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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30.9.2009-15.2.2010
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:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Fluidized Bed Combustion (FBC) Bottom Ash
- Substance type: technical product
- Physical state: solid
- Appearance: Light grey solid powder with some small black particle
- Chemical structure: Complex product of oxides
- Main components: SiO2 - 27,78%, Fe2O3 - 5,98%, CaO (total) - 35,65%, Na2O - 0,25%, P2O5 - 0,38%, CO2 - 0,5%, CaO (free) - 19,91%, Al2O3 - 11,16%, TiO2 - 2,65%, MgO - 0,44%, K2O - 0,42%, SO3 (sulphate) - 2,84%
- Impurities: Metals: As, Be, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Sb, Se, Tl, V, Zn - sum < 0,1%
- Lot/batch No.: FBC/230309/T2
- Expiration date of the lot/batch: 03/2024
- Stability under test conditions: stable
- Storage: The substance was stored in PE container at room temperature.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760152
- Age at study initiation: 9 weeks
- Fasting period before study: none
- 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.
- Water (e.g. ad libitum): drinking water
- Acclimation period: 7 days

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

STUDY TIME SCHEDULE
Date of animal arrival: 23. 9. 2009
Start of administration: 30. 9. 2009
End of administration: males 27. 10. 2009, females 12. 11. 2009
Clinical examination: 30.9. – 12. 11. 2009
Necropsy and biometry of organs: males 28. 10. 2009, females 09. – 13. 11. 2009

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.

VEHICLE
- Olive oil (pharmaceutical quality) - Batch No.: L 803142, 4683401
Details on mating procedure:
- M/F ratio per cage: Mating 1 : 1 (one male to one female) was used.
- Proof of pregnancy: Day 0 of pregnancy was defined as the day the sperms were found in vaginal smears.
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
- males and females – 2 weeks prior to the mating period and then during the mating period
- pregnant females - during pregnancy and till the 3rd day of lactation
- males - after mating period – totally for 28 days
- non-pregnant females (mated females without parturition) - for 26 days after the confirmed mating.

Frequency of treatment:
7 days per week at the same time
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
800 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 females and 10 males per group

Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels for study – 50, 200 and 800 mg/kg/day were chosen on the basis of the results of the Study No. 66/09/8: Fluidized Bed Combustion (FBC) Bottom Ash – Repeated Dose 90-day Oral Toxicity (dose-range finding experiment with 28-day application period).

Examinations

Parental animals: Observations and examinations:
HEALTH CONDITION CONTROL: Yes
- Time schedule: daily - during the acclimatization and the experimental part

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: males and females - daily during the administration period

BODY WEIGHT: Yes
- Time schedule for examinations: males - weekly; females - weekly in premating period, during pregnancy: 0., 7th, 14th, 20th day, during lactation: 0. or 1st and 4th day

FOOD CONSUMPTION:
- Males and females in premating period - (grams/animal/day) calculated from average values of each group (except the mating period)
- Females in pregnancy and lactation period - average individual values (grams/animal/day) - (non-pregnant females not included in calculation)
- Time schedule: males - weekly (except the mating period); females - weekly during premating period, during pregnancy and lactation – on the same days as body weight

MORTALITY:
- Time schedule: daily (for vitality or mortality changes)
Sperm parameters (parental animals):
Parameters examined in male parental generations: sperm motility and sperm morphology.
Litter observations:
CLINICAL OBSERVATION OF PUPS
- Time schedule: as soon as possible after delivery and then daily
- Detailed examination of each litter was performed as soon as possible after delivery (day 0 or 1 post-partum) and the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.

BODY WEIGHT OF LITTER
- Time schedule: 0. or 1st and 4th day
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: at the end of the administration period – after 28 days of administration
- Maternal animals: parental females - on the 4th day of lactation. Mated females without delivery - 27th day after confirmed mating.

GROSS NECROPSY
- Gross necropsy consisted of macroscopically examination for any pathological changes with special attention to the organs of the reproductive systems.

HISTOPATHOLOGY
- The following tissue and organs were collected for further histopathological evaluation: relevant gross lesions, pituitary gland, coagulation gland, prostate gland, seminal vesicles, epididymis and testes (fixed in Davidson´s solution), cervix of uterus, ovaries, uterus and vagina.

