Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 21 May 2014 and 17 April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
See below.
Principles of method if other than guideline:
Deviations from Study Plan
Deviation No.1
Necropsy
Due to macroscopic observations noted during the necropsy of the first set of females treated
with 1000 mg/kg bw/day, the kidneys, liver, spleen and stomach were retained at necropsy from
all remaining animals.

Deviation No 2
Animal Husbandry
Environment
The Study Plan target values for temperature and relative humidity (RH) were 22 ± 3ºC and 50 ±
20% respectively. While there were no deviations from the target range for temperature, the
target range for relative humidity was exceeded for extended periods of time during the study
due to adverse weather conditions. The majority of the deviations for relative humidity were less
than 80% RH but on some occasions this value was also exceeded, although these instances were
generally transient in nature.
There were no findings for control animals that indicated that animal health had been affected in
any way by these extended periods of high humidity and therefore the study was still considered
to represent a valid assessment of Test Item toxicity. While it is accepted that these deviations
from the target range for relative humidity were less than ideal, overall it is considered that these
deviations had no adverse impact on the scientific purpose of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were
obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the
animals were examined for signs of ill-health or injury. The animals were acclimatized for at
least five days during which time their health status was assessed. A total of ninety six animals
(forty eight males and forty eight females) were accepted into the study. At the start of treatment
the males weighed 265 to 340g, the females weighed 191 to 232g, and were approximately
twelve weeks old.
Initially, all animals were housed in groups of four in solid floor polypropylene cages with
stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the
pairing phase, the animals were transferred to polypropylene grid floor cages suspended over
trays lined with absorbent paper on a one male: one female basis. Following evidence of
successful mating, the males were returned to their original cages. Mated females were housed
individually during gestation and lactation in solid floor polypropylene cages with stainless steel
mesh lids and softwood flakes.
The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad
Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. Mains drinking water was
supplied from polycarbonate bottles attached to the cage. Environmental enrichment was
provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd.,
Cheshire, UK) except for paired animals and mated females during final week of gestation and
lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and
lactation. The diet, drinking water, bedding and environmental enrichment were considered not
to contain any contaminant at a level that might have affected the purpose or integrity of the
study.
The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd.,
Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen
air changes per hour and the low intensity fluorescent lighting was controlled to give twelve
hours continuous light and twelve hours darkness. Environmental conditions were continuously
monitored by a computerized system, and print-outs of hourly temperatures and humidities are
included in the study records. The Study Plan target ranges for temperature and relative
humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets
were considered not to have affected the purpose or integrity of the study.
The animals were randomly allocated to treatment groups using a stratified body weight
randomization procedure and the group mean body weights were then determined to ensure
similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system
routinely used in these laboratories.

Justification
The rat was selected for this study as it is a readily available rodent species historically used in
safety evaluation studies and is acceptable to appropriate regulatory authorities.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Test Item Preparation
For the purpose of this study the test item was prepared at the appropriate concentrations as a
solution in Distilled water. The stability and homogeneity of the test item formulations were
determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services as part of Harlan
Study Number: 41304630. Results showed the formulations to be stable for at least twenty five
days. Formulations were therefore prepared every two weeks and stored at approximately 4 °C
in the dark.
Samples of the test item formulation were taken and analyzed for concentration of
FAT 20341/A TE at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results
indicate that the prepared formulations were within acceptable ranges for the purpose of this
study.
The test item was administered daily by gavage using a stainless steel cannula attached to a
disposable plastic syringe. Control animals were treated in an identical manner with 5 mL/kg of
Distilled water.
The volume of test and control item administered to each animal was based on the most recent
scheduled body weight and was adjusted at weekly intervals.
Details on mating procedure:
On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Introduction
The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC/UV) using an external standard technique.

Test item
The test item described in the main part of this study was also used as the analytical standard.

Analytical procedure
Preparation of standard solution
Stock solutions of test item in water were prepared for external standard calibration. An aliquot, 100 mg test item was accurately weighed into a 100 mL volumetric flask and brought to volume with water to yield a solution with a concentration of 1 mg/mL. Aliquots of this stick standard solution were used to prepare working standard solutions in water with a concentration of 0.01 mg/mL.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.

Analysis of samples
The formulations received were diluted with water. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with water this was then shaken to dissolve. Where necessary, sample solutions were further diluted with water to achieve the working concentration.

Preparation of accuracy samples
The accuracy determinations were performed under Harlan Laboratories Ltd. study number 41304630.

Preparation of line standards
The accuracy determinations were performed under Harlan Laboratories Ltd. study number 41304630.

Instrumental set up
HPLC: Agilent Technologies 1200, incorporating auto-sampler and workstation
Column: Gemini C18 3 µ (100 x 4.6 mm id)
Column: 40 °C
Mobile phase:
Eluent A: 20 g/L TBAB: water (10:90 v/v)
Eluent B: 20 g/L TBAB: acetonitrile (10:90 v/v)
Time %B
0 50
5 100
6 100
8 100
Flow rate: 1 mL/min
UV detector wavelength: 295 nm
Injection volume: 100 µL
Retention time: ~3.5 – 5 mins

Study samples and storage
Representative samples were dispatched to the analytical laboratories internally (under ambient conditions) and stored at room temperature until analysis.

Results
Validation of analytical methods
Specificity
The control dose samples and an analysed solvent blank showed no significant interfering response at the retention time of the test item. The standard solutions contained a peak specific for the test item whose area changed accordingly with known concentration; hence the specificity of the method by retention time was confirmed.

Linearity
The linearity determinations were performed under Harlan Laboratories Ltd. study number 41304630.

Accuracy
The accuracy determinations were performed under Harlan Laboratories Ltd. study number 41304630.

Test item formulations
The formulations investigated during the study were found to comprise test item in the range of 99% - 111%.
The test item was found to be stable in the formulations during Harlan Laboratories Ltd. study number 41304630.
In conclusion the results obtained during Harlan Laboratories Ltd. study number 41304630 indicate the accurate use of the test item and Distilled Water as vehicle during this study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was proven.

Discussion
The detection system was found to have an acceptable linearity. The analytical procedure had acceptable recoveries of test item in the vehicle. The method of analysis was validated and proven to be suitable for use.
Duration of treatment / exposure:
Males: 42 days
Females: 46 days (to Day 4 post partum)
Frequency of treatment:
Daily
Details on study schedule:
Chronological Sequence of Study
i. Groups of twelve male and twelve female animals were treated daily at the appropriate
dose level throughout the study (except for females during parturition where applicable).
The first day of dosing was designated as Day 1 of the study.
ii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a
maximum of fourteen days.
iii. Following evidence of mating (designated as Day 0 post coitum) the males were returned
to their original cages and females were transferred to individual cages.
iv. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post
partum. Litter size, offspring weight and sex, surface righting and clinical signs were
also recorded during this period.
v. The male dose groups were killed and examined macroscopically on Day 43.
vi. At Day 5 post partum, all surviving females and surviving offspring were killed and
examined macroscopically.
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
None specified
Positive control:
None specified
Parental animals: Observations and examinations:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change
immediately before dosing, soon after dosing, and one hour after dosing during the working
week (except for females during parturition where applicable). All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males
until termination and weekly for females until pairing. During the pairing phase females were
weighed daily until mating was confirmed. Body weights were then recorded for females on
Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults
until pairing. This was continued for males after the mating phase. For females showing
evidence of mating, food consumption was recorded for the periods covering post coitum Days
0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the
lactation period (Days 1-4).
Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males
and for females during the pre-pairing phase. Due to offspring growth and milk production for
lactation, food efficiency for females could not be accurately calculated for females during
gestation and lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.

