Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

one study available: Screening for reproduction toxicity and developmental effects. No effects observed.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08. Mar. 2016 - 30. Jan. 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
not relevant
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TY2031
- Expiration date of the lot/batch: 17 February 2017
- Purity test date:
RADIOLABELLING INFORMATION (if applicable)
-not applicable
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In freezer (≤ -15°C) protected from light
- Stability under test conditions: Stability for at least 4 hours at room temperature
protected from light is confirmed over the concentration
range 1 to 200 mg/mL; stability for at least 2 days in the
refrigerator and 1 week in the freezer is confirmed at
200 mg/mL;
- Solubility and stability of the test substance in the solvent/vehicle: stable for limited timing and
soluble as determined in trial formulations
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: see above
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
none
Species:
rat
Strain:
Wistar
Details on species / strain selection:
This species and strain of rat has been recognized as
appropriate for general and reproduction toxicity studies.
Charles River Den Bosch 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 and environmental conditions:
TEST ANIMALS
- Source: Female animals: Charles River Deutschland, Sulzfeld,
Germany.
Male animals: Charles River Laboratories UK, Margate, UK.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 - 12 weeks
- Weight at study initiation: males: mean 318-323 g (depending of treatment group)
females: mean 219 -229 g (depending of treatment group)
- Fasting period before study: none
- Housing: Pretest Females were housed in groups of 5 females/cage in Macrolon
plastic cages (MIV type, height 18 cm).
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).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF®
Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period: at least 5 days
DETAILS OF FOOD AND WATER QUALITY:
see above
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24 °C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 10 per hour
- Photoperiod (hrs dark / hrs light): 12 / 12
IN-LIFE DATES: From: 14 March 2016 (randomization dose
range finding study) To: 05 July 2016 (end of in-life phase)
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 4 hours prior to
dosing and were homogenized to a visually acceptable level.
No adjustment was made for specific gravity/density of the test
item. Adjustment was made for specific gravity of the vehicle.
No correction was made for the purity/composition of the test
item.
- VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations pe
rformed at Charles River Den Bosch
- Concentration in vehicle: 1 - 10 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated
according to the latest body weight.
- Lot/batch no. (if required):
- Purity:
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 was
confirmed, the males and females were separated.
Detection of mating was not confirmed for animal no. 56 which
did deliver live offspring. The mating date of this animal was
estimated at 21 days prior to the actual delivery date. This day
was designated as Day 0 post-coitum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on two occasions during the treatment phase (11 May and 04 July
2016), according to a validated method (Test Facility Study No.511265). Samples of
formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy
of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations
were 85-115% of the 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 that delivered were exposed for 50-55 days, i.e. during
2 weeks prior to mating (with the objective of covering at least
two complete estrous cycles), the variable time to conception,
the duration of the pregnancy and at least 13 days after delivery
up to and including the day before scheduled necropsy.
Females which failed to deliver healthy offspring were exposed
for 41-44 days.
Female nos. 45 (Group 1), 51, 54 (Group 2), 66, 68 (Group 3),
71 and 78 (Group 4) were not dosed on one occasion as these
females was littering at the time of dosing. The omission of one
day of dosing over a period of several weeks was not
considered to affect the toxicological evaluation.
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.
Details on study schedule:
see mating procedures
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on dose range finding study
- Rationale for animal assignment (if not random): Before initiation of pretest (females) or prior to
commencement
of treatment (males), by computer-generated random algorithm
according to body weight, with all animals within ± 20% of the
sex mean.
- Rationale for selecting satellite groups: no satelite group
- Post-exposure recovery period in satellite groups: no satelite group
- Section schedule rationale (if not random): basicly random: 5 animals of each group/sex were
