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Effects on fertility

Description of key information

The method: OECD Test Guideline No. 421 Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on July 27th 1995. GLP study.

In Reproduction/Developmental Toxicity Screening Test the following dose levels 160, 400 and 1000 mg/kg/day were administered.

 

Negative influence of the test substance on sperm motility and sperm morphology was observed in males of the highest dose level (limit); but this affection had no negative effect on mating and reproduction parameters of males at the highest dose level.

The test substance did not affect number of pups. Different total number of pups was not influenced by the test substance, but by number of pregnant females (average number was well-balanced).

Number of females achieving pregnancy, durations of mating and pregnancy, number of pups, sex ratio, average weight of litter, average body weight and postnatal development of pups were unaffected by the test substance treatment. The test substance did not affect fertility of treated males and females and development of pups.

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9.9.2009 – 25.3.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1995
Deviations:
no
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
other: Wistar Han
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Koleč u Kladna, Czech Republic
- sex: males and virgin females
- Age at study initiation: 9 weeks
- Fasting period before study: no

- 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:
Complete peleted diet for rats and mice in SPF breeding - ST 1 BERGMAN. Diet was sterilised before using.

- Water:
Free access to drinking water (ad libitum). Water was sterilised before using.

- Acclimation period: 14 days

- Identification:
Identification of animals was made by colour marks on fur (system 1 – 10), each cage was marked with the number of study, number of animals, sex, number of cage, name and dose of the test substance and mark of group.

- Additional Information: The standard pelleted laboratory animal diet are analysed for nutrients (once a year) and bacteriologically examined (every two months) on a regular basis. Results are retained in the CETA archives. Reports of analysis of water (twice a year) are retained in the CETA archives. Results of sterilizer effectivity control (performed once a year) are retained in the CETA archives.
Analysis of diet and water and steriliser control, did not reveal any findings that could affect study integrity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): a relative humidity of 30-70%
- Air changes (per hr): Animals were housed in controlled environment, in which optimal conditions were considered to be 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: 9.9.2009
Start of administration: 23.9. 2009
End of administration: males 20.10.2009, females 8.11.2009
Clinical examination: 9.9. – 9.11.2009
Necropsy and biometry of organs: males 21.10.2009, females 2.11. – 9.11.2009
Histopathological examination: 9.11.2009 – 24.2.2010
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
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), manufacturer: Dr.Kulich Pharma s.r.o.
- Lot/batch no. (if required): 4683401
Details on mating procedure:
- M/F ratio per cage: Mating 1 : 1 (one male to one female) was used in this study.
- Length of cohabitation: until the presence of spermatozoa
- Proof of pregnancy: Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found.

- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The test substance consists of various oxides insoluble in the application form (olive oil). A suitable analytical method cannot be found for homogeneity and stability testing. Since undissolved particles of the test substance are easily visible in the application form, homogeneity was checked by eye (suspension were mixed for 10 minutes by magnetic stirrer). Stability of the test substance in the application form cannot be verified but there is no indication that a mixture of rigid oxides would be unstable in its solution (in olive oil) for that short time period (1 hour).
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 - 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 26 days after the confirmed mating.
Frequency of treatment:
The animals were treated 7 days per week at the same time (8.00 – 10.00 am).
Dose / conc.:
160 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
3 treated groups (dose 160, 400, 1000 mg/kg body weight/day) and one control group (vehicle only). Each group consisted of 10 males and 10 females.

Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels for study – 160, 400 and 1000 mg/kg bw/day were chosen on the basis of the results of the Study No. 67/09/8: Semi Dry Absorption (SDA) Product – Repeated Dose 90-day Oral Toxicity Study (dose-range finding experiment with 28-day application period).
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 (in grams). Nonpregnant females were not included in calculation of averages in pregnancy and lactation period.
males - weekly
females - weekly in premating and mating 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
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):
It was not monitored.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
In all males of all groups surviving to scheduled necropsy the sperm parameters were examined: sperm motility and sperm morphology.