ORGAN WEIGHTS (absolute weight)
- Males - testes, epididymis, prostate gland and pituitary gland
- Females - ovaries, uterus (incl. uterine tube and cervix) and pituitary gland
Postmortem examinations (offspring):
SACRIFICE
- On the 4th day of lactation
- These animals were subjected to postmortem examinations (macroscopic examination) as follows: external examination of the cranium, and to macroscopic examination of the thoracic and abdominal tissues and organs.

- Dead pups were sexed and externally examined; the stomach was examined for the presence of milk.
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. Treated groups were compared with control group.
Reproductive indices:
LOSS OF OFFSPRING
- Pre-implantation loss
- Post-implantation loss
- Post-natal loss

FERTILITY PARAMETERS
- Percentage mating
- Fertility index
- Conception index
- Gestation index



Offspring viability indices:
- Percentage of postnatal loss days 0-4 post partum
- Viability index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significantly decreased food consumption with dose dependence was recorded at all dose levels during lactation in females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significantly decreased food consumption with dose dependence was recorded at all dose levels during lactation in females.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

MORTALITY (PARENTAL ANIMALS)
The application of the test substance did not cause the death of any female or male.

CLINICAL SIGNS (PARENTAL ANIMALS)
In parental males negative influence of the test substance on clinical status was not found out. In females clinical changes were observed only sporadically and these changes were without toxicological importance.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
The test substance did not affect growth of parental males of all dose levels. Average body weight and average body weight increment of parental females were decreased at the highest dose level from the 14th day of pregnancy and during lactation in females. Statistically significantly decreased food consumption at the dose level 800 mg/kg was recorded during lactation in females.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The test substance had not negative effect on sperm quality of treated males.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Number of females achieving pregnancy and accompanying conception index was well balanced at the control group and at all dose levels. Duration of mating of treated groups was similar to control group.

ORGAN WEIGHTS (PARENTAL ANIMALS)
MALES: Biometry of organs proved only slight difference in the prostate glands weight at the highest dose level in comparison with control, but this decrease was caused by markedly reduced prostate glands in two males. No statistically significant differences were detected.
FEMALES: Biometry of organs also proved significant differences without dose dependence in treated animals.

HISTOPATHOLOGY (PARENTAL ANIMALS)
MALES: Only vacuolation of cytoplasm of spermatogonium in testes and vacuolation of cells in pituitary gland increased incidence at all treated males and lymphocyte infiltration in epididymides of all males. Microscopical changes diagnosed in testes and in pituitary gland were not considered as changes of toxicological significance.
FEMALES: Increased occurrence of cysts in ovaries in treated females – this finding was not dose dependent. Hyperplasia, hypertrophy and atrophy of cervical epithelial cells were detected at all treated females with dose-response relationship. These affections had no negative effect on reproduction.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effect on reproduction in the highest dose

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
No pups died at the control group and at the lowest and middle dose levels during lactation period. At the highest dose level one pup died during lactation period.

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 and had an increasing trend during whole lactation period.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic abnormalities were detected sporadically.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
800 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effect on development in the highest dose

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The test substance did not have effect on reproduction in parental males and females and development in pups. The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION and DEVELOPMENT was established as 800 mg/kg body weight/day.
Executive summary:

Introduction

The test substance, Fluidized Bed Combustion (FBC) Bottom Ash, 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.

Methods

Wistar rats of SPF quality were used for testing. The test substance was administered dissolved in olive oil using a stomach tube; oral application of rats was made daily.

The concentrations of solutions at all dose levels were adjusted to ensure the administered volume of 1 mL per 100 g of body weight. Each group consisted of 10 males and 10 females. The dose levels for study – 50, 200 and 800 mg/kg/day were chosen on the basis of the results of the Study No. 66/09/8: Fluidized Bed Combustion (FBC) Bottom Ash - Repeated Dose 90-day Oral Toxicity Study (dose-range finding experiment with 28-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 26 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. Fertility parameters and loss of offspring parameters were caclutated.

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.

Conclusion

The test substance did not have effect on reproduction in parental males and females and development in pups. The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION and DEVELOPMENT was established as 800 mg/kg body weight/day.