chosen. criteria was randomly assigned animal number.
Positive control:
none
Parental animals: Observations and examinations:
Mortality / Viability At least twice daily (early in the morning and close to the end
of the working day).
Clinical signs At least once daily from treatment onwards up to the day prior
to necropsy, detailed clinical observations were made in all
animals, at least immediately (0-30 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. Signs were graded for severity and the maximum
grade was predefined at 3 or 4. Grades were coded as slight
(grade 1), moderate (grade 2), severe (grade 3) and very severe
(grade 4). For certain signs, only its presence (grade 1) or
absence (grade 0) was scored. In the data tables, the scored
grades were reported, as well as the percentage of animals
affected in summary tables.
Functional Observations The following tests were performed on the selected
5 animals/sex/group:
• hearing ability (HEARING), pupillary reflex (PUPIL L/R),
static righting reflex (STATIC R) (Score 0 =
normal/present, score 1 = abnormal/absent).
• fore- and hind-limb grip strength, recorded as the mean of
three measurements per animal (Series M4-10, Mark-10
Corporation, J.J. Bos, Gouda, The Netherlands).
• 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 are reported.
Ambulations represent movements characterized by a
relocation of the entire body position like walking, whereas
total movements represent all movements made by the
animals, including ambulations but also smaller or more
fine movements like grooming, weaving or movements of
the head.
The selected males were tested during Week 4 of treatment and
the selected females were tested once during the last week of
lactation (i.e. PND 6-13). These tests were performed after
observation for clinical signs (including arena observation, if
applicable) and started at least immediately (0-30 min) after
dosing.
Body weights Males and females were weighed on the first day of exposure
(prior to first exposure) and weekly thereafter. Mated females
were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum
and during lactation on PND 1, 4, 7 and 13.
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 post-coitum and during lactation
on PND 1, 4, 7 and 13.
Water consumption Subjective appraisal was maintained during the study, but no
quantitative investigation was introduced as no treatment
related effect was suspected.
Estrous cycle
determination Daily vaginal lavage was performed to determine the stage of
estrous beginning 14 days prior to treatment (pretest), the first
14 days of treatment and during mating until evidence of
copulation was observed. Vaginal lavage continued for those
females with no evidence of copulation until termination of the
mating period.
On the day of scheduled necropsy, a vaginal lavage was taken
to determine the stage of estrus.
General reproduction data Male number paired with, mating date, confirmation of
pregnancy, and delivery day were recorded. Palpation was used
to aid in confirmation of pregnancy. Cage debris of pregnant
females were examined for evidence of premature delivery.
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.
Clinical Laboratory Investigations (F0-Generation only)
Blood samples were collected at the end of the treatment period on the day of scheduled
necropsy from the selected 5 F0 animals/sex/group under anaesthesia using isoflurane (Abbott
B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m.
The animals were deprived of food overnight (with a maximum of 24 hours) before blood
sampling, but water was available. Blood samples were drawn from the retro-orbital sinus
and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with K3-
EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and
tubes treated with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional
blood sample (0.25 mL) was collected into serum tubes for determination of bile acids. The
following parameters were determined:
Haematology
The following haematology parameters were determined in blood prepared with K3-EDTA as
an anti-coagulant, using the ADVIA® 2120i Hematology System (Siemens Healthcare
Diagnostics B.V., Den Haag, The Netherlands):
see table section
The following clotting parameters were determined in plasma prepared with citrate as anticoagulant,
using the STA Compact® (Diagnostica Stago S.A.S., Asnières, France):
see table section
Clinical Biochemistry
The following clinical biochemistry parameters were determined using the AU400 (Beckman
Coulter Nederland B.V., Woerden, The Netherlands). All parameters were determined in
plasma, except for bile acids which were determined in serum:
see table section
Blood Sampling for Thyroid Hormone Analysis
Was performed on the following occasions:
F1-generation, PND 4 pups:
6
From 2 surplus pups per litter at culling (when possible). Blood samples from the 2 pups per
litter were collected into one serum tube. If only 1 surplus pup per litter was available at
culling, as much as possible blood was collected from this single pup.