Reproductive Performance

Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to
fourteen days. Cage tray-liners were checked each morning for the presence of ejected
copulation plugs and each female was examined for the presence of a copulation plug in the
vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence
of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in
situ was taken as positive evidence of mating (Day 0 of gestation) and the males were
subsequently returned to their original holding cages (unless required for additional pairing).
Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the
period of expected parturition. Observations were carried out at approximately 0830 and
1230 hours at weekends and public holidays. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition
Oestrous cyclicity (parental animals):
Not specified
Sperm parameters (parental animals):
No specified
Litter observations:
Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was
recorded. Offspring were individually identified within each litter by tattoo on Day 1 post
partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated
retrospectively from this data)

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.
Postmortem examinations (parental animals):
Necropsy
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by
exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable
barbiturate agent followed by exsanguination on Day 5 post partum. Any females which failed to
achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.
For all surviving females, the uterus was examined for signs of implantation and the number of
uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by
staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora
lutea were also counted.
All adult animals and offspring, including those dying during the study, were subjected to a full
external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The epididymides and testes were removed from terminal kill adult males dissected free from fat
and weighed before fixation.

Histopathology
Samples of the following tissues were preserved from all animals from each dose group, in
buffered 10% formalin, except where stated:
Coagulating gland
Prostate
Epididymides ♦
Seminal vesicles
Ovaries
Testes ♦
Mammary gland (females only)
Uterus/Cervix
Pituitary
Vagina
All tissues were dispatched to the histology processing Test Site (Propath UK Ltd, Willow
Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal
Investigator: N Fower). The tissues from control and 1000 mg/kg bw/day dose group animals,
any animals dying during the study, and any animals which failed to mate or did not achieve a
pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and
stained with Hematoxylin and Eosin for subsequent microscopic examination. In addition,
sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic
Acid-Schiff (PAS) stain and examined. Due to macroscopic observations of discoloration in the
kidney, liver, spleen and stomach of females treated with 1000 mg/kg bw/day at necropsy, these
tissues were retained from all remaining animals (see deviations to Study Plan).
Initial histopathology examinations revealed microscopic findings in the testes of males treated
with 1000 mg/kg bw/day. These changes were considered to be related to the physical clearance
of the test item and were not associated with any other pathological change. As there were no
pathological changes associated with the finding the decision was taken, in collaboration with
the Sponsor, not to investigate this finding further. As a result of adverse kidney changes
apparent in previous toxicity studies with the test item, histopathological processing and
examination of the retained kidneys of animals of either sex from all dose groups, where
available, was conducted.
Postmortem examinations (offspring):
Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent.

All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
See below
Reproductive indices:
Reproductive Indices

Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of
the parental generation:

i. Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive
evidence of mating.

ii. Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals mated / Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females / Number of animals mated) x 100


Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and
parturition period of the parental generation:

i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating
and the start of parturition.

ii. Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of females delivering live offspring / Number of pregnant females) x 100
Offspring viability indices:
Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first
calculated for each litter and the group mean was calculated using their individual litter values.
Group mean values included all litters reared to termination (Day 5 of age).

i. Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for
each female/litter as follows:
Pre–implantation loss (%) = ((Number of corpora lutea - Number of implantation sites) / Number of corpora lutea) x 100
Post–implantation loss (%) = ((Number of implantation sites - Total number of offspring born) / Number of implantation sites) x100

ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1 / Number of offspring born) x 100
Viability Index (%) = (Number of offspring alive on Day 4 / Number of offspring alive on Day 1) x 100

iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:
(Total number of male offspring / total number of offspring) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See results
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See results
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See results
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See results
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Mortality
One female treated with 1000 mg/kg bw/day was found dead on Day 6. At necropsy, the animal
had a fluid filled chest cavity and the majority of tissues observed for this animal were
discolored black or blue. The cause of death for this animal was unknown but considered likely
to be a result of either dosing trauma or aspiration of the test item when dosed.

Clinical Observations
Animals of either sex treated with 1000 mg/kg bw/day had dark eyes from Day 16 onwards.
Neither the type, incidence or distribution of the remaining clinical signs apparent indicated an
adverse effect of treatment at 100, 300 or 1000 mg/kg bw/day.
Nine males and eight females treated with 1000 mg/kg bw/day showed episodes of increased
salivation post-dose from Day 13 and Day 20 respectively. One female treated with 300 mg/kg
bw/day had increased salivation post-dose on Day 19. Observations of this nature are commonly
experienced following the oral administration of an unpalatable or slightly irritant test item
formulation and in isolation are considered not to be of toxicological importance.
One female treated with 1000 mg/kg bw/day and one male treated with 300 mg/kg bw/day
showed isolated single incidences of noisy and laboured respiration and decreased respiratory
rate. One male and a further female treated with 1000 mg/kg bw/day also showed single
incidences of noisy respiration. It is likely that these observation arose as a consequence of
accidental aspiration of the test item rather than being indicative of systemic toxicity.
Animals of either sex treated with 1000 mg/kg bw/day had episodes of blue or black staining of
the fur throughout the majority of the study. Ten males and seven females treated with
300 mg/kg bw/day had episodes of blue stained fur from Day 8 onwards. One male and two
females treated with 100 mg/kg bw/day had blue stained fur on Day 8, with observations
continuing to Day 9 in males. In the cages of animals of either sex from all treatment groups, the
faecal matter was observed to be blue in colour. The bedding of animals of either sex treated
with 1000 mg/kg bw/day was also stained blue. These observations were considered to reflect
the dark coloration of the test item.

Body Weight
No effect of treatment on body weight development was apparent for males or for females during
the pre-pairing period. Body weight gain during the first week of treatment was statistically
significantly higher than controls for females treated with 300 mg/kg bw/day. An increase in
body weight gain is unlikely to indicate an adverse effect of treatment. During gestation,
females treated with 1000 mg/kg bw/day had statistically significantly lower body weight gain
between Day 14 and Day 20 probably reflecting lower litter size at this dosage.

Food Consumption
There were no obvious effects of treatment on food consumption or food efficiency apparent at 100, 300 or 1000 mg/kg bw/day.

Water Consumption
Daily visual inspection of water bottles did not indicate any overt differences in water consumption at 100, 300 or 1000 mg/kg bw/day.

Reproductive Performance
Mating
There were no treatment-related effects on mating performance.