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, no head, abnormal form of neck – were recorded.
Litter observations:
PARAMETERS EXAMINED
BODY WEIGHT: Yes
pups (litters) – 0. or 1st and 4th day

CLINICAL OBSERVATION: Yes
All pups were observed in natural conditions in their cages daily during the lactation. Changes in behavioural abnormalities were recorded. 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.








Postmortem examinations (parental animals):
SACRIFICE and PATHOLOGICAL EXAMINATION:
Parental males - at the end of the administration period – after 28 days of administration.
Parental females were killed on the 4th day of lactation. Mated females without delivery were killed 27th day after confirmed mating.
Then they were macroscopically examined for any pathological changes with special attention to the organs of the reproductive systems. All macroscopic abnormalities were recorded.

BIOMETRIC OF REPRODUCTIVE ORGANS: Yes
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.

HISTOLOGICAL TECHNIQUE
The following tissue and organs were collected from all killed males and females at necropsy and fixed in neutral 4% formaldehyde suspension (v/v) for further histopathological evaluation:
relevant gross lesions, pituitary gland, coagulation gland, prostate gland, seminal vesicles, epididymis and testes (fixed in Davidson´s suspension), cervix of uterus, ovaries, uterus and vagina.
For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used.

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 criteria mentioned in 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 criteria mentioned in 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):
SACRIFICE AND GROSS EXAMINATION OF DEAD PUPS
- on the 4th day of lactation
- these animals were subjected to external examination of the cranium, and to macroscopic examination of the thoracic and abdominal tissues and organs. All macroscopic changes were recorded.

- 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 and biometry of organs and number of pups. Control group with vehicle was compared with three treated groups.
The results statistically significant on probability level 0.05 are indicated by figures with asterisk in the summary tables.
Reproductive indices:
For each of parental females the following parameters were counted:
Pre-implantation loss, Post implantation loss, Post-natal loss
For each dose group reproduction parameters will be counted:
Percentage mating, Fertility index, Conception index, Gestation index
Offspring viability indices:
Percentage of postnatal loss days 0-4 post partum, Viability index
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
M: No statistically significant differences were detected.
F: see box Details on results (P0)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
M/F: see box Details on results (P0)
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
M/F: see box Details on results (P0)
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
M/F: see box Details on results (P0)
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
M/F: see box Details on results (P0)
The most of histopathological findings recorded in parental males and females responded to common findings in animals in reproductive period.
Histopathological findings: neoplastic:
not specified
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Negative influence of the test substance on sperm motility and sperm morphology was observed in males of the highest dose level (limit) - see box Details on results (P0) but this affection had no negative effect on mating and reproduction parameters of males at the highest dose level.
Reproductive performance:
no effects observed
Description (incidence and severity):
see box Details on results (P0)
MORTALITY (PARENTAL ANIMALS)
There were no unscheduled deaths at any dose levels and at the control group during whole study.

CLINICAL SIGNS (PARENTAL ANIMALS)
Males and females: 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. No clinical changes were observed in males of all dose levels after application of the test substance.

FOOD CONSUMPTION
Males - food consumption of males at all treated dose levels was lower than in the control group during the whole study. In comparison with the control group, statistically significant decrease of average food consumption was recorded at the middle and highest dose level in the 2nd week and at all treated groups in the 4th week of study.
Females
Pre-mating period - during pre-mating period, average food consumption was well-balanced in the control group and in the lowest treated group. In the groups 400 and 1000 mg/kg/day, average food consumption was decreased in comparison with the control group. This decrease was statistically significant in the 1st week of study.
Pregnancy - females without parturition (non pregnant or aborted females) were not included in evaluation of food consumption during pregnancy.
In the second and third week of pregnancy, average food consumption at all treated groups was relatively well-balanced but lower compared to the control group. In the 3rd week of pregnancy, this decrease was statistically significant in all treated group.
Lactation - only mothers (females with live pups) were included in evaluation of food consumption during lactation period. Average food consumption of treated mothers was well-balanced, but lower than in mothers of control group. Decrease of average food consumption of the highest dose level was statistically significant.