Blood samples were collected by decapitation, between 7.00 and 10.30 a.m.. Blood samples
from the 2 pups per litter (0.4 mL in total) were collected into one serum tube (Greiner Bio-
One GmbH, Kremsmünster, Austria) for possible future measurement of thyroxine (T4).
After clotting and centrifugation, serum samples were stored at ≤-75°C. Under these storage
conditions, the samples are stable for 6 months. Any samples remaining after 6 months will
be discarded.
F1-generation, PND 13-15 pups:
From 2 pups per litter (when possible from one male and one female) at planned necropsy.
As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture
under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands), between 7.00
and 10.30 a.m.. Blood was collected into serum tubes.
After clotting and centrifugation, serum from each sample was divided into 2 aliquots: 150
μL serum for measurement of thyroxine (T4) and the remaining volume of serum for
measurement of thyroid-stimulating hormone (TSH).
Serum samples were stored at ≤-75°C. Under these storage conditions, the samples are stable
for 6 months. Any samples remaining after 6 months will be discarded.
F0-generation, males and females:
End of study from all animals at planned necropsy; this included females on Day 14-16 of
lactation, all females that failed to deliver healthy pups and all males after at least 4 weeks of
treatment (including all males that failed to sire).
Blood samples were collected under anaesthesia using isoflurane between 7.00 and 10.30
a.m. The animals were deprived of food overnight (with a maximum of 24 hours) before
blood sampling, but water was available. Blood samples (0.9 mL) were drawn from the retroorbital
sinus and collected into serum tubes (Greiner Bio-One GmbH, Kremsmünster,
Austria).
After clotting and centrifugation, serum was used as listed below.
Males: 1 aliquot of 150 μL serum was used for measurement of thyroxine (T4), and the
remaining volume of serum for measurement of thyroid-stimulating hormone (TSH).
Females: The serum was stored for possible measurement of thyroxine (T4) and/or thyroidstimulating
hormone (TSH).
Serum samples were stored at ≤-75°C. Under these storage conditions, the samples are stable
for 6 months. Any samples remaining after 6 months will be discarded.
In general:
Total T4 and TSH was measured in serum using the Immulite 1000® (Siemens Healthcare
Diagnostics B.V., Den Haag, The Netherlands):
see table section
General reproduction data Male number paired with, mating date, confirmation of
pregnancy, and delivery day were recorded. Palpation was used
to aid in confirmation of pregnancy. Cage debris of pregnant
females were examined for evidence of premature delivery.
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):
Daily vaginal lavage was performed to determine the stage of
estrous beginning 14 days prior to treatment (pretest), the first
14 days of treatment and during mating until evidence of
copulation was observed. Vaginal lavage continued for those
females with no evidence of copulation until termination of the
mating period.
On the day of scheduled necropsy, a vaginal lavage was taken
to determine the stage of estrus.
Sperm parameters (parental animals):
none specific examinations
Litter observations:
Each litter was examined to determine the following, if practically possible:
Mortality / Viability The numbers of live and dead pups were determined on PND 1
and daily thereafter. If possible, defects or cause of death were
evaluated.
Clinical signs At least once daily, detailed clinical observations were made
for all animals. Only days on which clinical signs were present
between first and last litter check were presented in the
respective tables.
Body weights Live pups were weighed on PND 1, 4, 7 and 13.
Sex Sex was determined for all pups on PND 1 and 4.
Anogenital distance Anogenital distance (AGD) was measured for all live pups on
PND 1. The AGD was normalized to the cube root of body
weight.
Areola/nipple retention On PND 13, all males in each litter were examined for the
number of areola/nipples.
Postmortem examinations (parental animals):
F0-generation - Termination
All animals were deprived of food overnight (with a maximum of 24 hours) before blood
sampling, but water was available.
All animals surviving to the end of the observation period and all moribund animals were
deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and
subsequently exsanguinated.
Necropsy was conducted on the following days:
Condition Day of necropsy
Males Following completion of the mating period (a minimum of 28
days of dose administration).
Females which delivered PND 14-16.
Females which failed to
deliver5 (nos. 42, 46,55,
64, 70 and 77)
Post-coitum Days 26-27 (females with evidence of mating).
Euthanized in extremis (no.
47)
When pain, distress or discomfort was considered not transient
in nature or was likely to become more severe.
F0-generation – Macroscopic Examination
After sacrifice, all animals were subjected to a full post mortem necropsy, with special
attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities
were recorded.
