Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
other: read across from analogue substance
Adequacy of study:
key study
Study period:
From 2013, August 16 to 2014, April 2
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
other: read across from analogue substance
Adequacy of study:
key study
Study period:
From 2013, August 16 to 2014, April 2
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
test performed on analogue substance
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March 1996
Deviations:
yes
Remarks:
Devations are described below, The study integrity was not adversely affected by the deviations.
Principles of method if other than guideline:
List of protocol deviations:
1. On Day 4 of lactation, no clinical observations were registered in the computer for pup 5 of litter
56 (Group 2).
Evaluation: Sufficient information available. Moreover, no findings were noted on Day 3 and 5,
and its body weight was determined on Day 4.
2. Analysis of stability of the test substance under test conditions was not performed as part of
this study, since Total Organic Carbon (TOC) analysis is a general method that does not
distinguish original substance from hydrolysis product(s).
Evaluation: Data from the sponsor indicated stability in water for >24 hours at a concentration
of 5%. As all formulations (2, 6 and 20%) were prepared daily within 4 hours prior to dosing, it
can be assumed that the formulations under the current study conditions were also sufficiently
stable.
3. Carbon analysis of formulations were recorded electronically using Shimadzu TOC-Control V
version 2.10 (Shimadzu, Kyoto, Japan).
Evaluation: omission from the protocol. Appropriate method was used.
GLP compliance:
yes (incl. QA statement)
Limit test:
yes
Species:
rat
Strain:
other: Crl:WI(Han) (outbred, SPF-Quality).
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for
general and reproduction toxicity studies. WIL Research Europe B.V. has
general and reproduction/developmental historical data in this species
from the same strain and source. This animal model has been proven to
be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test system Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and non-pregnant
females and untreated animals were used at initiation of the study.

Source F0: Charles River Deutschland, Sulzfeld, Germany.

Age at start F0-treatment: Approximately 11 weeks.

Number of F0-animals 40 females and 40 males.

Acclimatization F0: At least 5 days prior to start of treatment.

Health inspection F0: Upon receipt of the animals.

Randomization F0: Prior to commencement of treatment, by computer-generated random
algorithm according to body weight, with all animals within ± 20% of the
sex mean.

Identification F0: Earmark and tattoo.

Mating procedures: Following a minimum of 14 days of exposure for the males and females,
one female was cohabitated with one male of the same treatment group,
avoiding sibling mating. Detection of mating was confirmed by evidence
of sperm in the vaginal lavage or by the appearance of an intravaginal
copulatory plug. This day was designated Day 0 post-coitum. Once
mating occurred, the males and females were separated.

Parturition: The females were allowed to litter normally. Day 1 of lactation was
defined as the day when a litter was found completed (i.e. membranes
and placentas cleaned up, nest build up and/or feeding of pups started).
Females that were littering were left undisturbed.

Number of pups: 430 pups.

Identification of pups: On Day 1 of lactation, all pups were individually identified by means of
subcutaneous injection of Indian ink.

Conditions: Environmental controls for the animal room were set to maintain 18 to
24°C, a relative humidity of 40 to 70%, approximately 15 room air
changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to
these conditions were maintained in the raw data and had no effect on
the outcome of the study.

Accommodation
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic
cages (MIV type, height 18 cm).

Mating: Females were caged together with males on a one-to-one-basis in
Macrolon plastic cages (MIII type, height 18 cm).

Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type,
height 18 cm) with a maximum of 5 animals/cage. Females were
individually housed in Macrolon plastic cages (MIII type, height 18 cm).

Lactation: Pups were kept with the dam until termination in Macrolon plastic cages
(MIII type, height 18 cm). During locomotor activity monitoring of the dams
the pups were kept warm in their home cage using bottles filled with warm
water. In order to avoid hypothermia of pups, pups were not left without
their dam or a bottle filled with warm water for longer than 30-40 minutes.
General Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil,
France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm.
Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
During locomotor activity monitoring, animals were housed individually in a
Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x
20.3 cm) without cage-enrichment, bedding material, food and water.
Diet Free access to pelleted rodent diet (SM R/M-Z from SSNIFF®
Spezialdiäten GmbH, Soest, Germany).

