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Toxicological information

Toxicity to reproduction

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Administrative data

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)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): BLANCOLEN HP (Disodiumhydroxy(sulfonate)acetate)
- Substance type: Sulfonic acid derivative
- Physical state: solid
- Analytical purity: 99.92%
- Impurities (identity and concentrations): not specified
- Lot/batch No.: 09102303
- Expiration date of the lot/batch: 09 December 2018
- Stability under test conditions: stable
- Storage condition of test material: room temperature

Test animals

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) x6-8 wks
- Weight at study initiation: (P) Males: 201-225 g; Females: 151-175 g;
- Fasting period before study: no
- Housing: limited access rodent facility
- Use of restrainers for preventing ingestion (if dermal): N/A
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 20 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): daily

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 10, 30 and 100 mg/mL
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated in RTC Study no. 92500 in the range of 5 to 150
mg/mL.
Linearity, accuracy and precision were within the limits stated in RTC SOPs for suspensions
(r > 0.98; accuracy 95-105%; precision CV<5%).
Stability was verified in the range of 10 to 100 mg/mL, and was found to be at least 6 hours
at room temperature.
The proposed formulation procedure for the test item was checked during the pre-treatment
period in the range of 10 to 100 mg/mL by chemical analysis (concentration and
homogeneity) to confirm that the method was acceptable.
Final results for all levels were within the acceptability limits stated in the RTC SOPs for
concentration (90-110%) and homogeneity (CV<10%).
Final results for stability were within the acceptability limits stated in RTC SOPs for
concentration (90-110%) and homogeneity (CV<10%).
Samples of the formulations prepared on Day 1 and during Week 4 were analysed to check
the homogeneity and concentration. Results of the analyses were within the limits of
acceptance stated in RTC SOPs for suspensions (90-110% for concentration and CV <10%
for homogeneity). The results of all these analyses, as well as a description of the analytical
method, are presented in Addendum III of this report.
Duration of treatment / exposure:
Males
Animals were 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 were adjusted once per week for each animal according to the last recorded
body weight.
Females
Animals were 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 were adjusted once per week for each
animal according to the last recorded body weight.

During the gestation period, dose volumes were 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 remained constant.
Frequency of treatment:
once a day (7/7)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
300 mg/kg/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
nominal in water
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: two times / day


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): N/A
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: N/A
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: N/A

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): N/A
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning from the first day of treatment and up to the
end of the mating period, until a positive identification of mating was made. The vaginal
smear data were examined to determine the following:
a) anomalies of the oestrous cycle;
b) the pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Litter observations:
As soon as possible, after parturition was considered complete (Day 0 or 1 post partum), all
pups (live and dead) were counted, sexed and live pups were identified.
Live pups were individually weighed on Days 1 and 4 post partum.
Pups dying during the lactation period were weighed before the despatch to necropsy.
Observations were performed once daily for all litters.
Postmortem examinations (parental animals):
Euthanasia
Parental animals killed for humane reasons and those that had completed the scheduled test
period were killed with carbon dioxide.
Pups that had completed the scheduled test period (Day 4 post partum) were euthanised by
intraperitoneal injection of Thiopenthal.
Parental males:
The males were killed after the mating of all females, after at least 28 days of treatment
period.
Parental females:
The females with live pups were killed on Day 4 post partum.
One female with total litter loss was killed one day after the occurrence of total litter loss.
The females which had not given birth 25 days after the positive identification of mating
were killed shortly after.

Necropsy
The clinical history of the males and females of the parental generation was studied and a
detailed post mortem examination was conducted (including examination of the external
surface and orifices).
Changes were noted, the requisite organs weighed (excluding animals sacrificed for humane
reasons or found dead) and the required tissue samples preserved in fixative and processed
for histopathological examination.
Females:
All females were examined also for the following:
a) number of visible implantation sites (pregnant animals);
b) number of corpora lutea (pregnant animals).
Uteri of females with no visible implantations were immersed in a 10-20% solution of
ammonium sulphide to reveal evidence of implantation.
Pups:
All pups found dead in the cage were examined for external and internal abnormalities.
All live pups were killed and examined for external abnormalities and sex confirmation by
gonadal inspection.

