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Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
SPF quality
Sex:
male/female
Details on test animals and environmental conditions:
Supplier: SPF breeding, VELAZ s.r.o., Koleč u Kladna Czech Republic, RČH CZ 21760118
Sex: males and virgin females
Age at start of the study: males, females: sexually adult, 7-9 weeks on arrival
Acclimatization: 5 days
Total number of animals: 48 males and 48 females (12 females and 12 males per group)
Housing conditions: SPF conditions according to internal SOP No.12
Animal per cage: 2 rats of the same sex in one cage in pre-mating period, during mating period – one male and one female in one cage, pregnant females – individually,
Food: complete pelleted diet for rats and mice in SPF breeding Altromin for Rats/Mice, Manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany
Water: drinking water ad libitum, quality corresponding to the Regulation No. 252/2004 of Czech Coll. of Law
Light cycle: 12 hour light / 12 hour dark
Microclimate: 22 +-3°C, relative humidity 30-70%
Bedding: sterilized soft wood fibers Lignocel
Route of administration:
oral: gavage
Vehicle:
DMSO
Details on exposure:
Doses: 125, 500, 1000 mg/kg of body weight /day

Administration
Parental males (totally 49 days of administration):
1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration ) → 29th day – 42nd day ( mating ) → 43rd day – 63rd day (administration period) → 64th day (necropsy)

Parental females:
1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre -mating period, administration ) → 29th day – 42nd day ( mating )→ gestation → lactation → day 12 post partum

Non-pregnant females (without evidence of copulation):
1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre -mating period, administration ) → 29th day – 42nd day ( mating ) → 25 days after the end of mating period

Non-pregnant females (with evidence of copulation):
1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre -mating period, administration ) → 29th day – 42nd day ( mating ) → 25th day after confirmed mating
Details on mating procedure:
Animals were mated from the 29th day of study. Mating 1: 1 (one male to one female) was used in this study. 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.
Analytical verification of doses or concentrations:
not specified
Frequency of treatment:
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 12th day of lactation,
males – after mating period – totally for 49 days,
nonpregnant females (mated females without parturition) – for 25 days after the confirmed mating,
non-mated females – for 25 days after the end of mating period
After the end of administration period the animals of were sacrificed.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 males / 12 females
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
Health Condition Control:
All rats were observed pre-experimentally to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. During the administration period they were examined for behaviour changes each day before, during application and immediately after application.

Body weight: The body weight of animals was recorded on automatic balances with group mean computing module on specified days. All animals were weighed immediately before euthanasia.
Weight increment was computed as a mean per group per day (in grams). Non-pregnant females (females without parturition) were not included in calculation of means in pregnancy and lactation period.

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 mean values were calculated for each week of the study (except the mating period). Food consumption for animal/day was calculated from mean values of each group. The same way of calculation of mean food consumption was used for females in pre-mating period. In pregnancy and lactation period mean individual values (grams/animal/day) were calculated for each week of the study. Mean food consumption for each group was calculated from individual values. Nonpregnant females (females without parturition) were not included in calculation of mean food consumption in pregnancy and lactation period.

Clinical Observations of Males and Females:
All rats were observed daily during the administration period.
This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day
(11.00 – 13.00 p.m.) – at the time of expectation of probable maximal effect of the test substance. Animals were observed in natural conditions in their cages.

Clinical Observation of Pups:
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 1 post-partum) and on the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.

Laboratory examinations
Examination of Vaginal Smears
Vaginal smears were made from the 1st till the 14th day of study (pre-exposure period) for monitoring of the oestrous cycle of females. Only females with regular cyclicity were put into the study.
Each morning in the mating period vaginal smears were prepared from all the mated females. These smears were examined for presence of spermatozoa.
Vaginal smears were made also on necropsy day to determine the stage of oestrous cycle. Vaginal smears were prepared and examined according to internal SOP No. M/74.

Biochemical Examination
Blood samples from the day 13 pups and the parental males and females were assessed for serum levels of thyroid hormone thyroxine (T4) by ELISA kit. Pup blood was pooled by litter (one male and one female pups).