Fertility
Two control females, one female treated with 100 mg/kg bw/day, one female treated with
300 mg/kg bw/day and one female treated with 1000 mg/kg bw/day were non-pregnant. One
female treated with 1000 mg/kg bw/day had a total litter loss.
There were no treatment-related effects in conception rates for treated animals as assessed by the
number of females producing a litter.

Gestation Length
There were no differences in gestation lengths. The distribution for treated females was
compared to controls. Gestation lengths were between 22 and 23.5 days.

Pathology
Necropsy - Adults
The majority of animals treated with 1000 mg/kg bw/day and a proportion of animals treated
with 300 mg/kg bw/day had colored contents throughout most of the digestive tract.
Discoloration or dark tissue was apparent in the mammary gland, liver, spleen, kidney and
stomach of the majority of animals treated with 1000 mg/kg bw/day. Males at this dose level
also had discolored testes and epididymides, whilst the majority females had discolored uterus,
cervix and vagina. Discoloration of the kidneys extended to some animals treated with
300 mg/kg bw/day. The observations of discolouration are likely to be associated with the
physical properties of the test item. One male treated with 100 mg/kg bw/day had small, flaccid
testes; this male failed to induce pregnancy in its female partner and microscopic examination of the testes revealed marked diffuse tubular atrophy. In the absence of similar effects at
1000 mg/kg bw/day, this finding was considered to be incidental and of no toxicological
importance. One female treated with 1000 mg/kg bw/day had an enlarged, fluid filled uterus and
cervix, in the absence of similar findings in other females treated with 1000 mg/kg bw/day, this
finding was considered to have arisen spontaneously and was considered to be of no
toxicological significance.

Organ Weights
Males treated with 1000 mg/kg bw/day had a statistically significant increase in testes weight,
both absolute and relative to terminal body weight.
There were no adverse effects of treatment on the organ weights measured

Histopathology
Testes: Pigmented macrophages, particularly notable with PAS staining were present in
increased amounts in Group 4 animals.
Kidneys: At 1000 mg/kg bw/day the following changes were apparent in the kidneys. Tubular
degeneration was present in 12/12 males and 5/12 females. Fine brown pigment within the
tubular cells (probably lipofuscin) was present in 12/12 males and 11/12 females. Other
pigment, likely to be test item or a metabolite was present in 10/12 females. There was an
increase in incidence and severity of hyaline droplets in males. There were no changes related to
treatment in the kidneys from animals in Groups 2 and 3.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Increased pre-implantation loss, a lower number of implantations and a smaller litter size at birth and a subsequent decline to Day 4 post partum.
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
See results
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See results
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Litter Responses
In total 10 females from control, 11 females from the 100 and 300 mg/kg bw/day dose groups
and 9 females from the 1000 mg/kg bw/day dose group gave birth to a live litter and successfully
reared young to Day 5 of age. At 1000 mg/kg bw/day an additional littering female showed total
litter loss post partum. The following assessment of litter response is generally based on all
litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size, Sex Ratio and Viability
Females treated with 1000 mg/kg bw/day appeared to produce a smaller litter compared with
control, principally due to a lower number of implantations resulting from higher preimplantation
loss. Despite the lower litter size, offspring survival at this dosage was slightly
inferior to control with the resulting difference in live litter size attaining statistical significance
on Days 1 and 4 (p<0.05 and p<0.01 respectively). This increase in post-natal loss in litters
surviving to Day 4 was accompanied by one female that showed total litter loss post partum.
Sex ratio was similar in all groups indicating that there was no selective effect of maternal
treatment on survival for either sex.

Pre-implantation loss for females treated with 100 or 300 mg/kg bw/day was statistically
significantly lower than controls; a lower percentage of loss is considered not to represent an
adverse effect of treatment.

Offspring Growth and Development
Litter weights were statistically significantly lower on Day 1 and Day 4 for females treated with
1000 mg/kg bw/day. The lower litter weights were associated with the smaller litter size apparent
rather than a reduction in offspring weight as offspring weights for male and female offspring at
this dose level were comparable to controls on Day 1 and Day 4.
Clinical signs apparent for offspring on the study were typical of the age observed. Neither the
incidence or distribution of these observations indicated any adverse effect of maternal
treatment. Statistical analysis of surface righting reflex data did not reveal any significant
intergroup differences.

Pathology
Necropsy Offspring
Macroscopic necropsy findings for offspring on the study were typical for the age observed and
neither the incidence or distribution of these observations indicated any adverse effect of
maternal treatment on offspring development at 100, 300 or 1000 mg/kg bw/day.
Dose descriptor:
NOEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Survival, growth and development of offspring.
Reproductive effects observed:
not specified

Discussion

The oral administration of FAT 20341/A TE by gavage for a period of 42 consecutive days for

males and up to and including Day 4 post partum for females (including two weeks pre-pairing,

gestation and early lactation for females) at dose levels of 100, 300 and 1000 mg/kg bw/day

resulted in treatment related effects in animals of either sex treated with 1000 mg/kg bw/day.

Animals of either sex treated with 1000 mg/kg bw/day had dark eyes from Day 16 onwards.

There were no treatment-related effects on body weight gain or food consumption for males.

Females treated with 1000 mg/kg bw/day had statistically significantly lower body weight gains

from Day 14 to Day 20 of gestation and these females subsequently went on to produce a smaller

litter size in comparison to controls. The smaller litter size was a consequence of a lower

number of implantation sites resulting from increased pre-implantation loss. Despite the lower

litter size, offspring survival at this dosage was slightly inferior to control with the resulting live

litter size attaining statistical significance on Day 1 and 4. This increase in post-natal loss in

litters surviving to Day 4 was accompanied by one female that showed total litter loss post

partum. Litter weight on both Day 1 and Day 4 were statistically significantly lower than

controls, these intergroup differences were considered to reflect the smaller litter size for females

at 1000 mg/kg bw/day rather than a reduction in offspring weight as both male and female

offspring weight were comparable to controls on Day 1 and Day 4. There were no

histopathological changes in the reproductive tissues or associated tissues which accounted for

the variation in pre-implantation loss or litter size, however an association with treatment cannot

be discounted.

Microscopic examination of the kidneys revealed minimal to mild multifocal tubular

degeneration in all males and five females treated with 1000 mg/kg bw/day. Fine brown

pigment within the tubular cells (probably lipofuscin) was also present in the kidneys of all

males and eleven females at this dose level. Tubular degeneration and the presence of

lipofuscin, an indicator of degenerative change, are considered to be a direct effect of the test

item and represent an adverse effect of treatment. Males treated with 1000 mg/kg bw/day also

had increase in incidence and severity of hyaline droplet deposition in the tubules of the kidneys

when compared to controls. Hyaline droplet formation within the tubules is consistent with the

accumulation of alpha 2u-globulin, a common finding in untreated male rats. This finding is

considered to be specific to the male rat and does not represent a risk to humans. There were no

treatment-related changes in the kidneys of animals of either sex treated with 100 or 300 mg/kg

bw/day.

