Registration Dossier

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:
24.2.2010-9.7.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP 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
Details on test material:
- Physical state: solid
- Composition of test material, percentage of components: Fe total 57.64% (mainly as oxides), CaO 8.89%, Zn 4.16%, MgO 3.64%, C 0.69%, SiO2 1.56%, Mn 0.57 %, K2O 0.281%, Na2O 0.251%, Al2O3 0.18%
- Lot/batch No.: 21.10.2009
- Expiration date of the lot/batch: unlimited
- Storage condition of test material: stored in PE container at room temperature

Test animals

Species:
rat
Strain:
other: Wistar Han
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: VELAZ, s.r.o., Koleč u Kladna, Czech Republic (SPF [Specified Pathogen Free] quality guaranteed)
- Age at study initiation: 10 weeks
- Weight at study initiation: (P) Males: cca 316 g; Females: cca 225 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 Mrkvička - Výroba krmných směsí, 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 7 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): 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
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
900 mg/kg/day
Basis:
actual ingested
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 dose-range finding experiment with 14-day application period for study No. 107/09/7: Dust, steelmaking - Repeated Dose 28-day Oral Toxicity Study

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 of study.

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.

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).
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.
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
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.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Effect were observed, but considered negligible.
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
see Details on results
Reproductive performance:
no effects observed
Description (incidence and severity):
Effect were observed, but considered negligible.

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
In the 2nd, 3rd and 4th week of application the animal body weight at all treated group was slightly increased. The body weight increment at all dose levels was relatively well-balanced compared with control group during the whole time of application period. No statistically significant differences were detected.
Pregnancy
Average body weight and body weight increment at all dose levels were relatively well-balanced with control group.
Lactation
Average body weight on the 4th day of lactation was slightly decreased at the lowest dose level and the highest dose level compared to the control. Average body weight increment was decreased in the all treated groups with dependence on the dose level. No statistically significant differences were detected.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
At the middle and lowest dose level increased impaired sperm motility was observed. Percentage portion of morphologically changed sperms was increased at all treated groups comparing with control group without dependence on dose level.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
All control females and all treated females were mated. Number of females achieving pregnancy and accompanying conception index was decreased at the lowest dose level. Duration of mating of treated groups was similar to control group, only slight prolonged duration of mating in one female at the highest dose levels was recorded. Fertility index was mildly decreased at the lowest and middle dose level. Duration of pregnancy of females at the treated groups was similar to control group. The duration of pregnancy in treated groups not exceeded 23 days what was similar to the control group. Gestation index at the middle and highest dose levels was similar to control group. At the lowest dose level decreased of gestation index was recorded (three aborted female).

ORGAN WEIGHTS (PARENTAL ANIMALS)
Males
Absolute and relative weights of all observed organs were similar in treated and control males. Slight increase of absolute weight of testes and prostate gland was observed in treated group without dependence on the dose. Relative weights of testes at the lowest and highest dose level were also increased. Slightly increased absolute and relative weights of pituitary gland were recorded at the middle dose level. No statistically significant differences were detected.
Females
Slightly decreased absolute weights of ovaries were recorded in treated groups. In pituitary gland absolute and relative weights were decreased at the middle dose level. All changes were without statistical significance.

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 group. In abdominal cavity reduced seminal vesicles were recorded in one male at the highest dose level and reduced prostate gland was recorded in one male at the middle dose level and in one male at the highest dose level.
Females
Examination of the external surface of the body revealed no changes. In thoracic, abdominal cavity and pelvic cavity no changes were observed. Only in liver - marked structure was observed in one female at the highest dose level. Dilatation of uterus was recorded at the control group, the lowest dose level and the middle dose level in nonpaired or nonpregnant females or aborted females.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Males
Histopathological affections of male reproductive system were detected at all dose levels. Slight lymphocyte infiltrations and exocytosis (especially interstitial) were described in epididymis of all males. In testes sporadical atrophy and degeneration of germ epithelium and marked atrophy and degeneration of germ epithelium in one male was described. Also in testes vacuolation of cytoplasm of spermatogonia in most of treatment males were diagnosed. Lymphocyte infiltration in prostate gland was recorded in several males. Other changes in prostate gland - atrophy of epithelium, lymphocyte in interstitium and suspension hyperplasia were observed only sporadically. Also sporadically were observed changes in seminal vesicles - suspension hyperplasia and in epididymides - decreased count of sperm.
Female
No histopathological changes were detected. In mothers a typical status of the 4th day after parturition was found out in sexual organs: ovarium - follicles and corpora lutea in different phases of development, degeneration of some follicles; uterine cervix - vacuolated and apoptotic epithelial cells, atrophy of germinal area, 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 nonpregnat and aborted females a various phase of oestrous cycle was recorded. In uterus accumulation of ovulatory intraluminal fluid (hydrometra) was found out. During the histopathological examination pregnancy was confirmed in female No. 172 (the female had achieved pregnancy but then she aborted).