BODY WEIGHT
Males: no statistically significant differences were detected.
The animal body weights at all dose levels were relatively well-balanced with the control group during whole application period.
Body weight increments at all treated animals were well-balanced, but were a little bit lower in comparison with control group.

Females
Pre-mating period - average body weight increments of all dose levels were relatively well-balanced, but a little bit lower in comparison with the control group. Only in the 2nd week, average body weight increment of females in the lowest dose level (160 mg/kg/day) was higher than in the control group. No statistically significant changes during the whole pre-mating application period were found out.
Pregnancy - females without parturition (non pregnant or aborted females) were not included in evaluation of the body weight increments during pregnancy.
Only in the 1st week of pregnancy, average body weight increment of females at the dose level 160 mg/kg/day persisted higher compared to the control group. The second and third week of pregnancy, average body weight increment of all treated groups was well balanced but lower than in control the group.
Lactation - only mothers (females with live pups) were included in evaluation of body weight increment during lactation period.
Average weight increments of treated mothers were markedly lower in comparison with mothers of control group.

SPERM QUALITY
The test substance had negative effect on sperm quality of treated males. Sperm motility was affected in males of all treated groups (0-40-30-60% impaired motility); mostly in males of the highest dose level 1000 mg/kg. Microscopical examination of sperms revealed increased percentage portion of morphologically changed sperms in males of all dose levels (5-6.1-5.8-6.9% affected sperms), mostly in males of the highest dose level 1000 mg/kg.
These findings were not accompanied by damage of spermiogenesis in testicular tubules and had no effect on reproduction.

BIOMETRY OF REPRODUCTIVE ORGANS
Males
Absolute and relative weights of all observed organs were similar in control males and in males of the highest dose level.
Decreased weight of testes was recorded in males of the dose level 160 mg/kg/day; decreased weight of epididymis was recorded in males of the dose levels 160 and 400 mg/kg/day. On the contrary, increased weight of prostate gland was recorded at the dose level 400 mg/kg/day.
No statistically significant differences were detected.
Females
Nonpregnant females and females with abortion were not used for calculation of means and evaluation of organs weight.
Absolute weight of uterus at the dose level 160 mg/kg/day was lower in comparison with control group; at the dose level 400 mg/kg/day and the dose level 1000 mg/kg/day, weight of uterus was higher than at the control group. At these dose levels, also relative weight of uterus was higher.
Absolute and relative weight of ovaries was similar at the control group and the highest dose level. Absolute weight of ovaries at the lowest and middle dose levels was lower in comparison with the control group.
Absolute and relative weight of pituitary gland of all treated groups was lower compared to weight of pituitary gland at the control group. This difference was not dependent on dose level.
No statistically significant differences of relative and absolute weights of reproductive organs were detected.

REPRODUCTION DATA
Number of females achieving pregnancy and accompanying conception index was well balanced at the control group and at the lowest and middle dose levels. At the highest dose level, these parameters were higher than in other groups. Duration of mating and pregnancy of treated groups was similar to control group.
Number of females bearing live pups was higher at the dose level 1000 mg/kg/day, in the rest of the treated group and control group, this number was similar.
Number of pregnant females was similar at the control group and lowest and the middle dose level; the highest number of pregnant females was recorded at the highest dose level. At the lowest dose level and at the middle dose level 2 females aborted. None abortion was observed at the control and the highest dose level.
Pre-implantation losses were relative well balanced among treated groups. At the lowest dose level abortion in two females together with increase of post-implantation losses were recorded. There were no gross or microscopic findings that could be related to treatment and theirs abortion was considered spontaneous without toxicological importance. At the middle and the highest dose level post-implantation loss were well-balanced with the control group. Slight disbalance of other fertility parameters recorded in parental females was without toxicological importance.