In terms of the changes apparent in the reproductive indices, the significant changes in the

kidneys of males and females, evident in a number of animals, may have caused an increase in

the overall stress on the females and could have had an effect on the reproductive performance as

determined by slightly lower litter sizes at 1000 mg/kg bw/day.

Pigmented macrophages were present in the testes of males treated with 1000 mg/kg bw/day;

however this finding did not appear to be associated with any other pathological process. The

presence of the pigmented macrophages may be related to the clearance of the specific type of

test item and, in the absence of a pathological change, was considered to be of no toxicological

importance. Males treated with 1000 mg/kg bw/day also had a statistically significant increase

in testicular weight, both absolute and relative to terminal body weight however all individual

values were within the historical control ranges for rats of the strain and age used. Although it is

unlikely that the presence of the pigmented macrophages in the testes of these males resulted in

the increase in testicular weight for these males, a correlation cannot be completely ruled out.

Conclusions:
The oral administration of FAT 20341/A TE to male rats by gavage for a period of 42
consecutive days and to females by gavage up to and including Day 4 post partum (including a
two week pre-pairing phase, pairing, gestation and early lactation) at dose levels of 100, 300 and
1000 mg/kg bw/day resulted in treatment-related effects in animals of either sex treated with
1000 mg/kg bw/day.
Degenerative changes in the kidneys of animals of either sex treated with 1000 mg/kg bw/day
were considered to be treatment-related and adverse. Therefore, the No Observable Adverse
Effect Level (NOAEL) for systemic toxicity for adult animals of either sex was considered to be
300 mg/kg bw/day.
Increased pre-implantation loss, a lower number of implantations and a smaller litter size at birth
and a subsequent decline to Day 4 post partum were considered to preclude the 1000 mg/kg
bw/day dose level from consideration as the No Observed Effect Level (NOEL) for reproductive
toxicity.
Therefore the clear `No Observed Effect Level' (NOEL) for reproductive toxicity for animals of
either sex and survival, growth and development of offspring was considered to be 300 mg/kg
bw/day.
Executive summary:

Introduction

The study was performed to screen for potential adverse effects of the test item on reproduction,

including offspring development, and provides an initial hazard assessment for effect on

reproduction. The study is compatible with the requirements of the recommendations of the

OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity

Screening Test” (adopted 27 July 1995).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008

of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the

European Parliament and of the Council on the Registration, Evaluation, Authorisation and

Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve

female Wistar Han™:RccHan™:WIST strain rats; males were treated for 42 consecutive days

and females were treated up to and including Day 4 post partum (including a two week prepairing

phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and

1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with

vehicle alone (Distilled water).

Clinical signs, body weight change, dietary intake and water consumption were monitored

during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis

within each treatment group on Day 15 of the study, with females subsequently being allowed to

litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring,

together with litter size and offspring weights and assessment of surface righting reflex.

Surviving adult males were terminated on Day 43, followed by the termination of all surviving

females and offspring on Day 5 post partum. Any female which did not produce a pregnancy

was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy

examination and histopathological evaluation of reproductive tissues was performed.

Results

Adult Responses

Mortality

One female treated with 1000 mg/kg bw/day was found dead on Day 6. The cause of death for

this animal was likely to be a result of either dosing trauma or aspiration of the test item when

dosed.

Clinical Observations

Animals of either sex treated with 1000 mg/kg bw/day had dark eyes from Day 16 onwards.

Neither the type, incidence or distribution of the remaining clinical signs indicated an adverse

effect of treatment at 100, 300 or 1000 mg/kg bw/day.

Body Weight

No effect of treatment on body weight development was apparent for males throughout treatment

or for females during the pre-pairing period.

During gestation, females treated with 1000 mg/kg bw/day had statistically significantly lower

body weight gain between Day 14 and Day 20.

No such effects were seen in females treated with 300 or 100 mg/kg bw/day.

Food Consumption

There were no obvious effects of treatment on food consumption or food efficiency apparent at

100, 300 or 1000 mg/kg bw/day.

Water Consumption

Daily visual inspection of water bottles did not indicate any overt differences in water

consumption at 100, 300 or 1000 mg/kg bw/day.

Reproductive Performance

Mating

There were no treatment-related effects on mating performance.

Fertility

There were no treatment-related effects in conception rates for treated animals as assessed by the

number of females producing a litter.

Gestation Length

There were no differences in gestation lengths. The distribution for treated females was

compared to controls. Gestation lengths were between 22 and 23½ days.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

Females treated with 1000 mg/kg bw/day appeared to produce a smaller litter compared with

control, principally due to a lower number of implantations resulting from higher preimplantation

loss. Despite the lower litter size, offspring survival at this dosage was slightly

inferior to control with the resulting live litter size attaining statistical significance on Day 1

and 4. This increase in post-natal loss in litters surviving to Day 4 was accompanied by one

female that showed total litter loss post partum. Sex ratio was similar in all groups indicating

that there was no selective effect of maternal treatment on survival for either sex.

Offspring Growth and Development

Litter weights were statistically significantly lower on Day 1 and Day 4 for females treated with

1000 mg/kg bw/day.

Statistical analysis of surface righting reflex data did not reveal any significant intergroup

differences.

Offspring Observations

Clinical signs apparent for offspring on the study were typical of the age observed. Neither the

incidence or distribution of these observations indicated any adverse effect of maternal

treatment.

Pathology

Necropsy

Macroscopic necropsy findings for offspring on the study were typical for the age observed and

neither the incidence or distribution of these observations indicated any adverse effect of

maternal treatment on offspring development at 100, 300 or 1000 mg/kg bw/day.

Organ Weights

The majority of animals treated with 1000 mg/kg bw/day and a proportion of animals treated

with 300 mg/kg bw/day had colored contents throughout most of the digestive tract.

Discoloration or dark tissue was apparent in the mammary gland, liver, spleen, kidney and

stomach of the majority of animals treated with 1000 mg/kg bw/day. Males at this dose level

also had discolored testes and epididymides, whilst the majority females had discolored uterus,

cervix and vagina. Discoloration of the kidneys extended to some animals treated with

300 mg/kg bw/day.

Histopathology

Testes: Pigmented macrophages, particularly notable with PAS staining were present in

increased amounts in Group 4 animals. Testes were not examined from animals in Groups 2

and 3.

Kidneys: At 1000 mg/kg bw/day the following changes were apparent in the kidneys. Tubular

degeneration was present in 12/12 males and 5/12 females. Fine brown pigment within the

tubular cells (probably lipofuscin) was present in 12/12 males and 11/12 females. Other

pigment, likely to be test item or a metabolite was present in 10/12 females. There was an

increase in incidence and severity of hyaline droplets in males. There were no changes related to

treatment in the kidneys from animals in Groups 2 and 3.