Anogenital Distance (AGD) Measurement
The AGD of each pup was measured on day 4 of lactation. For measuring digital calliper was used. The AGD was normalized to a measure of pup size. Corrected AGD was calculated according to the formula: AGD divided by the cube root of body weight.

Nipples Examination
The presence of nipples in male pups were counted on day 13 of lactation.

Pathological Examination
During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. Organs for consequent histopathological examination were taken out and stored in containers with fixative (buffered 4% formaldehyde). Testes and epididymides were fixed in modified Davidson’s fixative.

Observation of Sperm
Oestrous cyclicity (parental animals):
Before beginning of treatment, oestrous cycle of all females was monitored. Vaginal smears of all females were monitored daily for two weeks. Only females with regular cyclicity were select for the reproduction part of study. No female with irregular cycle was detected.
Sperm parameters (parental animals):
In all males the following sperm parameters were examined: sperm motility and sperm morphology. Sperm specimens were prepared and examined according to internal SOP No. M/82.
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 solution. 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 was assessed from these samples. A smear from the sperm solution was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination.
Litter observations:
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 1 post-partum) and on 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):
At the end of study, the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected.
The absolute weights of testes, epididymis, prostate gland with seminal vesicles, 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.
From all adult males and females and one male and female day 13 pup from each litter thyroid glands were preserved in fixation medium. The thyroid weight was determined after fixation.

The tissues 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 with seminal vesicles, epididymis and testes (fixed in Davidson´s solution), cervix of uterus, ovaries, uterus (incl. uterine tube and cervix) and vagina. For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used.
The full histopathology of the preserved organs and tissues was performed for tvelve/five high dose and control males and females. Organs with macroscopical changes (liver and seminal vesicles) were examined also at the middle and low dose level groups.
Detailed histological examination was performed on testes of males (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure). Spermatogenesis and spermatogenic cycle were evaluated according to the related guidance.
Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female (dose level 1000 mg/kg bw) died after protracted parturition.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The differences in weight of animals on the first weighing before application are caused by the gradual sequencing of animals into the study.

Males: The body weight of all treated males was decreased in a dose-dependent manner compared with control group.
The body weight at the dose level 1000 mg/kg/day was statistically significantly decreased throughout the study.
Statistically significant differences in necropsy body weight were found in treated males at the dose levels 500 and 1000 mg/kg/day.
The body weight increments in treated males at the dose levels 500 and 1000 mg/kg/day were decreased during the 1st,2nd, 6th weeks of study and necropsy body weight compared to the control animals.

Females:
1) Before mating period
The differences in weight of animals on the first weighing before application are caused by
the gradual sequencing of animals into the study.
The mean body weight of treated females was decreased at the dose level 1000 mg/kg/day before mating period. Mean body weight increments of treated females were variable without dose-dependent significance.

2) Pregnancy
Females without parturition (non pregnant or females with abortion) were not included in the evaluation of mean body weight and body weight increments during pregnancy.
The mean body weight and body weight increment of all treated group was decreased than at the control group during whole pregnancy. At the dose level 125 and 1000 mg/kg/day the statistically significantly decreased body weight was recorded at the last week.

3) Lactation
The mean body weight of all treated mothers was significantly decreased during whole lactation period. Statistically significantly decreased necropsy body weight was found in females at the dose levels 500 and 1000 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Male: The mean food consumption was decreased at the dose levels 500 and 1000 mg/kg/day compared with the control animals.
Female:
1) Pre-mating period
The mean food consumption of treated females at the dose level 1000 mg/kg/day was lower than at the control group during the 1st and 2nd week of study.
2) Pregnancy
Females without parturition (non-pregnant or females with abortion) were not included in the evaluation of food consumption during pregnancy.
The mean food consumption of females at all dose levels was decreased during the whole pregnancy in the dose-dependent manner in the 1st and 3rd week of pregnancy.
3) Lactation
Only mothers (females with live pups born) were included in evaluation of food consumption during lactation period.
The mean food consumption of mothers at all dose levels was decreased in a dose-dependent manner compared with the control animals during lactation period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males: Blood samples from the all adult parental males were assessed for serum levels of thyroid hormone thyroxine (T4).
Mean concentration of T4 was significantly increased in males at the dose level 1000 mg/kg/day. Concentration of other dosed groups were similar to the control group.