PATHOLOGY - MACROSCOPIC AND MICROSCOPIC FINDINGS
Males
Histopathological examination of reproductive system of parental males showed increased incidence of tubules with destructed epithelium in testes of males in the dose levels 400 and 1000 mg/kg/day; increased incidence of lymphocyte infiltration in prostate gland in males of the dose levels 160 and 400 mg/kg/day. Increased incidence of vacuolation of spermatogoium in testes was recorded in males at all treated groups.

Females
Macroscopic structure of reproductive organs of treated parental females was not markedly affected by treatment of the test substance.
Histopathological examination of reproductive system of parental females revealed increased incidence of microscopic affections in ovaries – follicular cysts in females of the dose level 1000 mg/kg/day, but this affection was recorded also sporadically at the control group and at the middle dose level. Atrophy of epithelium of vagina was recorded in two females of the highest dose level. Affections (atrophy, hypertrophy, hyperplasia) in cervix were also observed in females of treated groups, especially in the highest dose level. These findings had no negative effect on parental females.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The test substance did not affect fertility of treated males and females and development of pups
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
see box Details on results (F1)
Body weight and weight changes:
no effects observed
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
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
BODY WEIGHT
In comparison with the control group, the average weights of litter at all treated groups on 0/1 day after parturition were lower. Average body weight of pup per litter was relatively well-balanced.
On the 4th day of lactation, average weights of litter were still lower in comparison with the control group, but average body weight of pup per litter was slightly higher at all treated groups compared to average body weight of pup at the control group.

DEVELOPMENT AND ABNORMAL
No pups died at the control group and at the dose levels 160 mg/kg/day during lactation period. At the dose level 400 and 1000 mg/kg/day one pup of each dose level died during lactation period.
No differences in development of pups were observed at the control group and at all treated groups.

VIABILITY (OFFSPRING)
The test substance did not affect number of pups. The total number of live pups was the highest at the highest dose level but average number of pups per litter in comparison with the control group was lower.
Marked decrease of total number of live pups and average number of pups per litter (in the day of parturition/1st day after parturition and the day 4th after parturition) in comparison with control group were recorded at the lowest and middle dose levels. This decrease is not influenced by the test substance, but by number of pregnant females (average number was well-balanced).

GROSS PATHOLOGY (OFFSPRING)
The macroscopic examination was performed in all pups. No pathologic findings were recorded at control group and at the all dose levels.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: The test substance did not affect fertility of treated males and females and development of pups
Based on:
not specified
Sex:
not specified
Remarks on result:
not determinable
Key result
Reproductive effects observed:
no
Conclusions:
Effect on average food consumption (decrease) was observed in males of the middle and the highest dose level. The same effect on average food consumption (decrease) was recorded in females of the highest dose level. Marked decrease of average body weight increment was recorded in all treated mothers on 4th day of lactation.
Negative influence of the test substance on sperm motility and sperm morphology was observed in males of the highest dose level (limit); but this affection had no negative effect on mating and reproduction parameters of males at the highest dose level.
The most of histopathological findings recorded in parental males and females responded to findings of animals in reproductive period.
The test substance did not affect number of pups. Different total number of pups was not influenced by the test substance, but by number of pregnant females (average number was well-balanced).
Number of females achieving pregnancy, durations of mating and pregnancy, number of pups, sex ratio, average weight of litter, average body weight and postnatal development of pups were unaffected by the test substance treatment.
The test substance did not affect fertility of treated males and females and development of pups.
The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION of males was established as 1000 mg/kg body weight/day.
The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION of females and pups DEVELOPMENT was established as 1000 mg/kg body weight/day.
Executive summary:

Introduction

The test substance, Semi Dry Absorption (SDA) Product, 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 suspensions at all dose levels were adjusted to ensure the administered volume of 1 mL per 100 g of body weight. Four groups of animals were included in the study - 3 treated groups (doses 160, 400, 1000 mg/kg of body weight/day) and one control group (vehicle only). Each group consisted of 10 males and 10 females. The dose levels for study – 160, 400 and 1000 mg/kg/day were chosen on the basis of the results of the Study No. 67/09/8: Semi Dry Absorption (SDA) Product – 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,

non-pregnant 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.