Water: Free access to tap-water.
Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or
nutrients was performed according to facility standard procedures. There were no findings that could
interfere with the study.
Route of administration:
oral: gavage
Details on route of administration:
Formulations were placed on a
magnetic stirrer during dosing.
Vehicle:
water
Details on oral exposure:
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to
the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
As the validated method was not finalized yet, formulation samples were collected during in-life (31
October 2013) and stored in the freezer at ≤-15°C until analyses. However, as the NPOC method was
not suited to measure the test substance in these frozen samples, additional samples were collected
after the in-life phase (19 December 2013). These formulations were prepared in the same manner as
used during the study.
For quantitative analysis of the test substance in formulation, Total Organic Carbon (TOC) analysis
was used (see project 503334). Samples of formulations were analyzed for homogeneity (highest and
lowest concentration) and accuracy of preparation (all concentrations). Stability could not be
determined since TOC analysis is a general method that does not distinguish original substance from
hydrolysis product(s); therefore results on stability assessment were not reported.
Accuracy of preparation was considered acceptable if mean measured concentrations were 90-110%
of target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during
mating, and up to the day prior to scheduled necropsy. Females were
exposed for 41-47 days, i.e. during 2 weeks prior to mating, during
mating, during post-coitum, and during at least 4 days of lactation (up to
the day prior to scheduled necropsy). Female no. 47 (Group 1) was not
dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day
with a maximum of 6 hours difference between the earliest and latest
dose.
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animal per sex per dose
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
Parental animals
Mortality / Viability: At least twice daily.

Clinical signs: Daily from treatment onwards up to the day prior to necropsy, detailed
clinical observations were made in all animals, at least 1 hour (± 30 min)
after dosing. Once prior to start of treatment and at weekly intervals
during the treatment period this was also performed outside the home
cage in a standard arena. The time of onset, grade and duration of any
observed sign was recorded.

Functional Observations The following tests were performed on the selected 5 animals/sex/group:
- hearing ability
- pupillary reflex
- static righting reflex
- grip strength
- locomotor activity (recording period: 1-hour under normal laboratory
light conditions, using a computerized monitoring system, Kinder
Scientific LLC, Poway, USA). Total movements and ambulations
were reported.

Body weights: Males and females were weighed on the first day of exposure and weekly
thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and
20 post-coitum and during lactation on Days 1 and 4.

Food consumption: Weekly, except for males and females which were housed together for
mating and for females without evidence of mating. Food consumption of
mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum
and on Days 1 and 4 of lactation.

Water consumption: Subjective appraisal was maintained during the study, but no quantitative
investigation was introduced as no treatment related effect was
suspected.

General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and
delivery day were recorded. Pregnant females were examined to detect
signs of difficult or prolonged parturition, and cage debris of pregnant
females was examined to detect signs of abortion or premature birth. Any
deficiencies in maternal care (such as inadequate construction or
cleaning of the nest, pups left scattered and cold, physical abuse of pups
or apparently inadequate lactation or feeding) were examined.

Pups:

Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily
thereafter were determined. If possible, defects or cause of death were
evaluated.

Clinical signs: At least once daily, detailed clinical observations were made for all
animals.

Body weights: Live pups were weighed on Days 1 and 4 of lactation.

Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Clinical laboratory investigations:
Blood samples were collected at the end of the treatment period on the day of scheduled necropsy
from the selected 5 animals/sex/group under anaesthesia using isoflurane (Abbott B.V., Hoofddorp,
The Netherlands) between 7.00 and 10.30 a.m.