Conclusion

The oral administration of FAT 20341/A TE to male rats by gavage for a period of 42

consecutive days and to females by gavage up to and including Day 4 post partum (including a

two week pre-pairing phase, pairing, gestation and early lactation) at dose levels of 100, 300 and

1000 mg/kg bw/day resulted in treatment-related effects in animals of either sex treated with

1000 mg/kg bw/day.

Degenerative changes in the kidneys of animals of either sex treated with 1000 mg/kg bw/day

were considered to be treatment-related and adverse. Therefore, the No Observable Adverse

Effect Level (NOAEL) for systemic toxicity for adult animals of either sex was considered to be

300 mg/kg bw/day.

Increased pre-implantation loss, a lower number of implantations and a smaller litter size at birth

and a subsequent decline to Day 4 post partum were considered to preclude the 1000 mg/kg

bw/day dose level from consideration as the No Observed Effect Level (NOEL) for reproductive

toxicity.

Therefore the clear `No Observed Effect Level' (NOEL) for reproductive toxicity for animals of

either sex and survival, growth and development of offspring was considered to be 300 mg/kg

bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Study Klimisch 1.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The available report (project no. 41304631) was conducted according to OECD 421 under GLP condition. FAT 20341/A TE was administred by daily oral gavage to male and female at dose levels of 100, 300 and 1000 mg/kg. Males were treated for 42 consecutive days and females were treated up to and including Day 4 post partum (including a two week prepairing phase, pairing, gestation and early lactation for females).

Degenerative changes in the kidneys of animals of either sex treated with 1000 mg/kg bw/day were considered to be treatment-related and adverse. Therefore, the No Observable Adverse Effect Level (NOAEL) for systemic toxicity for adult animals of either sex was considered to be300 mg/kg bw/day.

Increased pre-implantation loss, a lower number of implantations and a smaller litter size at birth and a subsequent decline to Day 4 post partum were considered to preclude the 1000 mg/kg bw/day dose level from consideration as the No Observed Effect Level (NOEL) for reproductive toxicity.

Therefore the clear `No Observed Effect Level' (NOEL) for reproductive toxicity for adult animals of either sex and survival, growth and development of male and female offspring was considered to be 300 mg/kg bw/day.


Short description of key information:
The "No Observed Effect Level" (NOEL) for reproductive toxicity for animals of either sex and survival, growth and development of offspring was considered to be 300 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:
Only his study is available

Effects on developmental toxicity

Description of key information
The "No Observed Effect Level" (NOEL) for developmental toxicity for animals of either sex was considered to be 300 mg/kg bw/day.
At 1000 mg/kg effects were observed in the development.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 21 May 2014 and 17 April 2015.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
other: OECD 421
Deviations:
yes
Remarks:
See below
Principles of method if other than guideline:
Deviations from Study Plan
Deviation No.1
Necropsy
Due to macroscopic observations noted during the necropsy of the first set of females treated with 1000 mg/kg bw/day, the kidneys, liver, spleen and stomach were retained at necropsy from all remaining animals.

Deviation No 2
Animal Husbandry
Environment
The Study Plan target values for temperature and relative humidity (RH) were 22 ± 3ºC and 50 ± 20% respectively. While there were no deviations from the target range for temperature, the target range for relative humidity was exceeded for extended periods of time during the study due to adverse weather conditions. The majority of the deviations for relative humidity were less than 80% RH but on some occasions this value was also exceeded, although these instances were
generally transient in nature.
There were no findings for control animals that indicated that animal health had been affected in any way by these extended periods of high humidity and therefore the study was still considered to represent a valid assessment of Test Item toxicity. While it is accepted that these deviations from the target range for relative humidity were less than ideal, overall it is considered that these deviations had no adverse impact on the scientific purpose of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were
obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the
animals were examined for signs of ill-health or injury. The animals were acclimatized for at
least five days during which time their health status was assessed. A total of ninety six animals
(forty eight males and forty eight females) were accepted into the study. At the start of treatment
the males weighed 265 to 340g, the females weighed 191 to 232g, and were approximately
twelve weeks old.
Initially, all animals were housed in groups of four in solid floor polypropylene cages with
stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the
pairing phase, the animals were transferred to polypropylene grid floor cages suspended over
trays lined with absorbent paper on a one male: one female basis. Following evidence of
successful mating, the males were returned to their original cages. Mated females were housed
individually during gestation and lactation in solid floor polypropylene cages with stainless steel
mesh lids and softwood flakes.
The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad
Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. Certificates of
analysis of the batches of diet used are given in Appendix 21. Mains drinking water was
supplied from polycarbonate bottles attached to the cage. Environmental enrichment was
provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd.,
Cheshire, UK) except for paired animals and mated females during final week of gestation and
lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and
lactation. The diet, drinking water, bedding and environmental enrichment were considered not
to contain any contaminant at a level that might have affected the purpose or integrity of the
study.
The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd.,
Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen
air changes per hour and the low intensity fluorescent lighting was controlled to give twelve
hours continuous light and twelve hours darkness. Environmental conditions were continuously
monitored by a computerized system, and print-outs of hourly temperatures and humidities are
included in the study records. The Study Plan target ranges for temperature and relative
humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets
were considered not to have affected the purpose or integrity of the study; see deviations from
Study Plan.
The animals were randomly allocated to treatment groups using a stratified body weight
randomization procedure and the group mean body weights were then determined to ensure
similarity between the treatment groups. The cage distribution within the holding rack was also
randomized. The animals were uniquely identified within the study by an ear punching system
routinely used in these laboratories.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Test Item Preparation
For the purpose of this study the test item was prepared at the appropriate concentrations as a
solution in Distilled water. The stability and homogeneity of the test item formulations were
determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services as part of Harlan
Study Number: 41304630. Results showed the formulations to be stable for at least twenty five
days. Formulations were therefore prepared every two weeks and stored at approximately 4 °C
in the dark.
Samples of the test item formulation were taken and analyzed for concentration of
FAT 20341/A TE at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results
indicate that the prepared formulations were within acceptable ranges for the purpose of this
study.
The test item was administered daily by gavage using a stainless steel cannula attached to a
disposable plastic syringe. Control animals were treated in an identical manner with 5 mL/kg of
Distilled water.
The volume of test and control item administered to each animal was based on the most recent
scheduled body weight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Introduction
The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC/UV) using an external standard technique.

Test item
The test item described in the main part of this study was also used as the analytical standard.

Analytical procedure
Preparation of standard solution
Stock solutions of test item in water were prepared for external standard calibration. An aliquot, 100 mg test item was accurately weighed into a 100 mL volumetric flask and brought to volume with water to yield a solution with a concentration of 1 mg/mL. Aliquots of this stick standard solution were used to prepare working standard solutions in water with a concentration of 0.01 mg/mL.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.

Analysis of samples
The formulations received were diluted with water. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with water this was then shaken to dissolve. Where necessary, sample solutions were further diluted with water to achieve the working concentration.

Preparation of accuracy samples
The accuracy determinations were performed under Harlan Laboratories Ltd. study number 41304630.

Preparation of line standards
The accuracy determinations were performed under Harlan Laboratories Ltd. study number 41304630.