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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliability 1

Effects on developmental toxicity

Description of key information

The method: OECD Test Guideline No. 421 Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on July 27th 1995. GLP study.

In Reproduction/Developmental Toxicity Screening Test the following dose levels: 160, 400 and 1000 mg/kg/day were administered.

Number of females achieving pregnancy, durations of mating and pregnancy, number of pups, sex ratio, average weight of litter, average body weight and postnatal development of pups were unaffected by the test substance treatment.

The test substance did not affect fertility of treated males and females and development of pups.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23.08.2017 - 14.12.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
Published in O.J.L. 142, 2008
Deviations:
yes
Remarks:
See Any other information (no impact)
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Adopted on 22nd January 2001
Deviations:
yes
Remarks:
See Any other information (no impact)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
CRL (SPF quality - guaranteed)
Details on test animals and environmental conditions:
TEST ANIMALS
sexually adult females (males – only for mating)
- Source: Charles River SPF breeding, supplied via VELAZ s.r.o., Czech Republic, RČH CZ 11760500
- Age at study initiation: 11 weeks
- Weight at study initiation: 201.8 - 306.3 g
- Housing: in plastic cages containing sterilised clean shavings of soft wood or sterilized LIGNOCEL in a controlled environment
- Diet (e.g. ad libitum): Complete peleted diet for rats and mice in SPF breeding (manufacturer: Altromin Spezialfutter GmbH&Co.KG, Im Seelenkamp 20, D-3271, Lage, Germany), sterilized before using.
- Water (e.g. ad libitum): drinking water ad libitum; water was sterilised before using
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 30 - 70 %
- Air changes: 15 per hour
- Photoperiod: 12 h light / 12 h dark
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance consists of various components insoluble in vehicle (olive oil). A suitable analytical method cannot be found for homogeneity and stability testing. Since undissolved particles of the test substance are easily visible in the application form, homogeneity will be checked by eye (suspension will be mixed for 10 minutes by magnetic stirrer). Stability of the test substance in the application form cannot be verified analytically. But the application form will be prepared just before the application and there is no indication that a mixture of rigid components would be unstable in the suspension (in olive oil) for that short time period (1 hour).

The application forms of the test substance (suspensions in olive oil) were prepared daily just before administration. The mixtures were mixed by the magnetic stirrer for 10 minutes and then during administration.

VEHICLE
- Concentration in vehicle: The concentrations of suspensions at all dose levels were adjusted to ensure the administration of 1mL per 100 g of body weight. The vehicle control group was administered by olive oil in the same volume.
- Amount of vehicle (if gavage):
- Lot/batch no.: 8002182001
- Purity: pharmaceutical quality
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Stability of the test substance in the application form cannot be verified analytically. But the application form will be prepared just before the application and there is no indication that a mixture of rigid components would be unstable in the suspension (in olive oil) for that short time period (1 hour).
Details on mating procedure:
Měla Lenka:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 M / 2 F
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy




- Impregnation procedure: [artificial insemination / purchased timed pregnant / cohoused]
- If cohoused:
- M/F ratio per cage: 1 M / 2 F
- Length of cohabitation:
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- Verification of same strain and source of both sexes: [yes / no (explain)]
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy





- Any other deviations from standard protocol:




Duration of treatment / exposure:
5th - 19th day after fertilisation
Frequency of treatment:
7 days per week at the same time (8.00 – 10.00 am)
Duration of test:
20 days
No. of animals per sex per dose:
24 pregnant females for each dose level (total number of animals before mating: 100 females and 25 males)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels for study – 160, 400 and 1000 mg/kg/day – have been chosen with respect to the information given in the Study No. 67/09/18: Semi Dry Absorption (SDA) Product - Reproduction/Developmental Toxicity Screening Test.