Haematology:
White blood cells (WBC) 109/L
Differential leucocyte count:
Neutrophils %WBC
Lymphocytes %WBC
Monocytes %WBC
Eosinophils %WBC
Basophils %WBC
Red blood cells 1012/L
Reticulocytes %RBC
Red blood cell distribution width (RDW) %
Haemoglobin mmol/L
Haematocrit L/L
Mean corpuscular volume (MCV) fL
Mean corpuscular haemoglobin (MCH) fmol
Mean corpuscular haemoglobin concentration (MCHC) mmol/L
Platelets 109/L

Prothrombin Time (PT) s
Activated Partial Thromboplastin Time (APTT) s

Clinical biochemistry:
Alanine aminotransferase (ALAT) U/L
Aspartate aminotransferase (ASAT) U/L
Alkaline Phosphatase (ALP) U/L
Total protein g/L
Albumin g/L
Total Bilirubin μmol/L
Bile acids μmol/L
Urea mmol/L
Creatinine μmol/L
Glucose mmol/L
Cholesterol mmol/L
Sodium mmol/L
Potassium mmol/L
Chloride mmol/L
Calcium mmol/L
Inorganic Phosphate (Inorg. Phos) mmol/L
Sacrifice and pathology:
Pathology
Necropsy parental animals
All males and the selected 5 females/group were deprived of food overnight (with a maximum of 24
hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of
food.
All animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands)
and subsequently exsanguinated and subjected to a full post mortem examination, with special
attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were
recorded.

The numbers of former implantation sites and corpora lutea were recorded for all paired females.

Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral
phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
Selected 5 animals/sex/group:
Identification marks: not processed Ovaries
Adrenal glands (Pancreas)
(Aorta) Peyer's patches [jejunum, ileum] if detectable
Brain - cerebellum, mid-brain, cortex Pituitary gland
Caecum Preputial gland
Cervix Prostate gland
Clitoral gland Rectum
Colon (Salivary glands - mandibular, sublingual)
Coagulation gland Sciatic nerve
Duodenum Seminal vesicles
Epididymides 1 Skeletal muscle
Eyes (with optic nerve (if detectable) and Harderian
gland) 1
(Skin)
Spinal cord -cervical, midthoracic, lumbar
Female mammary gland area Spleen
Femur including joint Sternum with bone marrow
Heart Stomach
Ileum Testes 1
Jejunum Thymus
Kidneys Thyroid including parathyroid if detectable
(Lacrimal gland, exorbital) (Tongue)
(Larynx) Trachea
Liver Urinary bladder
Lung, infused with formalin Uterus
Lymph nodes - mandibular, mesenteric Vagina
(Nasopharynx) All gross lesions
(Esophagus)
All remaining animals and females which failed to deliver:
Cervix Preputial gland
Clitoral gland Prostate gland
Coagulation gland Seminal vesicles
Epididymides Testes
Female mammary gland area Uterus
Ovaries Vagina

Identification marks: not processed All gross lesions

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of
toxicity were noted at macroscopic examination.

Necropsy pups
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation. All pups
were sexed and descriptions of all external abnormalities were recorded.

Organ weights
Terminal body weight were recorded from all males and the selected 5 females/sex/group. The
following organ weights were recorded from the following animals on the scheduled day of necropsy:
Selected 5 animals/sex/group:
Adrenal glands Spleen
Brain Testes
Epididymides Thymus
Heart Uterus (including cervix)
Kidneys Prostate1
Liver Seminal vesicles including coagulating glands
Ovaries Thyroid including parathyroid


All remaining males:
Epididymides
Testes

Histotechnology
All organ and tissue samples, as defined under Histopathology (following section), were processed,
embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin
(Klinipath, Duiven, The Netherlands).
From the selected 5 males of the control and high dose group, and all males suspected to be infertile,
additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were
processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The
Netherlands).