The number of implantation sites 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 (see Allocation) and control female no. 47 (killed in extremis):
Identification marks: not processed (Nasopharynx)
Adrenal glands (Esophagus)
(Aorta) Ovaries
Brain - cerebellum, mid-brain, cortex (7-levels) (Pancreas)
Caecum Peyer's patches [jejunum, ileum] if
Cervix detectable
Clitoral gland Pituitary gland
Colon Preputial gland
Coagulation gland Prostate gland
(Cowper’s gland) Rectum
Duodenum (Salivary glands - mandibular, sublingual)
Epididymides6 Sciatic nerve
Eyes (with optic nerve (if detectable) and
Harderian gland)6
Seminal vesicles
Skeletal muscle
Mammary gland area (males and females) (Skin)
Femur including joint Spinal cord -cervical, midthoracic, lumbar
(Glans penis) Spleen
(Levator ani plus bulbocavernosus muscle
complex (LABC))
Sternum with bone marrow
Stomach
Heart Testes6
Ileum Thymus
Jejunum Thyroid including parathyroid if detectable
Kidneys (Tongue)
(Lacrimal gland, exorbital) Trachea
(Larynx) Urinary bladder
Liver Uterus
Lung, infused with formalin Vagina
Lymph nodes - mandibular, mesenteric All gross lesions
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no
signs of toxicity were noted at macroscopic examination.
All remaining animals, males that failed to sire and females which failed to deliver7.
Cervix Preputial gland
Clitoral gland Prostate gland
Coagulation gland Seminal vesicles
Cowper’s glands Testes8
Epididymides8 Thyroid including parathyroid if detectable
Glans penis Uterus
Levator ani plus bulbocavernosus muscle
complex (LABC)
Vagina
All gross lesions
Mammary gland area (males and females) Identification marks: not processed
Ovaries
Reproductive organs were only examined by the pathologist for males that failed to sire and
all females that failed to deliver healthy pups. Female mammary gland area was not
examined by the pathologist as there was no female with total litter loss.
F0-generation – Organ Weights
The following organ weights and terminal body weight were recorded from the following
animals on the scheduled day of necropsy:
Selected 5 animals/sex/group (see Allocation):
Adrenal glands
Brain
Cowper’s glands
Epididymides
Glans penis
Heart
Kidneys
Levator ani plus bulbocavernosus muscle complex (LABC)
Liver
Ovaries
Prostate
Seminal vesicles including coagulating glands
Spleen
Testes
Thymus
Thyroid
Uterus (including cervix)
All remaining animals:
Cowper’s glands
Epididymides
Glans penis
Levator ani plus bulbocavernosus muscle complex (LABC)
Testes
Thyroid
Absolute organ weights and organ to body weight ratios are reported.
F0-generation - Histotechnology
All organ and tissue samples, as defined under Histopathology (following section), were
processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained
with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). The additional slides of
the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin
(Klinipath, Duiven, The Netherlands).
F0-generation – 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 (see second table below) to examine staging of
spermatogenesis.
• The preserved organs and tissues of the animal that was killed in extremis.
• All gross lesions of all animals (all dose groups).
• Thyroid gland and kidneys of all selected 5 males of Groups 2 and 3, based on
(possible) treatment-related changes in these organs in Group 4.
• Liver of all selected 5 animals of Groups 2 and 3 (males and females), based on
(possible) treatment-related changes in this organ in Group 4.
• The reproductive organs9 of all males that failed to sire and all females that failed to
deliver healthy pups (presented in the table below):
Group Female/Male nos. Reason
1 42/--a
46/6
Not pregnant
Implantation sites only
2 55/15 Not pregnant
3 64/24
70/30
Implantation sites only
Implantation sites only
4 77/37 Not pregnant
a Male no. 2 was already selected for assessment of reproductive organs.
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.
Postmortem examinations (offspring):
F1-generation Pups - Termination
Pups, younger than 7 days were killed by decapitation.
All remaining pups (PND 7-15) were sacrificed using Euthasol® 20% (AST Farma B.V.,
Oudewater, The Netherlands) by intraperitoneal (ip) injection.
On PND 4 (at culling), from 2 pups per litter blood samples (0.4 mL in total) were collected
by decapitation between 7.00 and 10.30 a.m.. Blood samples from the 2 pups per litter were
collected into one serum tube. For further details, see section 5.9.
On PND 13-15, from 2 pups per litter (if possible from one male and one female) blood
samples were collected at planned necropsy. As part of the necropsy procedure, blood
samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane (Abbott
B.V., Hoofddorp, The Netherlands), between 7.00 and 10.30 a.m., followed by
exsanguination. Blood was collected into serum tubes. For further details, see section 5.9.
Necropsy was conducted on the following days:
Condition Day of necropsy
Culling PND 4.
Terminal sacrifice PND 13-15.
Spontaneous deaths As soon as possible after death and always within 24 hours.

