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Toxicity to reproduction

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
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
Constituent 2
Reference substance name:
Slimes and Sludges, blast furnace and steelmaking
EC Number:
266-006-2
EC Name:
Slimes and Sludges, blast furnace and steelmaking
Cas Number:
65996-73-8
Molecular formula:
not applicable
IUPAC Name:
Slimes and sludges, blast furnace and steelmaking
Details on test material:
- Physical state: solid
- Composition of test material, percentage of components: Fe (total) 54.29% (mainly as oxides), CaO 10.17%, Zn 5.25%, MgO 4.32%, C 2.06%, SiO2 1.64%, Mn 0.69%, K2O 0.126%, Na2O 0.239%, Al2O3 0.16%
- Lot/batch No.: 21.10.2009
- Expiration date of the lot/batch: unlimited
- Stability under test conditions: unlimited
- Storage condition of test material: stored in PE container at room temperature

Test animals

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

Administration / exposure

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).
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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

Details on results (P0)

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.

Effect levels (P0)

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)

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

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

Applicant's summary and conclusion

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.