Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco, Lecco, Italy
- Age at study initiation: (P) 6 to 8 wks;
- Weight at study initiation: (P) Males: 201-225 g; Females: 151-175 g;
- Housing: The animals will be housed in a limited access rodent facility.
- Diet (e.g. ad libitum): laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): poor water solubility of the test item
- Amount of vehicle (if gavage): 5 mL/kg body weight
Details on mating procedure:
- M/F ratio per cage: 5/5
- Length of cohabitation: 14 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy:
Mating will be monogamous (one male to one female). A vaginal smear will be taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).
The female will be paired with the same male until positive identification occurs or 14 days have elapsed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations are verified by HPLC.
Duration of treatment / exposure:
Males
Animals will be dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing up to the day before necropsy.
Dose volumes will be adjusted once per week for each animal according to the last recorded body weight.

Females
Animals will be dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum or the day before sacrifice. Dose volumes will be adjusted once per week for each animal according to the last recorded body weight.
During the gestation period, dose volumes will be calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes will remain constant.
Frequency of treatment:
once a day
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
nominal in diet
No. of animals per sex per dose:
10 males and 10 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: on the basis of information from previous toxicity studies
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: yes

BODY WEIGHT: Yes
- Time schedule for examinations: yes

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data
Oestrous cyclicity (parental animals):
Gestation length will be calculated as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring are first detected in the cage will be considered Day 0 post partum.
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations:
The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) will be performed.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively.

Adrenal glands
Brain (cerebrum, cerebellum, medulla/pons)
Coagulating glandsa
Clitorids
Epididymides
Kidneys
Liver
Ovaries
Penis with prepurtial glands
Prostate gland
Spleen
Seminal vesicles with coagulating glands
Testes
Thymus
Thyroid
Uterus with cervix
Vagina
Postmortem examinations (offspring):
All pups found dead in the cage or sacrificed for humane reasons will be examined for external and internal abnormalities.
All live pups will be killed and examined for external abnormalities and sex confirmation by gonadal inspection. All pups with abnormalities will be retained in an appropriate fixative at the discretion of the Study Director.
Statistics:
Standard deviations will be calculated as appropriate. For continuous variables the significance of the differences amongst group means will be assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings will be carried out by means of the non-parametric Kolmogorov-Smirnov test if n is more than 5.
The non-parametric Kruskal-Wallis analysis of variance will be used for the other parameters. Intergroup differences between the control and treated groups will be assessed by the non-parametric version of the Williams test. The criterion for statistical significance will be p<0.05.
Further tests will be used as considered appropriate. Details of all tests used and the data to which they are applied will be included in the Final Report.
Reproductive indices:
The following reproductive indices will be calculated:

Males
Copulatory Index (%) = (no. of animals mated/no. of animals paired) x 100

Fertility Index (%) = (no. of males which induced pregnancy/no. of males paired) x 100

Females
Copulatory Index (%) = (no. of animals mated/no. of animals paired) x 100

Fertility Index (%) = (no. of pregnant females/no. of females paired) x 100

Males and females
Pre coital Interval = Mean number of days between pairing and mating

Females
Pre-birth loss will be calculated as a percentage from the formula:
((No. of visible implantations - total litter size at birth )/No. of visible implantations) x 100

Pup loss at birth will be calculated as a percentage from the formula:
((Total litter size - live litter size)/Total litter size) x 100

Cumulative pup loss on Day 4 post partum will be calculated as a percentage from the formula:
((Total litter size at birth - live litter size at Day 4)/Total litter size at birth) x 100

Pre-implantation loss will be calculated as a percentage from the formula:
((No. of corpora lutea – no. of implantation)/No of corpora lutea) x 100