F1-generation pups - Macroscopic Examination
All pups were sexed by both external as well as internal examination. Descriptions of all
abnormalities were recorded. Particular attention was paid to the external reproductive
genitals to examine signs of altered development.
At terminal sacrifice (PND 13-15), the thyroid from 2 pups per litter, i.e. the same pups as
selected for blood sampling (see also section 5.9), was preserved in 10% buffered formalin.
The stomach of pups not surviving to the scheduled necropsy date was examined for the
presence of milk, if possible. If possible, defects or cause of death were evaluated.
Statistics:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test
(Ref. 2; many-to-one t-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 (Ref. 3; many-to-one rank test) was applied if the data could not be
assumed to follow a normal distribution.
• The Fisher Exact-test (Ref. 4) was applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test (Ref. 5) was applied to motor
activity data to determine intergroup differences.
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:
Mating index (%)
Fertility index (%)
Conception index (%)
Gestation index (%)
Duration of gestation
Offspring viability indices:
Percentage live males at First Litter Check
Percentage live females at First Litter Check
Viability index (%)
Lactation index (%)
Sex ratio (percentage males)
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant clinical signs were noted during the observation period.
Salivation seen after dosing among a few animals at 50 mg/kg and in all animals at 150 and
500 mg/kg was considered to be a physiological response rather than a sign of systemic
toxicity considering the nature and severity of the effect and its time of occurrence (i.e. after
dosing).
Incidental findings that were noted included rales, scabs, alopecia, swelling and scales. These
findings occurred within the range of background findings to be expected for rats of this age
and strain which were housed and treated under the conditions in this study. At the incidence
observed, these were not considered to be signs of toxicological relevance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female from the control group (no. 47) was killed in extremis on Day 18 of study
immediately after the dosing procedure, because this animal broke the dosing cannula and
swallowed it. Macroscopic examination revealed that the esophagus and stomach contained
the dosing tube, many dark red foci were seen in the glandular mucosa of the stomach and
several dark ref foci were found in the thymus. As this was an accidental death of a control
animal, it was not related to treatment of the test item.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At 500 mg/kg, bodyweight gain of males was statistically significantly lower on the second
week of treatment when compared to controls. As this decrease was only slight and recovered
throughout the remaining of the treatment period, it was not considered toxicologically
relevant.
The statistically significant changes noted in females during the mating or lactation periods
were considered to be of no toxicologically relevance as no dose response and/or slight
increases were noted.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
At 500 mg/kg, food consumption of males was slightly decreased in the first week of
treatment, which explained the reduced bodyweight gain for these animals in the second
week. As it recovered during treatment, it was not considered toxicologically relevant.
The statistically significant changes noted in females during the post-coitum or lactation
periods were considered to be of no toxicologically relevance as no dose response and/or
non-adverse slight increases were noted.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Haematological parameters of treated rats were not considered to be affected by treatment.
The statistically significant changes noted for prothrombin time, mean corpuscular volume
and mean corpuscular hemoglobin were not considered to be toxicologically relevant as they
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:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg, total protein and albumin values were increased in males and albumin level
was increased in females and were considered to be related to the treatment.
Total bilirubin concentrations in males were statistically significantly decreased in groups 2,
3 and 4 of which the values at 150 and 500 mg/kg were outside the historical control data
(value of 1.6 μmol/L in this study vs. P5-P95 range of 1.8 - 2.6 μmol/L.
Any other statistically significant changes at 150 and 500 mg/kg were not considered to be
toxicologically significant as they occurred in the absence of a treatment-related distri
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 statistically significant changes noted for forelimb grip strength were not considered
toxicologically relevant as all values were within normal limits and control male values were
at the upper end. Furthermore, no clear dose response was noted, no effects on the hind limbs
and no effects on other functional observations or clinical signs were seen in any of the sexes.
Moreover, an opposite response was seen in both sexes (i.e. decrease in males and increase in
females).
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.
Immunological findings:
effects observed, treatment-related
Description (incidence and severity):
Thyroid hormone analyses:
At 500 mg/kg, serum level of T4 in F0 males was slightly but statistically significantly
decreased.
TSH values were increased for F0-males at 150 and 500 mg/kg. This was not statistically
