant study records
Reference
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
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
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:
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).
Dose / conc.:
160 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
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.
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.
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).
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
Dose descriptor:
NOAEL
Effect level:
< 160 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
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.
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
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
not specified

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.

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

The signs of reproduction toxicity were described at highest dose level (1000 mg/kg/day). Effect on motility of sperms and microscopic changes in testes in males, decreased body weight in females. Reduced body weight of pregnant females was observed at prenatal developmental toxicity study in all treated females but it is not entirely clear whether this is the sign of reproductive toxicity or symptom of general toxicity. This effect was not accompanied by other significant symptoms of the reproduction or developmental toxicity.

The developmental toxicity has not been confirmed. 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.

Observed effects were not evaluated as sufficient for classification as they are not sufficiently severe on reproduction parameters or development of foetus, and therefore the test substance have not classified for reproductive toxicity according to Regulation (EC) No. 1272/2008.

Additional information