Sex ratios will be calculated at birth and on Day 4 post partum and will be presented as the percentage of males per litter.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation and/or piloerection were observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant changes in body weight were observed in the treated animals when compared to controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significant changes in body weight were observed in the treated animals when compared to controls.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Test substance intake: No effects on food consumption were observed in the treated animals of either sexes.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No treatment-related anomalies were noted in the oestrus cycle of the treated females when compared to controls.
Reproductive function: sperm measures:
not specified
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
A copulatory index of 100% was observed for both sexes from all groups. A fertility index of 80% was found in the high dose group.
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Mortality and fate of females
One high dose female (no. 94680077) was found on Day 22, during parturition. No clinical signs
were observed in this animal during the study.
At post mortem examination, swollen liver and red staining of the urogenital region were observed.
No cause of death was identified in this animal, due to the limited findings observed at the
macroscopic and microscopic observations.
All females mated. Only two high dose females (nos. 94680065 and 94680069) were sacrificed 26
days post coitum and found not pregnant at necropsy.
The number of females with live pups on Day 4 post partum was: 10 in the control group, 9 in the
low dose group, 10 in the mid-dose group, 7 in the high dose group. Total litter loss was observed
in a single female from the low dose group (no. 94680037).
Clinical signs
Salivation and/or piloerection were observed in male animals from the mid- and high dose groups,
with a dose-related incidence during the whole study.
Salivation was also observed in the females from all treated groups. Incidence increased with the
dose level, showing a peak during the post coitum phase of the study.
No other signs of toxicological significance were observed.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight and body weight gain
No significant changes in body weight were observed in the treated animals when compared to
controls.
Food consumption
No effects on food consumption were observed in the treated animals of either sexes.


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
no data available

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No treatment-related anomalies were noted in the oestrus cycle of the treated females when
compared to controls.
The mean pre-coital interval and the number of copulation plugs were similar between control and
treated groups.


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
no data available


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
All females mated. However, two females of the high dose group (nos. 94680065 and 94680069)
were found not pregnant.
A copulatory index of 100% was observed for both sexes from all groups. A fertility index of 80%
was found in the high dose group.
A reduction in the number of females with live pups on Day 4 post partum was observed in the
high dose females (7) when compared to the other groups.


ORGAN WEIGHTS (PARENTAL ANIMALS)
Treatment-related findings were observed at post mortem microscopic examination in the liver
(hepatocytic hypertrophy) of most of the females dosed at 1000 mg/kg/day and in two females
dosed at 300 mg/kg/day. These confirmed the increases in liver weight seen in the females of the
same dose groups.


GROSS PATHOLOGY (PARENTAL ANIMALS)
Adrenal glands
Brain(cerebrum, cerebellum, medulla/pons)
Coagulating glandsa
Clitorids
Epididymides
Kidneys
Liver
Ovaries
Penis with prepurtial glands
Prostate gland
Spleen
Seminal vesicles with coagulating glands
Testes
Thymus
Thyroid
Uterus with cervix
Vagina
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Remarks:
reproduction and fertility
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Litter size, Sex ratios, Pre-weaning clinical signs of pups, Necropsy findings in pups
Reproductive effects observed:
no
Conclusions:
The effects of the test item, Bruggolen M12, on fertility, pregnancy and early lactation of the
offspring were investigated when administered orally to male and female Sprague Dawley rats.
Groups of 10 males and 10 females received the test item by gavage at dosages of 100, 300 and
1000 mg/kg/day.
One female dosed at 1000 mg/kg/day was found dead on Day 22, during parturition. No cause of
death was identified in this animal, due to the limited findings (swollen liver and red staining of the
urogenital region) observed at the macroscopic and microscopic observations.
With the exception of salivation, seen in the mid- and high dose animals, no treatment-related
effects were detected in males and in the remaining females from the high dose and the other
treated groups during the in vivo phase.
No treatment-related anomalies were noted in the oestrus cycle of the treated females when
compared to controls.
A copulatory index of 100% was observed for both sexes from all groups. However, a fertility
index of 80% was found in the high dose group.
Parturition, lactation, implantation, litter data and sex ratio did not show any changes of
toxicological relevance.
Necropsy findings in pups did not reveal any treatment-related effect.
Treatment-related findings were observed at post mortem microscopic examination in the liver
(hepatocytic hypertrophy) of most of the females dosed at 1000 mg/kg/day and in two females
dosed at 300 mg/kg/day. These confirmed the increases in liver weight seen in the females of the
same dose groups.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to
the integrity of the various cell types within the different stages and a regular layering in the
germinal epithelium was described.
On the basis of the above results, treatment-related effects indicating some systemic toxicity
(mainly in the liver) were observed in pregnant females dosed at 1000 mg/kg/day. These changes
were also observed in 2 females dosed at 300 mg/kg/day.
No significant changes were observed in males at any dose and in females dosed at 100
mg/kg/day.
Therefore, the dose level of 1000 mg/kg/day may be considered the NOAEL (No Observed
Adverse Effect Level) for systemic toxicity for males and the dose level of 300 mg/kg/day the
NOAEL for females.
Although fertility index resulted slightly reduced in the high dose group (80%), no other effects on
sexual function or in developmental parameters and lactation were observed in animals from this
and the other dose groups. Therefore this reduction was not considered adverse.
The NOAEL (No Observed Adverse Effect Level) for reproduction and fertility is 1000
mg/kg/day.
Executive summary:

The effects of the test item, Bruggolen M12, on fertility, pregnancy and early lactation of the

offspring were investigated when administered orally to male and female Sprague Dawley

rats. Groups of 10 males and 10 females received the test item by gavage at dosages of 100,

300 and 1000 mg/kg/day.

One female dosed at 1000 mg/kg/day was found dead on Day 22, during parturition. No

cause of death was identified in this animal, due to the limited findings (swollen liver and red

staining of the urogenital region) observed at the macroscopic and microscopic observations.

With the exception of salivation, seen in the mid- and high dose animals, no treatmentrelated

effects were detected in males and in the remaining females from the high dose and

the other treated groups during the in vivo phase.

No treatment-related anomalies were noted in the oestrus cycle of the treated females when

compared to controls.

A copulatory index of 100% was observed for both sexes from all groups. However, a

fertility index of 80% was found in the high dose group.

Parturition, lactation, implantation, litter data and sex ratio did not show any changes of

toxicological relevance.

Necropsy findings in pups did not reveal any treatment-related effect.

Treatment-related findings were observed at post mortem microscopic examination in the

liver (hepatocytic hypertrophy) of most of the females dosed at 1000 mg/kg/day and in two

females dosed at 300 mg/kg/day. These confirmed the increases in liver weight seen in the

females of the same dose groups.

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle

and to the integrity of the various cell types within the different stages and a regular layering

in the germinal epithelium was described.

On the basis of the above results, treatment-related effects indicating some systemic toxicity

(mainly in the liver) were observed in pregnant females dosed at 1000 mg/kg/day. These

changes were also observed in 2 females dosed at 300 mg/kg/day.

No significant changes were observed in males at any dose and in females dosed at 100

mg/kg/day.

Therefore, the dose level of 1000 mg/kg/day may be considered the NOAEL (No Observed

Adverse Effect Level) for systemic toxicity for males and the dose level of 100 mg/kg/day

the NOAEL for females.

Although fertility index resulted slightly reduced in the high dose group (80%), no other

effects on sexual function or in developmental parameters and lactation were observed in

animals from this and the other dose groups. Therefore this reduction was not considered

adverse.

The NOAEL (No Observed Adverse Effect Level) for reproduction and fertility is 1000

mg/kg/day.

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:
guideline study;
GLP compliant study.
Additional information

Justification for selection of Effect on fertility via oral route:
Experimental study result available.

Effects on developmental toxicity

Description of key information
OECD 421:
NOAEL (male) > 1000 mg/kg bw/day
NOAEL (female) > 100 mg/kg bw/day
NOAEL (reproduction and fertility) > 1000 mg/kg bw/day

Toxicity to reproduction: other studies

Additional information

Result of the OECD 421 test:

The dose level of 1000 mg/kg/day may be considered the NOAEL (No Observed

Adverse Effect Level) for systemic toxicity for males and the dose level of 100 mg/kg/day the

NOAEL for females.

Although fertility index resulted slightly reduced in the high dose group (80%), no other effects on

sexual function or in developmental parameters and lactation were observed in animals from this

and the other dose groups. Therefore this reduction was not considered adverse.

The NOAEL (No Observed Adverse Effect Level) for reproduction and fertility is 1000

mg/kg/day.

Justification for classification or non-classification

The results of the conducted screening tests do not give reason to the assumption, that the substance has to be classified into one of the categories of the hazard class "Reproductive toxicity".