Female: Blood samples from the all adult parental females were assessed for serum levels of thyroid hormone thyroxine (T4).
Mean concentration of females was similar to the control group.
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
In treated males of all dose levels no signs of diseases were recorded during the check-in and acclimatisation period. Piloerection was observed at the dose levels 500 mg/kg/day (from 7th to 23rd day of administration period) and 1000 mg/kg/day (from 7th day of administration period to the end of the study).
In treated females of all dose levels no signs of diseases were recorded during the check-in and acclimatisation period. Piloerection was observed at the dose level 500 mg/kg/day (from 7th to 23rd day of administration period) and 1000 mg/kg/day (from 7th day of administration period to the end study).
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males:
The incidence of affected males is expressed in numeric form and ranged in sequence of the dose levels of 0-125-500-1000 mg/kg/day further in the text. In males at the dose levels 125 and 500 mg/kg/day only livers and seminal vesicles were examined because histopathological examination of males at the dose level 1000 mg/kg/day showed severe changes only in these organs.
In 5-11-4-2 males no histological findings were diagnosed.
Atrophy was recorded in seminal vesicles of 0-1-5-6 males. Significant findings were observed in liver: apoptosis in 0-0-0-2 males, bile duct hyperplasia in 0-0-6-5 males, focal necrosis in 0-0-0-2 males and fibrosis in 0-0-4-1 males.
Histopathological findings in epididymis, kidneys and prostate gland were incidental, without treatment relationship: focal inflammation in epididymides of 2-/-/-1 males, spermatogranuloma in epididymides of 1-/-/-1 males, focal chronic inflammation in prostate gland in 1-/-/-0 male and hydronephrosis in kidneys of 4-/-/-0 males.

Females:
The incidence of affected females is expressed in numeric form and ranged in sequence of the dose levels of 0-125-500-1000 mg/kg/day further in the text. In females at the dose levels 125 and 500 mg/kg/day only livers were examined because histopathological examination of females at the dose level 1000 mg/kg/day showed severe changes only in this organ.

In 1-5-0-2 females no histological findings were diagnosed.
Significant findings were observed in liver: bile duct hyperplasia in 0-0-5-5 females, slight periductal fibrosis in 0-0-0-5 females, presence of hemosiderin in 0-0-0-2 females and focal necrosis in 0-0-1-2 females. All these hepatic lesions related to the test substance treatment.
Mild focal acute inflammation in the submucosa of glandular stomach was observed in 0-/-/-2 females. Origin of these findings is unclear and its relation to the test substance treatment is not probable.
No treatment-related changes were found in the kidneys: mild hydronephrosis in 3-/-/-2 females, solitary corticomedullary mineralization in 5-/-/-4 females and mild chronic pyelitis in 0-/-/-2 females.
Presence of different amount of the hemosiderin in the uterus mucosa and focal accumulation of lipophages and siderophages in the mesometrium of 11-/-/-5 females and mild focal fibrosis in the uterine submucosa of 2-/-/-1 females is related to previous gravidity. Hydrometra, observed in 0-/-/-4 females, was in relation to the status of their oestrous cycle.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Detected worse sperm motility and morphology in all treated groups in a dose-dependent manner and higher percentage of affected sperms compared to control animals. The most frequent change of sperm morphology was flattened head.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
All control females and treated females were mated: presence of sperm was found in all 12 females at the dose level 500 mg/kg/day and in the control group. Two females without presence of sperm were observed at the dose level 1000 and one female without presence of sperm was observed at the dose level 125 mg/kg/day.
The number of females achieving pregnancy was lower in the all treated groups. Duration of pregnancy was similar in all groups. The duration of mating of the females from the dose level 1000 mg/kg/day was longer in comparison with the other groups.
The number of females bearing live pups and females with live pups at day 4 and day 13 after parturition was the lowest in females treated at 1000 mg/kg/day. The number of females with total cannibalism was increased in a dose-dependent manner. Number off stillborn pups was the highest at the dose level 1000 mg/kg/day.
The numbers of corpora lutea and implantation in treated females were lower in comparison with the control animals