significant due to the high variation between the males.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related higher liver weights (absolute and relative to body weights) were noted in
the 150 and 500 mg/kg group males and females and test item-related higher kidney weights
(relative to body weights) were noted in the 500 mg/kg group males.
The statistically significant lower heart weight at 500 mg/kg in males was considered not to
be a sign of toxicity as relative heart weight was unaffected and no microscopic correlate was
noted.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related macroscopic findings were present in the thyroid gland and kidneys in
males and in the liver of both sexes, consisting of:
• Enlarged thyroid gland in 2/10 and in 1/10 males at 150 and 500 mg/kg, respectively.
• Black-brown discoloration of the liver in 1 male and 1 female at 150 mg/kg and in 4 males
at 500 mg/kg.
• Enlarged liver in 5/10 males and 4/10 females at 500 mg/kg.
• Accentuated lobular pattern of the liver in a male at 500 mg/kg.
• Accentuated pattern in the kidneys in 1 and 2 males at 150 and 500 mg/kg, respectively.
The incidence of other 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 dose-related incidence trend. These necropsy findings were therefore not
considered to be toxicologically relevant.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were present in the thyroid glands, liver and kidneys
at 150 and 500 mg/kg. Microscopic findings consisted of:
• An increased incidence and/or severity to slight degree of follicular cell hypertrophy of the
thyroid gland in 2/5 males at 150 mg/kg and in 5/6 males at 500 mg/kg.
• Centrilobular hepatocellular hypertrophy of the liver at a minimal degree was observed in
3/5 males and 2/5 females at 150 mg/kg and slight degree was noted for 4/6 males and5/5
females at 500 mg/kg. This finding was recorded up to moderate degree in 2/6 males at
500 mg/kg.
• Increased severities of hyaline droplet accumulation (up to marked) of the kidneys were
found in males at 150 and 500 mg/kg. This finding was recorded for 1, 1, 3 and 1 animal
at slight degree and for 0, 0, 1 and 4 animals at moderate degree in the control, 50, 150 and
500 mg/kg dose groups, respectively. At 500 mg/kg, marked hyaline droplet accumulation
was recorded for one male. At this dose level there were accompanying degenerative
findings such as an increased incidence and/or severity of tubular basophilia and/or
presence of tubular degeneration and/or granular casts. Therefore, these microscopic
findings were regarded as adverse in male rats at 500 mg/kg.
Hyaline droplet accumulation, recorded in all male kidneys, most likely represents
alpha2uglobulin, a normal protein in male rats, not present in female rats or human, which
undergoes re-absorption in the proximal cortical tubules. A range of chemicals is known to
increase hyaline droplet formation (Ref. 7). At 150 mg/kg, the observed hyaline droplet
accumulation (up to moderate) was present above background levels (up to slight) but was
without additional treatment-related degenerative changes and therefore considered as a nonadverse
finding. At 500 mg/kg the severity increased up to marked degree, could be
correlated with higher relative kidney weight (15% increase) and was accompanied by the
following degenerative changes: increased severities of tubular basophilia (slight) and/or the
presence of granular casts and/or tubular degeneration (both minimal degree). Therefore, at
500 mg/kg, this range of morphologic findings was considered as adverse in the male rat.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
All females had a regular 4-day estrous cycle during the pretest and treatment period until
mating occurred.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There were 2/10 couples of the control group, 1/10 couple at 50 mg/kg/day, 2/10 couples at
150 mg/kg/day and 1/10 couple at 500 mg/kg/day which did not have offspring. No
abnormalities were seen in the reproductive organs, which could account for their lack of
healthy offspring and spermatogenic profiles were normal for all males examined.
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no effects observed
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment.
Incidental clinical symptoms of pups consisted of lean appearance, discoloured spots on
different parts of the body and swelling on the back or mouth. The nature and incidence of
these clinical signs remained within the range considered normal for pups of this age, and
were therefore not considered to be toxicologically relevant.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of live offspring on Day 4 before culling compared to the number of offspring on
Day 1 was not affected by treatment.
One pup each at 150 and 500 mg/kg was found dead between lactation Days 2 and 4. No
toxicological relevance was attributed to these dead pups since the mortality incidence
remained within the range considered normal for pups of this age.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were not considered to be affected by treatment.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 and TSH levels in male and female PND 13-15 pups were considered not to be
affected by treatment up to 500 mg/kg.
The statistically significant change for TSH at 150 mg/kg (male pups) was not considered to
be toxicologically relevant as this occurred in the absence of a dose-related distribution and
remained within the range considered normal for rats of this age and strain.
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
Sex ratio
Sex ratio was not considered to be affected by treatment.