Histopathology
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 and all males
suspected to be infertile or which died before mating to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The heart of all selected 5 males of Groups 2 and 3 and the adrenal glands of all selected 5
females of Groups 2 and 3, based on (possible) treatment-related changes in these organs in
Group 4.
- The reproductive organs of all males that failed to sire and all females that failed to deliver
healthy pups:


All abnormalities were described and included in the report. An attempt was made to correlate gross
observations with microscopic findings.
A peer review on the histopathology data was performed by a second pathologist.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed tofollow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to
determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data
(scores) in the summary tables. Test statistics were calculated on the basis of exact values for means
and pooled variances. Individual values, means and standard deviations may have been rounded off
before printing. Therefore, two groups may display the same printed means for a given parameter, yet
display different test statistics values.
Clinical signs:
no effects observed
Description (incidence and severity):
No toxicologically significant clinical signs were noted during the observation period.
Incidental findings that were noted included piloerection and rales. These findings occurred within the
range of background findings to be expected for rats of this age and strain which are housed and
treated under the conditions in this study. At the incidence observed, these were considered signs of
no toxicological relevance.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in body weights and body weight gain were noted.
The statistically significant changes in body weight gain noted for males at 100 mg/kg were not
considered toxicologically relevant as no dose response relationship was observed and the individual
values were within normal limits.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after allowance for body weight was similar between treated and control
animals.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
The statistically significantly decreased prothrombin time for males treated at 300 mg/kg was
considered to be of no toxicological relevance as this occurred in the absence of a treatment-related
distribution and remained within the range considered normal for rats of this age and strain.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Clinical biochemistry parameters of treated rats were considered not to have been affected by
treatment up to 1000 mg/kg.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected
animals.
The variation in motor activity did not indicate a relation with treatment.
All groups showed a similar habituation profile with very high activity in the first interval that decreased
over the duration of the test period.
The statistically significant increase noted for number of ambulations for males at 100 mg/kg was not
considered toxicologically relevant as the individual values were within normal limits and no dose
response relationship was seen.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, increased weights (absolute and relative) of seminal vesicles and uterus were noted.
In addition, relative kidneys weights were higher for males treated at 300 and 1000 mg/kg.
The statistically significantly decreased absolute liver weights for males treated at 100 mg/kg was
considered to be of no toxicological relevance as this occurred in the absence of a treatment-related
distribution and remained within the range considered normal for rats of this age and strain.
Female number 69 (treated at 300 mg/kg) showed an enlarged liver macroscopically which was
confirmed by a high liver weight. At this single occurrence and without a dose response, it was not
considered toxicologically relevant.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have
arisen as a result of treatment.
The incidence of incidental findings among control and treated animals was within the background
range of findings that are encountered among rats of this age and strain, and did not show a doserelated
incidence trend.
In addition, one female (no. 64) treated at 300 mg/kg had cannibalised one of her pups during transfer
from the animal room to the necropsy room. At this single occurrence, it was not considered
toxicologically significant.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Possible treatment-related findings were recorded in the heart of males and adrenal glands of
females.
Heart, males:
- an increase in severity (slight degree) of mononuclear inflammation in 1/5 males treated at 1000
mg/kg, compared to minimal degrees in 1/5 males treated at 0 mg/kg, in 1/5 males at 100 mg/kg and
in 1/5 males at 300 mg/kg.
- an increase in incidence and/or severity of myofiber degeneration (2/5, up to slight) in males treated
at 1000 mg/kg, compared to minimal degree in 1/5 males treated at 300 mg/kg and absence of this
finding in control males and males treated at 100 mg/kg.
Adrenal glands, females:
an increase in incidence of vacuolation of the zona glomerulosa in 4/5 females treated at 1000 mg/kg,
compared to 1/5 females treated at 0 mg/kg, 1/5 females at 100 mg/kg and 1/5 females at 300 mg/kg.
The remainder of microscopic findings recorded, were within the normal range of background
pathology encountered in Wistar (Han) rats of this age.
In each group one or two females were not pregnant. No treatment-related findings were recorded in
the reproductive organs of those females and their matching males.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Conclusions:
Treatment with analogue substance with oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg revealed parental toxicity for males at 1000 mg/kg. Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: 300 mg/kg for males, and at least 1000 mg/kg for females
Executive summary:

Analogue substance 1 was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks

prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41-47 days). Formulation analysis showed that the formulations were prepared accurately and homogenously.