- Rationale for animal assignment: randomly
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the 1st day of pregnancy and then on the 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: uterus (incl. the cervix) was removed and weighed.
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 early resorptions: Yes
- Number of late resorptions: Yes

Uteri of non-pregnant females were examined to confirm the non-pregnant status
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Sex and individual body weights of foetuses were recorded.
Each foetus was examined for external alterations: symmetry of fore and hind limbs, number of fingers, closing or opening of eye fissures and external auditory canal, symmetry of head, integrity of superior palatum, status of umbilicus and genital papilla were observed.

One half of each litter (one half of female and male foetuses) was examined for soft tissue alterations using careful gross dissection.
Second half of each litter was processed and microscopically examined for skeletal and cartilage alterations according to the internal SOP.
Single staining displayed only ossified skeletal structures was used: the foetuses were fixed in ethanol, macerated in potassium hydroxide solution, stained with Alizarin red and placed in glycerine-based solution.
The skeletal examination was performed using a stereomicroscope and included examination of skull, clavicle, scapula, sternebra and sternum, ribs, vertebrae, pelvic girdle, forelimb/hindlimb.
Statistics:
For statistical evaluation the software Statgraphic® Centurion (version XV, USA) was used. The data from control group were compared with data from treated groups.
The results statistically significant on probability level 0.05 are indicated in the summary tables.

The parametric tests were used for statistical evaluation of:
• body weight of females (5th, 8th, 11th , 14th , 17th , 20th day of pregnancy)
• corrected body weight (subtraction weight of uterus from surgery body weight of females)
• food consumption (per interval)
• mean weight of foetuses (males, females, both sex)
• biometry of uteri (absolute and relative weight )
• preimplantation (IUDE) and postimplantation (IUDL) losses

As the first step the test for normality (Shapiro-Wilk test) was performed. If the data were not normally distributed the transformation of data was performed (Box-Cox transformation). If the data were not normal distributed after transformation,the non-parametric tests (Kruskal-Wallis Test and Mann-Whitney test) for comparison of the medians were performed.
If data were normally distributed after transformation, the Variance check (Levene’s test) to verify standard deviations within each group was used. One-Way ANOVA (probability level 0.05) was used to detect whether there were any significant differences amongst the means and then the post hoc statistical testing (Fisher's least significant difference - LSD test) for only statistical significant differences was performed.

The non-parametric tests were used for statistical evaluation of following parameters:
• number of corpora lutea, number of implantations, number of resorptions
• number of live foetuses (males, females, both sex)
The two-groups Mann-Whitney test (probability level 0.05) was applied.