Anogenital distance
Anogenital distance (absolute and normalized for body weight) in male and female pups was
not considered to be affected by treatment.

Areola/nipple retention
Treatment up to 500 mg/kg/day had no effect on areola/nipple retention. For none of the
examined male pups nipples were observed at PND 13.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to
treatment.
Advanced autolysis was noted for one pup that was found dead at first litter check. No other
macroscopic findings were recorded.
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
Serum T4 and TSH levels in male and female PND 13-15 pups were considered not to be
affected by treatment up to 500 mg/kg.
The statistically significant change for TSH at 150 mg/kg (male pups) was not considered to
be toxicologically relevant as this occurred in the absence of a dose-related distribution and
remained within the range considered normal for rats of this age and strain.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Key result
Reproductive effects observed:
no
Conclusions:
Based on the results obtained in this study, the following No Observed Adverse Effect Levels
(NOAEL) were derived:
Parental NOAEL: 150 mg/kg in males due to kidney findings and at least 500 mg/kg in females.
Reproduction NOAEL: at least 500 mg/kg.
Developmental NOAEL: at least 500 mg/kg.
Executive summary:

Study outline

The test item, formulated in propylene glycol, was administered daily by oral gavage to SPFbred

Wistar Han rats. One control group and three treated groups were tested, each consisting

of 10 males and 10 females.

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to

termination. Females that delivered were exposed for 50-55 days, i.e. during 2 weeks prior to

mating, during mating, during post-coitum, and during 13-15 days of lactation. Females

which failed to deliver healthy offspring were exposed for 41-44 days.

Evaluated parameters

The following observations and examinations were evaluated: mortality / viability, clinical

signs (daily), functional observations and locomotor activity (end of treatment), body weight

and food consumption (at least at weekly intervals), estrous cycle determination (14 days

prior to treatment, 14 days of treatment and during mating until evidence of mating, and on

the day of necropsy), clinical pathology (end of treatment), measurement of thyroid hormone

T4 and TSH (F0-males at the end of treatment, PND 13-15 pups), macroscopy at termination,

organ weights and histopathology on a selection of tissues. In addition, the following

reproduction/developmental parameters were determined: mating, fertility and conception

indices, precoital time, number of implantation sites, gestation index and duration,

parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical

signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy).