Parental results:

Parental toxicity was observed for males at 1000 mg/kg. This consisted of a subtle increase in severity of mononuclear inflammation and in incidence and/or severity of myofiber degeneration in the heart.

Both findings were slightly above the range of background findings of Wistar (Han) rats of this age.

Therefore a possible test item-related effect cannot be excluded.

For females at 1000 mg/kg, an increase in incidence of vacuolation of the zona glomerulosa of the adrenal glands was noted. However, based on the minimal degree and absence of other test itemrelated findings in the females, this finding was considered not to be adverse.

At 1000 mg/kg, increased weights (absolute and relative) of seminal vesicles and uterus were noted.

In addition, relative kidneys weights were higher for males treated at 300 and 1000 mg/kg. In the absence of corroborative findings in blood parameters and histopathological examination, these changes were not considered toxicologically significant.

No treatment-related changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, and macroscopic examination).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March 1996
Deviations:
yes
Remarks:
Devations are described below, The study integrity was not adversely affected by the deviations.
Principles of method if other than guideline:
List of protocol deviations:
1. On Day 4 of lactation, no clinical observations were registered in the computer for pup 5 of litter 56 (Group 2).
Evaluation: Sufficient information available. Moreover, no findings were noted on Day 3 and 5, and its body weight was determined on Day 4.
2. Analysis of stability of the test substance under test conditions was not performed as part of this study, since Total Organic Carbon (TOC) analysis is a general method that does not distinguish original substance from hydrolysis product(s).
Evaluation: Data from the sponsor indicated stability in water for >24 hours at a concentration of 5%. As all formulations (2, 6 and 20%) were prepared daily within 4 hours prior to dosing, it can be assumed that the formulations under the current study conditions were also sufficiently stable.
3. Carbon analysis of formulations were recorded electronically using Shimadzu TOC-Control V version 2.10 (Shimadzu, Kyoto, Japan).
Evaluation: omission from the protocol. Appropriate method was used.
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Reference substance name:
Castor oil, sulfated, sodium salt
EC Number:
269-123-7
EC Name:
Castor oil, sulfated, sodium salt
Cas Number:
68187-76-8
Molecular formula:
Not applicable
IUPAC Name:
Castor Oil, sulfated, sodium salt
Test material form:
liquid

Test animals

Species:
rat
Strain:
other: Crl:WI(Han) (outbred, SPF-Quality
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for
general and reproduction toxicity studies. WIL Research Europe B.V. has
general and reproduction/developmental historical data in this species
from the same strain and source. This animal model has been proven to
be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test system Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and non-pregnant
females and untreated animals were used at initiation of the study.

Source F0: Charles River Deutschland, Sulzfeld, Germany.

Age at start F0-treatment: Approximately 11 weeks.

Number of F0-animals 40 females and 40 males.

Acclimatization F0: At least 5 days prior to start of treatment.

Health inspection F0: Upon receipt of the animals.

Randomization F0: Prior to commencement of treatment, by computer-generated random
algorithm according to body weight, with all animals within ± 20% of the
sex mean.

Identification F0: Earmark and tattoo.

Parturition: The females were allowed to litter normally. Day 1 of lactation was
defined as the day when a litter was found completed (i.e. membranes
and placentas cleaned up, nest build up and/or feeding of pups started).
Females that were littering were left undisturbed.

Number of pups: 430 pups.

Identification of pups: On Day 1 of lactation, all pups were individually identified by means of
subcutaneous injection of Indian ink.

