Registration Dossier

Toxicological information

Endpoint summary

Currently viewing:

Administrative data

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:
24.2.2010-9.7.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP 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:
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
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).
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
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.
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: 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.
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).
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
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.
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.
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
Reproductive effects observed:
not specified
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.           

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
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 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.

The Reproductive screening test result does not confirmed effect on female fertility and offspring development. Due to the unambiguous effect on male fertility was proposed to perform only the Prenatal development toxicity (teratogenicity) study (EU guideline B.31), to investigate the impact of substances on the development of pups and pregnant females.


Short description of key information:
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. The test was carried out under GLP conditions.
There is no other study for effect on fertility of Dust, steelmaking.

Effects on developmental toxicity

Description of key information
The test substance, Dust, steelmaking was tested for prenatal developmental toxicity using the Method B.31, Prenatal Developmental Toxicity Study, Council Regulation (EC) No. 440/2008, Published in O.J. L142, 2008 and OECD Test Guideline No. 414 Prenatal Developmental Toxicity Study, Adopted by the Council on January 22nd 2001. The test was carried out under GLP conditions.
There is no other study for effect on fertility of Dust, steelmaking.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2.1.2013 - 8.3.2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Velaz, s.r.o., Koleč u Kladna, Czech Republic (SPF Breeding)
- Age at study initiation: 11 weeks
- Housing: Animals were housed in plastic, individually ventilated cages containing sterilised clean shavings of soft wood. Before mating 2 or 3 rats of the same sex in one cage, during mating period – one male and two females in one cage were housed. During pregnancy 1 female was housed in one cage.
- Diet (e.g. ad libitum): complete peleted diet for rats and mice in SPF breeding (ST 1 BERGMAN)
- Water (e.g. ad libitum): sterilised drinking water (ad libitum).
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 30 - 70 %
- Air changes (per hr): cca 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark
Route of administration:
oral: gavage
Vehicle:
olive oil
Remarks:
pharmaceutical quality
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The treated and control females were administered daily by gavage – from the 5th to the 19th day of pregnancy. The animals were treated 7 days per week at the same time (8.00 – 10.00 am).
The concentrations of suspensions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. The application form (test substance suspension in olive oil) was prepared daily just before administration. The vehicle control group was administered by olive oil in the same volume. The application form was mixed during application by magnetic stirrer (10 minutes). The procedure was taken over from analytical report Determination of homogeneity and stability of Dust, steelmaking in vehicle (Annex 1 of Final report, Study No.107/09/7: Dust, steelmaking – Repeated Dose 28-day Oral Toxicity Study, VUOS-CETA Report No. 10-239, 2010).

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

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

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

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

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

PATHOLOGICAL FINDINGS:
- Macroscopic changes were evaluated in all females (including females without foetuses). The dilatations of uterus was detected in two control females.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
- Records: see Table No. 3
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
- Records: see Table No. 4
Statistics:
The ANOVA software - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis.
This statistical analysis was used for the results of body weight of pregnant females at the end of pregnancy, absolute and relative weight of uterus, number of corpora lutea and implantation, number of foetuses, number of male foetuses, number of female foetuses, weight of foetuses, weight of male foetuses and weight of female foetuses.
Control group with vehicle was compared with three treated groups.
Details on maternal toxic effects:
Maternal toxic effects:no effects
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
< 160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Examination of foetal skeleton indicated delayed development of the skeleton at treated dose levels. The increased frequency of presence of incomplete cranial bones (occipital, parietal, interparietal, frontal and their combinations) was detected. The incomplete ossification of parietal bone or parietal bone in combination with interparietal, frontal bone was found in all groups. The increased number of foetuses with decreased number of ossification sites of sternum was recorded also in all groups. These findings (in cranium and sternum) were related with decreased body weights of foetuses. The contrasting increased number of ossification sites of sternum was found only at the highest dose level. Anomaly of ribs – deformation of the false ribs (letter V) was most detected in litters at the highest dose level.
Examination of ribs revealed changes related with treatment not with delayed development. The wavy ribs were observed at all dose levels (not in control). The other findings in ribs -increased number of floating ribs was recorded only in litters at the middle dose level so it did not concern with treatment.
The incomplete ossification of sacral vertebrae (relationship with fetal body weight) was recorded at the all groups (including control group) but in litters at the middle dose level the increase in incidence of this variation (20 %) in comparison with control (11.11 %) was observed.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
No toxicity in maternal animals administered by test substance was observed. Delayed development of foetuses (delayed ossification of cranial, sternum skeleton and sacral vertebrae) was detected at all dose levels. These findings were related with decreased individual body weights of some foetuses. The test substance treatment evoked occurrence of variations – wavy ribs in all treated groups so it indicates teratogenic properties of the test substance (it did cause morphologic changes of skeleton) on early prenatal development of organism in uterus.

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

The test substance, Dust, steelmaking was tested for prenatal developmental toxicity using the Method B.31, Prenatal Developmental Toxicity Study, Council Regulation (EC) No. 440/2008, Published in O.J. L142, 2008 and OECD Test Guideline No. 414 Prenatal Developmental Toxicity Study, Adopted by the Council on January 22nd 2001.

Wistar rat females of SPF quality were used for testing. After acclimatization the females were mated with males. The test substance was then administered to pregnant females - daily from the 5th to the 19th day of pregnancy. The study included four groups of females – 3 treated groups and 1 control group (vehicle only). The test substance was administered suspended in olive oil (pharmaceutical quality) by stomach tube and the concentrations of suspensions at all dose levels were adjusted to ensure the administered volume of 1 mL per 100 g of body weight. 

The dose levels for study – 160, 400 and 1000 mg/kg/day, were chosen on the basis of results of preceding study No.107/09/18: “Dust, steelmaking – Reproduction/Developmental Toxicity Screening Test”, VUOS-CETA Report No. 10-238, 2010.

The health condition, clinical status after application, body weight and food consumption of maternal animals were monitored during developmental toxicity study. On the 20th day of pregnancy the maternal animals were killed, the uterine contents were examined and the foetuses were evaluated for soft tissue and skeletal changes.

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

Maternal animals:

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

Foetuses:

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

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

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

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

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
160 mg/kg bw/day
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
Additional information

No toxicity in maternal animals administered by test substance was observed.

The NOAEL (No Observed Adverse Effect Level) for toxicity in PREGNANT FEMALES is higher than 1000 mg/kg/day.

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

Justification for classification or non-classification

Although some adverse effects were identified during reproductive/developmental toxicity tests, these effects were not found sufficient for classification according to section 3.7.2.3.3 of Annex I to Regulation 1272/2008.

Additional information