Instrumental set up
HPLC: Agilent Technologies 1200, incorporating auto-sampler and workstation
Column: Gemini C18 3 µ (100 x 4.6 mm id)
Column: 40 °C
Mobile phase:
Eluent A: 20 g/L TBAB: water (10:90 v/v)
Eluent B: 20 g/L TBAB: acetonitrile (10:90 v/v)
Time %B
0 50
5 100
6 100
8 100
Flow rate: 1 mL/min
UV detector wavelength: 295 nm
Injection volume: 100 µL
Retention time: ~3.5 – 5 mins

Study samples and storage
Representative samples were dispatched to the analytical laboratories internally (under ambient conditions) and stored at room temperature until analysis.

Results
Validation of analytical methods
Specificity
The control dose samples and an analysed solvent blank showed no significant interfering response at the retention time of the test item. The standard solutions contained a peak specific for the test item whose area changed accordingly with known concentration; hence the specificity of the method by retention time was confirmed.

Linearity
The linearity determinations were performed under Harlan Laboratories Ltd. study number 41304630.

Accuracy
The accuracy determinations were performed under Harlan Laboratories Ltd. study number 41304630.

Test item formulations
The formulations investigated during the study were found to comprise test item in the range of 99% - 111%.
The test item was found to be stable in the formulations during Harlan Laboratories Ltd. study number 41304630.
In conclusion the results obtained during Harlan Laboratories Ltd. study number 41304630 indicate the accurate use of the test item and Distilled Water as vehicle during this study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was proven.

Discussion
The detection system was found to have an acceptable linearity. The analytical procedure had acceptable recoveries of test item in the vehicle. The method of analysis was validated and proven to be suitable for use.
Details on mating procedure:
On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
Duration of treatment / exposure:
Males: 42 days
Females: 46 days (to Day 4 post partum)
Frequency of treatment:
Daily
Duration of test:
Males: 42 days
Females: 46 days (to Day 4 post partum)
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Twelve
Control animals:
yes, concurrent vehicle
Details on study design:
Chronological Sequence of Study
i. Groups of twelve male and twelve female animals were treated daily at the appropriate
dose level throughout the study (except for females during parturition where applicable).
The first day of dosing was designated as Day 1 of the study.
ii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a
maximum of fourteen days.
iii. Following evidence of mating (designated as Day 0 post coitum) the males were returned
to their original cages and females were transferred to individual cages.
iv. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post
partum. Litter size, offspring weight and sex, surface righting and clinical signs were
also recorded during this period.
v. The male dose groups were killed and examined macroscopically on Day 43.
vi. At Day 5 post partum, all surviving females and surviving offspring were killed and
examined macroscopically.
Maternal examinations:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change
immediately before dosing, soon after dosing, and one hour after dosing during the working
week (except for females during parturition where applicable). All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males
until termination and weekly for females until pairing. During the pairing phase females were
weighed daily until mating was confirmed. Body weights were then recorded for females on
Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults
until pairing. This was continued for males after the mating phase. For females showing
evidence of mating, food consumption was recorded for the periods covering post coitum Days
0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the
lactation period (Days 1-4).
Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males
and for females during the pre-pairing phase. Due to offspring growth and milk production for
lactation, food efficiency for females could not be accurately calculated for females during
gestation and lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.

Reproductive Performance

Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to
fourteen days. Cage tray-liners were checked each morning for the presence of ejected
copulation plugs and each female was examined for the presence of a copulation plug in the
vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence
of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in
situ was taken as positive evidence of mating (Day 0 of gestation) and the males were
subsequently returned to their original holding cages (unless required for additional pairing).
Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the
period of expected parturition. Observations were carried out at approximately 0830 and
1230 hours at weekends and public holidays. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

Necropsy
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by
exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable
barbiturate agent followed by exsanguination on Day 5 post partum. Any females which failed to
achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.

All adult animals and offspring, including those dying during the study, were subjected to a full
external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The epididymides and testes were removed from terminal kill adult males dissected free from fat
and weighed before fixation.

Histopathology
Samples of the following tissues were preserved from all animals from each dose group, in
buffered 10% formalin, except where stated:
Coagulating gland
Prostate
Epididymides ♦
Seminal vesicles
Ovaries
Testes ♦
Mammary gland (females only)
Uterus/Cervix
Pituitary
Vagina
All tissues were dispatched to the histology processing Test Site (Propath UK Ltd, Willow
Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal
Investigator: N Fower). The tissues from control and 1000 mg/kg bw/day dose group animals,
any animals dying during the study, and any animals which failed to mate or did not achieve a
pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and
stained with Hematoxylin and Eosin for subsequent microscopic examination. In addition,
sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic
Acid-Schiff (PAS) stain and examined. Due to macroscopic observations of discoloration in the
kidney, liver, spleen and stomach of females treated with 1000 mg/kg bw/day at necropsy, these
tissues were retained from all remaining animals (see deviations to Study Plan).
Initial histopathology examinations revealed microscopic findings in the testes of males treated
with 1000 mg/kg bw/day. These changes were considered to be related to the physical clearance
of the test item and were not associated with any other pathological change. As there were no
pathological changes associated with the finding the decision was taken, in collaboration with
the Sponsor, not to investigate this finding further. As a result of adverse kidney changes
apparent in previous toxicity studies with the test item, histopathological processing and
examination of the retained kidneys of animals of either sex from all dose groups, where
available, was conducted.
Ovaries and uterine content:
For all surviving females, the uterus was examined for signs of implantation and the number of
uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by
staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora
lutea were also counted.
Fetal examinations:
Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was
recorded. Offspring were individually identified within each litter by tattoo on Day 1 post
partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated
retrospectively from this data)

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Necropsy
Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent.

All offspring, including those dying during the study, were subjected to a full
external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
See below.
Indices:
See below.
Historical control data:
No data
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: See results

Details on maternal toxic effects:
Mortality
One female treated with 1000 mg/kg bw/day was found dead on Day 6. At necropsy, the animal
had a fluid filled chest cavity and the majority of tissues observed for this animal were
discolored black or blue. The cause of death for this animal was unknown but considered likely
to be a result of either dosing trauma or aspiration of the test item when dosed.