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
< 100 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: reproduction
Dose descriptor:
NOAEL
Effect level:
900 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: reproduction

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
see Details on results
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Effect were observed, but considered negligible.
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.

Details on results (F1)

VIABILITY (OFFSPRING)
Total numbers of live pups at the lowest dose level and at the middle dose level were decreased compared with control group. At the highest dose level total number of live pups was increased. Increased average number of pups per litter was recorded at the lowest and highest dose level.
The number of dead pups in the day of parturition was negligible - only one pup was found dead at the highest dose level.
Increased number of males in litter was recorded at the lowest and the highest dose level. At the middle dose level slightly decreased number of males in litter was recorded. Number of females in litter was slightly increased at all dose levels with dependence on the dose.

CLINICAL SIGNS (OFFSPRING)
No differences in development of pups were observed at the control group and at all treated groups.

BODY WEIGHT (OFFSPRING)
The average body weights of pups at all dose levels were well-balanced with the control group. Only slightly decreased body weight of pups was recorded at the highest dose level.

DEVELOPMENT OF PUPS
No pup died at the control group and at the lowest dose level during lactation period. At the middle dose level three pups died during lactation period in two mothers and at the highest dose level one pup died on the 1st day of lactation period.

GROSS PATHOLOGY (OFFSPRING)
The macroscopic examination was performed in all pups. Stomach without milk was sporadically observed at the lowest, middle and highest dose level. Testes - focal haemorrhage was recorded in three males at the highest dose level in different mothers.

HISTOPATHOLOGY (OFFSPRING)
Histopathological examination of testes was performed in one male at the highest dose level. Haemorrhage under capsule of testes was observed.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
900 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: development

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL for REPRODUCTION for MALES is less than 100 mg/kg/day. The value was established on the basis of examination of sperm.
The NOAEL for REPRODUCTION for females was established as 900 mg/kg/day body weight/day.
The NOAEL for DEVELOPMENT was established as 900 mg/kg/day body weight/day.
Executive summary:

The test substance, Dust, 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. The dose levels for study - 100, 300 and 900 mg/kg/day were chosen on the basis of the results of the Study: Dust, steelmaking - Repeated Dose 28-day Oral Toxicity Study (dose-range finding experiment with 14-day application period).

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 27 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 application of the test substance did not cause the death of any female or male.

In parental males and females negative influence of the test substance on clinical status was not found out.

The test substance did not significantly affect growth of parental males of all dose levels. Average body weight increment of parental females of all treated groups on the 4th day was decreased with dependence on the dose level. No statistically significant differences were detected.

Histopathological examination of reproductive system of parental males showed vacuolation of cytoplasm of spermatogonium in testes of majority males in treated groups and lymphocyte infiltration in epididymides of all males. Also atrophy and degeneration of germ epithelium in testes and lymphocyte infiltration in interstitium of prostate gland was diagnosed in all groups. Other changes were recorded only sporadically. Microscopical changes diagnosed were not considered as changes of toxicological significance and they did not relate with the test substance. Biometry of organs proved only slight changes of weight of examined organs without statistical significance. 

Markedly decreased sperm motility was recorded at the lowest and middle dose levels. At the highest dose level only slightly increased sperm motility was recorded. The effect of the test substance on sperm motility declined with the dose level. Increased percentage of affected sperms was recorded at all treated groups.

Examination of reproductive system of parental females did not evidence any significant microscopical changes. Biometry of organs also proved no statistically significant and no dose dependent differences in treated animals.

Observation of pups - the number of pups, sex ratio, average weight of litter, average body weight of pups on the day of parturition/1st day after parturition and the 4th day of lactation and postnatal development of pups were unaffected by the test substance treatment. Only slight changes were recorded. Death of three pups at the middle dose level and one pup at the highest dose level was recorded in lactation period. Macroscopic abnormalities were detected sporadically. At the highest dose level focal haemorrhage was recorded in testes of pups of different mothers. Histopathological examination of testes was performed in one male. Haemorrhage under capsule of testes was observed.

Reproduction parameters - number of females achieving pregnancy and accompanying conception index, fertility index and gestation index were decreased at the lowest dose level. At the lowest dose level number females which aborted was also increased. These changes probably related to finding of impaired sperm quality recorded in males. Slightly decreased conception index and fertility index at the middle dose level were recorded.

Duration of mating of treated groups was similar to control group (except one female at the highest dose level). Other recorded changes of reproduction parameters were slight. 

The NOAEL for REPRODUCTION for MALES is less than 100 mg/kg/day. The value was established on the basis of examination of sperm.

The NOAEL for REPRODUCTION for females was established as 900 mg/kg/day body weight/day.           

The NOAEL (No Observed Adverse Effect Level) for DEVELOPMENT was established as 900 mg/kg/day body weight/day.