Formulations were analyzed twice during the study to assess accuracy and homogeneity.

Results/discussion

Accuracy and homogeneity of formulations were demonstrated by analyses.

Treatment up to 500 mg/kg was well-tolerated; no mortality related to the treatment with the

test item occurred, no treatment-related changes during functional observation tests were

seen, and no toxicologically relevant clinical signs or changes in body weights, food

consumptions or haematological parameters were noted.

At 500 mg/kg, a slightly higher mean value of total protein and albumin in males and slightly

higher albumin level in females occurred. As for females no adverse corroborative changes

were noted, the slightly higher albumin concentration in females was not regarded

toxicologically significant.

An increase in incidence and/or severity (up to slight) of follicular cell hypertrophy in the

thyroid was observed in males at 150 and 500 mg/kg and was in some rats correlated with the

gross finding enlarged thyroid and/or with decreased serum Thyroxin level (T4).

This follicular cell hypertrophy can be seen as a secondary event, after hepatocellular

microsomal enzyme induction and concomitantly hepatocellular hypertrophy as observed in

the present study. Microsomal enzyme induction in the liver can lead to a higher turnover of

T4, lowering the serum T4 levels (statistically significant decreased at 500 mg/kg/day and

minimal decreased at 150 mg/kg/day) resulting by feedback mechanisms to the hypothalamus

and pituitary gland into increased TSH (thyroid stimulating hormone, a subtle not statistically

significant increase of TSH in males at 150 and 500 mg/kg/day) leading to follicular cell

hypertrophy of the thyroid gland as observed at 150 and 500 mg/kg/day (Ref. 6). At the

recorded severity and in absence of other test item-related changes in the thyroid gland, the

thyroid findings were considered as adaptive and non-adverse (Ref. 7).

Centrilobular hepatocellular hypertrophy of the liver was observed at minimal degree in a

few male and female rats at 150 mg/kg and could be correlated with increased liver weights

(relative to bodyweight liver weights increased up to 22% in males and 14% in females

compared to the liver weight in the concurrent controls). At 500 mg/kg, the severity of the

hepatocellular hypertrophy increased up to moderate in males and up to slight in females, the

relative liver weights increased up to 52% in males and 37% in females and additional

correlating macroscopic findings were noted consisting of enlarged and/or accentuated

pattern. These liver findings might also be linked to the lower total bilirubin levels at all dose

levels. In absence of degenerative changes, the observed hypertrophy was regarded as an

adaptive change most likely due to microsomal enzyme induction, and non-adverse (Ref.

8).There was no microscopic correlate for the macroscopic finding black-brown

discoloration.

Hyaline droplet accumulation, recorded in all male kidneys, most likely represents

alpha2uglobulin, a normal protein in male rats, not present in female rats or human, which

undergoes re-absorption in the proximal cortical tubules. A range of chemicals is known to

increase hyaline droplet formation (Ref. 7). At 150 mg/kg, the observed hyaline droplet

accumulation (up to moderate) was present above background levels (up to slight) but was

without additional treatment-related degenerative changes and therefore considered as a nonadverse

finding. At 500 mg/kg the severity increased up to marked degree, could be

correlated with higher relative kidney weight (15% increase) and was accompanied by the

following degenerative changes: increased severities of tubular basophilia (slight) and/or the

presence of granular casts and/or tubular degeneration (both minimal degree). Therefore, at

500 mg/kg, this range of morphologic findings was considered as adverse in the male rat.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
one Guideline study available with reliability Klimisch 1
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

Description of key information

one study available: Screening for reproduction toxicity and developmental effects. No effects observed.

Study summary reported under section 7.8.1. Toxicity to reproduction

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
one Guideline study available with reliability Klimisch 1
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

At present, no evidence for Reproductive or Developmental toxicity present.

Therefore no mode of action can be established .

Justification for classification or non-classification

The available information is conclusive but not sufficient for classification.