Conditions: Environmental controls for the animal room were set to maintain 18 to
24°C, a relative humidity of 40 to 70%, approximately 15 room air
changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to
these conditions were maintained in the raw data and had no effect on
the outcome of the study.

Accommodation
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic
cages (MIV type, height 18 cm).

Mating: Females were caged together with males on a one-to-one-basis in
Macrolon plastic cages (MIII type, height 18 cm).

Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type,
height 18 cm) with a maximum of 5 animals/cage. Females were
individually housed in Macrolon plastic cages (MIII type, height 18 cm).

Lactation: Pups were kept with the dam until termination in Macrolon plastic cages
(MIII type, height 18 cm). During locomotor activity monitoring of the dams
the pups were kept warm in their home cage using bottles filled with warm
water. In order to avoid hypothermia of pups, pups were not left without
their dam or a bottle filled with warm water for longer than 30-40 minutes.
General Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil,
France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm.
Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
During locomotor activity monitoring, animals were housed individually in a
Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x
20.3 cm) without cage-enrichment, bedding material, food and water.
Diet Free access to pelleted rodent diet (SM R/M-Z from SSNIFF®
Spezialdiäten GmbH, Soest, Germany).

Water: Free access to tap-water.
Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or
nutrients was performed according to facility standard procedures. There were no findings that could
interfere with the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated.
Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started).
Females that were littering were left undisturbed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
As the validated method was not finalized yet, formulation samples were collected during in-life (31
October 2013) and stored in the freezer at ≤-15°C until analyses. However, as the NPOC method was
not suited to measure the test substance in these frozen samples, additional samples were collected
after the in-life phase (19 December 2013). These formulations were prepared in the same manner as
used during the study.
For quantitative analysis of the test substance in formulation, Total Organic Carbon (TOC) analysis
was used (see project 503334). Samples of formulations were analyzed for homogeneity (highest and
lowest concentration) and accuracy of preparation (all concentrations). Stability could not be
determined since TOC analysis is a general method that does not distinguish original substance from
hydrolysis product(s); therefore results on stability assessment were not reported.
Accuracy of preparation was considered acceptable if mean measured concentrations were 90-110%
of target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during
mating, and up to the day prior to scheduled necropsy. Females were
exposed for 41-47 days, i.e. during 2 weeks prior to mating, during
mating, during post-coitum, and during at least 4 days of lactation (up to
the day prior to scheduled necropsy). Female no. 47 (Group 1) was not
dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day
with a maximum of 6 hours difference between the earliest and latest
dose.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animal per sex per dose
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
Main observation and examinations have been described in the repeated-dose study.
General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect
signs of difficult or prolonged parturition, and cage debris of pregnant
females was examined to detect signs of abortion or premature birth. Any
deficiencies in maternal care (such as inadequate construction or
cleaning of the nest, pups left scattered and cold, physical abuse of pups
or apparently inadequate lactation or feeding) were examined.
Oestrous cyclicity (parental animals):
Not reported
Sperm parameters (parental animals):
From the selected 5 males of the control and high dose group, and all males suspected to be infertile,
additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were
processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The
Netherlands).
Litter observations:
Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily
thereafter were determined. If possible, defects or cause of death were
evaluated.
Clinical signs: At least once daily, detailed clinical observations were made for all
animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.
Postmortem examinations (parental animals):
See repeated dose toxicity
Postmortem examinations (offspring):
Necropsy pups
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation. All pups
were sexed and descriptions of all external abnormalities were recorded.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a
pooled variance estimate was applied for the comparison of the treated groups and the control groups
for each sex.
- The Steel-test was applied if the data could not be assumed tofollow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to
determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was ap
plied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data
(scores) in the summary tables. Test statistics were calculated on the basis of exact values for means
and pooled variances. Individual values, means and standard deviations may have been rounded off
before printing. Therefore, two groups may display the same printed means for a given parameter, yet
display different test statistics values.
Reproductive indices:
For each group, the following calculations were performed:
Mating index (%)
Fertility index (%)
Conception index (%)
Gestation index (%)
Duration of gestation Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Percentage live males at First Litter Check
Percentage live females at First Litter Check
Percentage of postnatal loss Number of dead pups before planned necropsy
Viability index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No toxicologically significant clinical signs were noted during the observation period.
Incidental findings that were noted included piloerection and rales. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in body weights and body weight gain were noted.
The statistically significant changes in body weight gain noted for males at 100 mg/kg were not
considered toxicologically relevant as no dose response relationship was observed and the individual
values were within normal limits.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after allowance for body weight was similar between treated and control
animals.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
The statistically significantly decreased prothrombin time for males treated at 300 mg/kg was
considered to be of no toxicological relevance as this occurred in the absence of a treatment-related
distribution and remained within the range considered normal for rats of this age and strain.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Clinical biochemistry parameters of treated rats were considered not to have been affected by
treatment up to 1000 mg/kg.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected
animals.
The variation in motor activity did not indicate a relation with treatment.
All groups showed a similar habituation profile with very high activity in the first interval that decreased
over the duration of the test period.
The statistically significant increase noted for number of ambulations for males at 100 mg/kg was not
considered toxicologically relevant as the individual values were within normal limits and no dose
response relationship was seen.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Possible treatment-related findings were recorded in the heart of males and adrenal glands of
females.
Heart, males:
- an increase in severity (slight degree) of mononuclear inflammation in 1/5 males treated at 1000
mg/kg, compared to minimal degrees in 1/5 males treated at 0 mg/kg, in 1/5 males at 100 mg/kg and
in 1/5 males at 300 mg/kg.
- an increase in incidence and/or severity of myofiber degeneration (2/5, up to slight) in males treated
at 1000 mg/kg, compared to minimal degree in 1/5 males treated at 300 mg/kg and absence of this
finding in control males and males treated at 100 mg/kg.
Adrenal glands, females:
an increase in incidence of vacuolation of the zona glomerulosa in 4/5 females treated at 1000 mg/kg,
compared to 1/5 females treated at 0 mg/kg, 1/5 females at 100 mg/kg and 1/5 females at 300 mg/kg.
The remainder of microscopic findings recorded, were within the normal range of background
pathology encountered in Wistar (Han) rats of this age.
In each group one or two females were not pregnant. No treatment-related findings were recorded in
the reproductive organs of those females and their matching males.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The spermatogenic staging profiles were normal for all males examined.
Reproductive performance:
no effects observed