Clinical Observations
Animals of either sex treated with 1000 mg/kg bw/day had dark eyes from Day 16 onwards.
Neither the type, incidence or distribution of the remaining clinical signs apparent indicated an
adverse effect of treatment at 100, 300 or 1000 mg/kg bw/day.
Nine males and eight females treated with 1000 mg/kg bw/day showed episodes of increased
salivation post-dose from Day 13 and Day 20 respectively. One female treated with 300 mg/kg
bw/day had increased salivation post-dose on Day 19. Observations of this nature are commonly
experienced following the oral administration of an unpalatable or slightly irritant test item
formulation and in isolation are considered not to be of toxicological importance.
One female treated with 1000 mg/kg bw/day and one male treated with 300 mg/kg bw/day
showed isolated single incidences of noisy and laboured respiration and decreased respiratory
rate. One male and a further female treated with 1000 mg/kg bw/day also showed single
incidences of noisy respiration. It is likely that these observation arose as a consequence of
accidental aspiration of the test item rather than being indicative of systemic toxicity.
Animals of either sex treated with 1000 mg/kg bw/day had episodes of blue or black staining of
the fur throughout the majority of the study. Ten males and seven females treated with
300 mg/kg bw/day had episodes of blue stained fur from Day 8 onwards. One male and two
females treated with 100 mg/kg bw/day had blue stained fur on Day 8, with observations
continuing to Day 9 in males. In the cages of animals of either sex from all treatment groups, the
faecal matter was observed to be blue in colour. The bedding of animals of either sex treated
with 1000 mg/kg bw/day was also stained blue. These observations were considered to reflect
the dark coloration of the test item.

Body Weight
No effect of treatment on body weight development was apparent for males or for females during
the pre-pairing period. Body weight gain during the first week of treatment was statistically
significantly higher than controls for females treated with 300 mg/kg bw/day. An increase in
body weight gain is unlikely to indicate an adverse effect of treatment. During gestation,
females treated with 1000 mg/kg bw/day had statistically significantly lower body weight gain
between Day 14 and Day 20 probably reflecting lower litter size at this dosage.

Food Consumption
There were no obvious effects of treatment on food consumption or food efficiency apparent at 100, 300 or 1000 mg/kg bw/day.

Water Consumption
Daily visual inspection of water bottles did not indicate any overt differences in water consumption at 100, 300 or 1000 mg/kg bw/day.

Reproductive Performance
Mating
There were no treatment-related effects on mating performance.

Fertility
Two control females, one female treated with 100 mg/kg bw/day, one female treated with
300 mg/kg bw/day and one female treated with 1000 mg/kg bw/day were non-pregnant. One
female treated with 1000 mg/kg bw/day had a total litter loss.
There were no treatment-related effects in conception rates for treated animals as assessed by the
number of females producing a litter.

Gestation Length
There were no differences in gestation lengths. The distribution for treated females was
compared to controls. Gestation lengths were between 22 and 23.5 days.

Pathology
Necropsy - Adults
The majority of animals treated with 1000 mg/kg bw/day and a proportion of animals treated
with 300 mg/kg bw/day had colored contents throughout most of the digestive tract.
Discoloration or dark tissue was apparent in the mammary gland, liver, spleen, kidney and
stomach of the majority of animals treated with 1000 mg/kg bw/day. Males at this dose level
also had discolored testes and epididymides, whilst the majority females had discolored uterus,
cervix and vagina. Discoloration of the kidneys extended to some animals treated with
300 mg/kg bw/day. The observations of discolouration are likely to be associated with the
physical properties of the test item. One male treated with 100 mg/kg bw/day had small, flaccid
testes; this male failed to induce pregnancy in its female partner and microscopic examination of the testes revealed marked diffuse tubular atrophy. In the absence of similar effects at
1000 mg/kg bw/day, this finding was considered to be incidental and of no toxicological
importance. One female treated with 1000 mg/kg bw/day had an enlarged, fluid filled uterus and
cervix, in the absence of similar findings in other females treated with 1000 mg/kg bw/day, this
finding was considered to have arisen spontaneously and was considered to be of no
toxicological significance.

Organ Weights
Males treated with 1000 mg/kg bw/day had a statistically significant increase in testes weight,
both absolute and relative to terminal body weight.
There were no adverse effects of treatment on the organ weights measured

Histopathology
Testes: Pigmented macrophages, particularly notable with PAS staining were present in
increased amounts in Group 4 animals.
Kidneys: At 1000 mg/kg bw/day the following changes were apparent in the kidneys. Tubular
degeneration was present in 12/12 males and 5/12 females. Fine brown pigment within the
tubular cells (probably lipofuscin) was present in 12/12 males and 11/12 females. Other
pigment, likely to be test item or a metabolite was present in 10/12 females. There was an
increase in incidence and severity of hyaline droplets in males. There were no changes related to
treatment in the kidneys from animals in Groups 2 and 3.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: See results

Details on embryotoxic / teratogenic effects:
Litter Responses
In total 10 females from control, 11 females from the 100 and 300 mg/kg bw/day dose groups
and 9 females from the 1000 mg/kg bw/day dose group gave birth to a live litter and successfully
reared young to Day 5 of age. At 1000 mg/kg bw/day an additional littering female showed total
litter loss post partum. The following assessment of litter response is generally based on all
litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size, Sex Ratio and Viability
Females treated with 1000 mg/kg bw/day appeared to produce a smaller litter compared with
control, principally due to a lower number of implantations resulting from higher preimplantation
loss. Despite the lower litter size, offspring survival at this dosage was slightly
inferior to control with the resulting difference in live litter size attaining statistical significance
on Days 1 and 4 (p<0.05 and p<0.01 respectively). This increase in post-natal loss in litters
surviving to Day 4 was accompanied by one female that showed total litter loss post partum.
Sex ratio was similar in all groups indicating that there was no selective effect of maternal
treatment on survival for either sex.

Pre-implantation loss for females treated with 100 or 300 mg/kg bw/day was statistically
significantly lower than controls; a lower percentage of loss is considered not to represent an
adverse effect of treatment.

Offspring Growth and Development
Litter weights were statistically significantly lower on Day 1 and Day 4 for females treated with
1000 mg/kg bw/day. The lower litter weights were associated with the smaller litter size apparent
rather than a reduction in offspring weight as offspring weights for male and female offspring at
this dose level were comparable to controls on Day 1 and Day 4.
Clinical signs apparent for offspring on the study were typical of the age observed. Neither the
incidence or distribution of these observations indicated any adverse effect of maternal
treatment. Statistical analysis of surface righting reflex data did not reveal any significant
intergroup differences.

Pathology
Necropsy Offspring
Macroscopic necropsy findings for offspring on the study were typical for the age observed and
neither the incidence or distribution of these observations indicated any adverse effect of
maternal treatment on offspring development at 100, 300 or 1000 mg/kg bw/day.
Abnormalities:
not specified
Developmental effects observed:
not specified

Discussion

The oral administration of FAT 20341/A TE by gavage for a period of 42 consecutive days for

males and up to and including Day 4 post partum for females (including two weeks pre-pairing,

gestation and early lactation for females) at dose levels of 100, 300 and 1000 mg/kg bw/day

resulted in treatment related effects in animals of either sex treated with 1000 mg/kg bw/day.

Animals of either sex treated with 1000 mg/kg bw/day had dark eyes from Day 16 onwards.

There were no treatment-related effects on body weight gain or food consumption for males.