Fertility parameters:
Mating indexes of both sexes were lower in animals at the dose level 1000 mg/kg/day, Fertility index was slightly reduced at the all dose levels and Gestation index was lower at the dose level 1000 mg/kg/day.
Pre-implantation losses were higher at the dose level 125 mg/kg/day, post-implantation losses were higher at the dose level 500 mg/kg/day, post-natal losses were higher at the dose levels 500 and 1000 mg/kg/day.
Dose descriptor:
NOAEL
Effect level:
ca. 125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Critical effects observed:
yes
Lowest effective dose / conc.:
125 mg/kg bw/day (nominal)
System:
other: reproductive system males and females
Organ:
other: reproductive system
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
not specified
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The total number of live pups and total number of pups at first check of litter after parturition, on the 4th day and 13th day of lactation were decreased in all treated litters compared to the control animals with statistical significance at the dose level 1000 mg/kg/day.
The mean number of live pups per litter (first check of litter after parturition) was decreased at all dose levels.
The total number of live pups at the 4th day and 13th day was decreased at the all dose levels compared to the control animals in a dose-dependent manner. This difference was marked at the dose level 1000 mg/kg/day.
The total numbers of stillborn and dead pups were increased at the dose level 1000 mg/kg/day compared with control group.

Postnatal losses
Control group: Six stillborn pups from two litters were found on the first day after delivery and two pups from two litters were cannibalized during the first week of lactation period.
125 mg/kg/day: One stillborn pup was found on the first day after parturition and one pup was found dead on the fifth day after delivery.
Two pups from two litters were cannibalized during the first week of lactation.
500 mg/kg/day: Five stillborn pups from two litters were found on the first day after parturition and eleven pups from three litters was cannibalized during the first four days after delivery.
1000 mg/kg/day: Thirteen stillborn pups from three litters were found on the first day after parturition and ten pups from three litters were found dead during lactation.
Thirteen pups from three litters were cannibalized during lactation period.

Increased of the total number of cannibalized pups and mortality of pups were observed at the dose levels 500 and 1000 mg/kg/day compared with control group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean weight of litter was decreased with statistical significance at the dose level 1000 mg/kg/day during whole lactation period, at the dose level 500 mg/kg/day from the 4th to 13th day of lactation period and at the dose level 125 mg/kg/day only at first check of litter after parturition. These changes were caused by the lower number of pups in litters in comparison with control animals.
The mean body weight of pups of all treated group was lower in comparison with control during whole lactation period. Marked decreasing was recorded at the dose level 1000 mg/kg/day at first check of litter after parturition. Decreasing in the dose-dependent manner was measured on the 12th day and 13th day of lactation.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Biochemical examination – hormone T4: No significant differences were recorded in pups from treated groups in comparison with the control pups.
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Nipples and anogenital distance
The presence of nipples in male pups was observed on day 13 of lactation - the presence of nipples in male pups was recorded in male pups at the dose levels 125 and 500 mg/kg/day.
The anogenital distance (AGD) of each pup was measured on day 4 of lactation.
No significant differences were recorded in pups from treated groups in comparison with the control pups.
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Reproductive effects observed:
yes
Lowest effective dose / conc.:
125 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
The NOAEL (No Observed Adverse Effect Level) for the REPRODUCTION and DEVELOPMENT was established as 125 mg/kg body weight/day.
Executive summary:

The ability of treated animals to mate and to produce viable offspring was influenced by the test substance treatment. The dose-response increase in prenatal and postnatal mortality of offspring was recorded.

Decrease of body weight and food consumption of treated males and females was observed. Quality of sperms of parental males were adversely influenced by the test substance treatment.Pathological changes associated with the test substance treatment were found in liver in males and females. Changes in reproductive parameters was observed in all treated animals. Development of pups were negatively influenced by the test substance treatment. Increased mortality of pups and consequently decreased survival index were recorded.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
125 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Acceptable data for assess the endpoint.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
125 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Acceptable for assess the endpoint.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results of the test performed according to OECD 421, the tested substance showed some effects both on parental animales and litters.
The NOAEL (No Observed Adverse Effect Level) for the REPRODUCTION and DEVELOPMENT was established
as 125mg/kg body weight/day.