Details on results (P0)

No toxicologically relevant effects on reproductive parameters were noted.
Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.
At 300 mg/kg, two females (nos. 63 and 70) had a relatively low number of corpora lutea, implantation sites and/or living pups at first litter check. As this was not noted at 1000 mg/kg and these low
numbers are occasionally seen historically in control data, it was not considered toxicologically relevant.
No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.
Parturition/maternal care: No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.
The statistically significantly change noted for sex ratio for pups at 300 mg/kg (71% female pups at 300 mg/kg compared to 52% female pups at the control group) was not considered toxicologically
significant as this change was not noted at the higher dose of 1000 mg/kg.
Incidental findings of pups consisted of blue spot on several body parts and scabbing of the snout.
The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
Mortality / viability:
no mortality observed
Description (incidence and severity):
Two pups at 100 mg/kg and one pup at 300 mg/kg were found dead or missing during lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
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

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg).
No toxicologically relevant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites).
Developmental results:
No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg).
No toxicologically relevant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).
In conclusion, treatment with analogue substance 1 by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg revealed parental toxicity for males at 1000 mg/kg. No reproduction and developmental toxicity was observed for treatment up to 1000 mg/kg.
Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: 300 mg/kg for males, and at least 1000 mg/kg for females
Reproduction NOAEL: at least 1000 mg/kg
Developmental NOAEL: at least 1000 mg/kg