Organ weights (parental generation)
From all animals completing the scheduled test period the organs indicated in section 4.4.6
were dissected free of fat and weighed. The ratios of organ weight to terminal body weight
were calculated for each animal.

Tissues fixed and preserved
Samples of all the tissues listed in section 4.4.6 were fixed and preserved in 10% neutral
buffered formalin (except testes and epididymides which were fixed in modified Davidson's
fluid) from all animals.

Histopathological examination and staging of spermatogenic cycle
The tissues required for histopathological examination are listed in section 4.4.6. After
dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre
thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides were cut at 2-3 micrometre thickness and stained
with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous
epithelium (staging of spermatogenic cycle) was performed.
The examination was restricted to the following:
a) Tissues specified in section 4.4.6 from all animals in the control and high dose group
killed at term.
b) Tissues specified in section 4.4.6 from all animals killed during the treatment period.
c) All abnormalities in all groups.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the
significance of the differences amongst group means were assessed by Dunnett’s test or a
modified t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings was carried out by means of the nonparametric
Kolmogorov-Smirnov test if n was more than 5.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters.
Intergroup differences between the control and treated groups were assessed by the nonparametric
version of the Williams test. The criterion for statistical significance was p<0.05.
Reproductive indices:
Group mean values were calculated for all parameters. Data from females which did not
mate or non-pregnant and from dams without live young were excluded from group mean
calculations as considered appropriate by the Study Director.

The following reproductive indices were calculated:
Males
Copulatory Index (%) = no. of animals mated x 100
no. of animals paired
Fertility Index (%) = no. of males which induced pregnancy x 100
no. of males paired
Females
Copulatory Index (%) = no. of animals mated x 100
no. of animals paired
Fertility Index (%) = no. of pregnant females x 100
no. of females paired
Males and females
Copulatory Interval = Mean number of days between pairing and mating
Females
Pre-birth loss was calculated as a percentage from the formula:
(No. of visible implantations - total litter size at birth ) x 100
No. of visible implantations
Pup loss at birth was calculated as a percentage from the formula:
(Total litter size - live litter size) x 100
Total litter size
Cumulative pup loss on Day 4 post partum was calculated as a percentage from the formula:
(Total litter size at birth - live litter size at Day 4) x 100
Total litter size at birth
Sex ratios were calculated at birth and on Day 4 post partum and were presented as the
percentage of males per litter.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Remarks:
systemic toxicity
Effect level:
> 300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Dose descriptor:
NOAEL
Remarks:
reproduction and fertility
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance

Results: F1 generation

General toxicity (F1)

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

Effect levels (F1)

Remarks on result:
not determinable because of methodological limitations
Remarks:
Study design of OECd 421 is not suitable to determine NOAEL values of F1 gneration.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The effects of the test item, BLANCOLEN HP, 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.
Four females dosed at 1000 mg/kg/day were sacrificed for humane reasons on Day 21/22 of
gestation due to the presence of a number of clinical signs (pale appearance, decreased
activity, piloerection and/or cold to touch). At necropsy examination they were all pregnant.
Treatment-related changes in the kidneys, stomach, spleen and thymus were seen at post
mortem macroscopic and microscopic observations in these animals.
No significant changes and 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.
Mean oestrus cycle was slightly reduced in the females dosed at 1000 mg/kg/day
(approximately 2 in the 15 day pre-mating period) when compared to controls
(approximately 3). This effect was not considered to be adverse.
Mating performance, gestation, parturition, lactation, implantation, litter data and sex ratio
were unaffected by treatment.
Necropsy findings in pups did not reveal any treatment-related effect.
No relevant changes were detected at post mortem examination in surviving animals dosed at
1000 mg/kg/day and those from the other treated groups, when compared with controls.
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 systemic toxicity were
observed in some pregnant females dosed at 1000 mg/kg/day.
No changes were observed in males at any dose and in females dosed at 100 and 300
mg/kg/day.
Therefore, the dose level of 300 mg/kg/day may be considered the NOEL (No Observed
Effect Level) for systemic toxicity.
No adverse effects on sexual function and fertility nor in developmental parameters and
lactation were observed at any of the dose levels investigated.
The NOAEL (No Observed Adverse Effect Level) for reproduction and fertility is 1000
mg/kg/day.