The categorical data (of serious findings - external and internal alteration) was not implemented by the reason of low or similar incidence of these findings at the treated group against the control.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean food consumption of all treated groups was comparable with control group from the 5th day to the 14th day pregnancy, except 8th day of pregnancy at the lowest dose level.
Statistically significant decrease of food consumption was observed on the 8th day (interval 5th – 8th day) in females of the dose level 160 mg/kg/day and on the 17th and 20th day of pregnancy (interval 14th – 20th day) in females of the dose levels 160 and 400 mg/kg/day.
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The increased absolute uterine weight was observed in females at dose level 1000 mg/kg/day. This increase was noted in relation to higher litter size and without clear dose response and thus did not relate to the test substance treatment. Statistically significant differences of uterus weights were not detected in females of any dose level.
Corrected body weight of all treated females (the necropsy body weight of female minus weight of uterus) was not statistically significantly changed compared to control females.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic examination was performed in all females (including females without foetuses).
The uterus dilatation (probably the change associated with the physiological oestrous cycle) was detected in all groups (2-3-1-1). No finding related with treatment was noted at necropsy in treated females. Only in one female No. 128 was detected dark brown vaginal discharge. This female became pregnant and then all implanted conceptuses in a uterus were totally resorbed.
Number of abortions:
no effects observed
Description (incidence and severity):
The mean number of implantations, corpora lutea and resorptions in treated females was not statistical significantly changed in comparison with the control females.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The numbers of implantations and corpora lutea were slightly higher at the highest dose level.
Preimplantation losses (IUDE) were slightly increased at the middle dose level (10.79 %) in comparison with control group (8.46 %).
Postimplantation losses (IUDL) were statistically insignificantly increased at the lowest (12.19 %) and at the middle (9.06 %) dose levels in comparison with control (5.10 %).
The mean number of implantations, corpora lutea and resorptions in treated females was not statistical significantly changed in comparison with the control females.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The numbers of resorptions were similar at all dose levels and comparable with control group. The mean number of implantations, corpora lutea and resorptions in treated females was not statistical significantly changed in comparison with the control females.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The mean number of implantations, corpora lutea and resorptions in treated females was not statistical significantly changed in comparison with the control females.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Dead foetuses were found out in treated groups as well in control group (1 – 3 – 1 – 0). The three dead foetuses at the lowest dose level came from one litter.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no mortality of females, no changes in health condition status, no pathological findings in the dams, no dose related changes in reproduction parameters
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weight of foetuses was increased with the dose dependence in all groups compared to the control. Although the variations between individual body weights of foetuses at all dose levels against control foetuses were observed, this difference was not statistically significant. Males were heavier than females in all groups (include control). This weight imbalance is common.
The foetal body weight was statistically insignificantly increased at all dose levels.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Although the variations between individual body weights of foetuses at all dose levels against control foetuses were observed, this difference was not statistically significant. Males were heavier than females in all groups (include control). This weight imbalance is common.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The total number of live foetuses in group was increased at the highest dose level compared to the control group but the average total number of foetuses in litter was well balanced with control.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The decreased number of male foetuses in litter at the middle dose and increased number of female foetuses in litter at the highest dose were statistically significant. Sex ratio (mean value) was similar in all groups, except sex ratio at the dose level 400 mg/kg/day (6 male : 8 female).
Changes in postnatal survival:
effects observed, non-treatment-related
Description (incidence and severity):
Dead foetuses were found out in treated groups as well in control group (1 – 3 – 1 – 0). The three dead foetuses at the lowest dose level came from one litter.
Test substance-related foetal mortality was not evident at any dose level.
External malformations:
no effects observed
Description (incidence and severity):
No macroscopic changes of soft tissues and external alteration were found out.
Detailed necropsy of foetuses did not reveal increase of external and visceral variations and malformations at any dose level.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Examination of foetal skeleton indicated mainly delayed development of the skeleton at all doses including control group.
During examination of foetal skeleton delayed ossification of skeleton at all doses including control group was observed. The occurrence of structural skeletal variations could not be considered as teratogenic effect of the test substance on early prenatal development of organism in uterus because incidence of these findings was accidental or comparable with control group.
Visceral malformations:
no effects observed
Description (incidence and severity):
Detailed necropsy of foetuses did not reveal increase of external and visceral variations and malformations at any dose level.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on no altered growth and no serious structural abnormality found in treated foetuses
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
There were no unscheduled deaths of females during the study at any dose level.
No adverse changes of health condition and no clinical symptoms of intoxication were found in females at any dose level after administration of the test substance.
During the control of body weight increments and food consumption in pregnant females at all dose levels: 160, 400 and 1000 mg/kg/day toxicologically significant treatment-related effects were not detected. Evaluation of uterine weights (incl. absolute and relative weight of uterus) did not reveal toxicologically significant treatment-related effects. Macroscopical structure of organs of pregnant females and values of reproduction parameters (number of females with live foetuses, number of live and dead foetuses, early and late resorptions and sex ratio of foetuses), were unaffected by treatment with the test substance.
Test substance-related foetal mortality was not evident at any dose level. The foetal body weight was statistically insignificantly increased at all dose levels. Detailed necropsy of foetuses did not reveal increase of external and visceral variations and malformations at any dose level.
During examination of foetal skeleton delayed ossification of skeleton at all doses including control group was observed. The occurrence of structural skeletal variations could not be considered as teratogenic effect of the test substance on early prenatal development of organism in uterus because incidence of these findings was accidental or comparable with control group.
The NOAEL (No Observed Adverse Effect Level) for toxicity in PREGNANT FEMALES was established as 1000 mg/kg/day. This NOAEL value is based on no mortality of females, no changes in health condition status, no pathological findings in the dams, no dose related changes in reproduction parameters.
The NOAEL (No Observed Adverse Effect Level) for PRENATAL DEVELOPMENT was established as 1000 mg/kg/day. This NOAEL is based on no altered growth and no serious structural abnormality found in treated foetuses.
Executive summary:

Introduction

The test substance, Product of Semi-Dry Absorption method of Flue Gas Desulphurization (SDA Product), was tested for prenatal developmental toxicity using the Method B.31, Prenatal Developmental Toxicity Study, Council Regulation (EC) No. 440/2008, Published in O.J. L. 142, 2008 and OECD Test Guideline No. 414, Prenatal Developmental Toxicity Study, Adopted by the Council on January 22nd 2001.

Study performance

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

The dose levels for study – 160, 400 and 1000 mg/kg/day – have been chosen with respect to the information given in the Study No. 67/09/18: Semi Dry Absorption (SDA) Product - Reproduction/Developmental Toxicity Screening Test (VUOS-CETA Report No. 1053, 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 euthanized, the uterine contents were examined and the foetuses were assessed for changes on soft tissues and skeleton.

Results

There were no unscheduled deaths of females during the study at any dose level.

No adverse changes of health condition and no clinical symptoms of intoxication were found in females at any dose level after administration of the test substance.

During the control of body weight increments and food consumption in pregnant females at all dose levels: 160, 400 and 1000 mg/kg/day, toxicologically significant treatment-related effects were not detected. Evaluation of uterine weights (incl. absolute and relative weight of uterus) did not reveal toxicologically significant treatment-related effects. Macroscopical structure of organs of pregnant females and values of reproduction parameters (number of females with live foetuses, number of live and dead foetuses, early and late resorptions and sex ratio of foetuses), were unaffected by treatment with the test substance.    

Test substance-related foetal mortality was not evident at any dose level. The foetal body weight was statistically insignificantly increased at all dose levels. Detailed necropsy of foetuses did not reveal increase of external and visceral variations and malformations at any dose level.

During examination of foetal skeleton delayed ossification of skeleton at all doses including control group was observed. The occurrence of structural skeletal variations could not be considered as teratogenic effect of the test substance on early prenatal development of organism in uterus because incidence of these findings was accidental or comparable with control group.

The NOAEL (No Observed Adverse Effect Level) for toxicity in PREGNANT FEMALES was established as 1000 mg/kg/day. This NOAEL value is based on no mortality of females, no changes in health condition status, no pathological findings in the dams, no dose related changes in reproduction parameters.

The NOAEL (No Observed Adverse Effect Level) for PRENATAL DEVELOPMENT was established as 1000 mg/kg/day. This NOAEL is based on no altered growth and no serious structural abnormality found in treated foetuses.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliability 1

Justification for classification or non-classification

According to the classification criteria the test substance, Product of Semi Dry Absorption (SDA) Product, is not classified.