Females treated with 1000 mg/kg bw/day had statistically significantly lower body weight gains

from Day 14 to Day 20 of gestation and these females subsequently went on to produce a smaller

litter size in comparison to controls. The smaller litter size was a consequence of a lower

number of implantation sites resulting from increased pre-implantation loss. Despite the lower

litter size, offspring survival at this dosage was slightly inferior to control with the resulting live

litter size attaining statistical significance on Day 1 and 4. This increase in post-natal loss in

litters surviving to Day 4 was accompanied by one female that showed total litter loss post

partum. Litter weight on both Day 1 and Day 4 were statistically significantly lower than

controls, these intergroup differences were considered to reflect the smaller litter size for females

at 1000 mg/kg bw/day rather than a reduction in offspring weight as both male and female

offspring weight were comparable to controls on Day 1 and Day 4. There were no

histopathological changes in the reproductive tissues or associated tissues which accounted for

the variation in pre-implantation loss or litter size, however an association with treatment cannot

be discounted.

Microscopic examination of the kidneys revealed minimal to mild multifocal tubular

degeneration in all males and five females treated with 1000 mg/kg bw/day. Fine brown

pigment within the tubular cells (probably lipofuscin) was also present in the kidneys of all

males and eleven females at this dose level. Tubular degeneration and the presence of

lipofuscin, an indicator of degenerative change, are considered to be a direct effect of the test

item and represent an adverse effect of treatment. Males treated with 1000 mg/kg bw/day also

had increase in incidence and severity of hyaline droplet deposition in the tubules of the kidneys

when compared to controls. Hyaline droplet formation within the tubules is consistent with the

accumulation of alpha 2u-globulin, a common finding in untreated male rats. This finding is

considered to be specific to the male rat and does not represent a risk to humans. There were no

treatment-related changes in the kidneys of animals of either sex treated with 100 or 300 mg/kg

bw/day.

In terms of the changes apparent in the reproductive indices, the significant changes in the

kidneys of males and females, evident in a number of animals, may have caused an increase in

the overall stress on the females and could have had an effect on the reproductive performance as

determined by slightly lower litter sizes at 1000 mg/kg bw/day.

Pigmented macrophages were present in the testes of males treated with 1000 mg/kg bw/day;

however this finding did not appear to be associated with any other pathological process. The

presence of the pigmented macrophages may be related to the clearance of the specific type of

test item and, in the absence of a pathological change, was considered to be of no toxicological

importance. Males treated with 1000 mg/kg bw/day also had a statistically significant increase

in testicular weight, both absolute and relative to terminal body weight however all individual

values were within the historical control ranges for rats of the strain and age used. Although it is

unlikely that the presence of the pigmented macrophages in the testes of these males resulted in

the increase in testicular weight for these males, a correlation cannot be completely ruled out.

Conclusions:
The oral administration of FAT 20341/A TE to male rats by gavage for a period of 42
consecutive days and to females by gavage up to and including Day 4 post partum (including a
two week pre-pairing phase, pairing, gestation and early lactation) at dose levels of 100, 300 and
1000 mg/kg bw/day resulted in treatment-related effects in animals of either sex treated with
1000 mg/kg bw/day.
Degenerative changes in the kidneys of animals of either sex treated with 1000 mg/kg bw/day
were considered to be treatment-related and adverse. Therefore, the No Observable Adverse
Effect Level (NOAEL) for systemic toxicity for adult animals of either sex was considered to be
300 mg/kg bw/day.
Increased pre-implantation loss, a lower number of implantations and a smaller litter size at birth
and a subsequent decline to Day 4 post partum were considered to preclude the 1000 mg/kg
bw/day dose level from consideration as the No Observed Effect Level (NOEL) for reproductive
toxicity.
Therefore the clear `No Observed Effect Level' (NOEL) for reproductive toxicity for animals of
either sex and survival, growth and development of offspring was considered to be 300 mg/kg
bw/day.
Executive summary:

Introduction

The study was performed to screen for potential adverse effects of the test item on reproduction,

including offspring development, and provides an initial hazard assessment for effect on

reproduction. The study is compatible with the requirements of the recommendations of the

OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity

Screening Test” (adopted 27 July 1995).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008

of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the

European Parliament and of the Council on the Registration, Evaluation, Authorisation and

Restriction of Chemicals (REACH).

Methods…….

The test item was administered by gavage to three groups, each of twelve male and twelve

female Wistar Han™:RccHan™:WIST strain rats; males were treated for 42 consecutive days

and females were treated up to and including Day 4 post partum (including a two week prepairing

phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and

1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with

vehicle alone (Distilled water).

Clinical signs, body weight change, dietary intake and water consumption were monitored

during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis

within each treatment group on Day 15 of the study, with females subsequently being allowed to

litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring,

together with litter size and offspring weights and assessment of surface righting reflex.

Surviving adult males were terminated on Day 43, followed by the termination of all surviving

females and offspring on Day 5 post partum. Any female which did not produce a pregnancy

was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy

examination and histopathological evaluation of reproductive tissues was performed.

Results…….

Adult Responses

Mortality

One female treated with 1000 mg/kg bw/day was found dead on Day 6. The cause of death for

this animal was likely to be a result of either dosing trauma or aspiration of the test item when

dosed.

Clinical Observations

Animals of either sex treated with 1000 mg/kg bw/day had dark eyes from Day 16 onwards.

Neither the type, incidence or distribution of the remaining clinical signs indicated an adverse

effect of treatment at 100, 300 or 1000 mg/kg bw/day.

Body Weight

No effect of treatment on body weight development was apparent for males throughout treatment

or for females during the pre-pairing period.

During gestation, females treated with 1000 mg/kg bw/day had statistically significantly lower

body weight gain between Day 14 and Day 20.

No such effects were seen in females treated with 300 or 100 mg/kg bw/day.

Food Consumption

There were no obvious effects of treatment on food consumption or food efficiency apparent at

100, 300 or 1000 mg/kg bw/day.

Water Consumption

Daily visual inspection of water bottles did not indicate any overt differences in water

consumption at 100, 300 or 1000 mg/kg bw/day.

Reproductive Performance

Mating

There were no treatment-related effects on mating performance.

Fertility

There were no treatment-related effects in conception rates for treated animals as assessed by the

number of females producing a litter.

Gestation Length

There were no differences in gestation lengths. The distribution for treated females was

compared to controls. Gestation lengths were between 22 and 23½ days.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

Females treated with 1000 mg/kg bw/day appeared to produce a smaller litter compared with

control, principally due to a lower number of implantations resulting from higher preimplantation

loss. Despite the lower litter size, offspring survival at this dosage was slightly

inferior to control with the resulting live litter size attaining statistical significance on Day 1

and 4. This increase in post-natal loss in litters surviving to Day 4 was accompanied by one

female that showed total litter loss post partum. Sex ratio was similar in all groups indicating

that there was no selective effect of maternal treatment on survival for either sex.

Offspring Growth and Development

Litter weights were statistically significantly lower on Day 1 and Day 4 for females treated with

1000 mg/kg bw/day.

Statistical analysis of surface righting reflex data did not reveal any significant intergroup

differences.

Offspring Observations

Clinical signs apparent for offspring on the study were typical of the age observed. Neither the

incidence or distribution of these observations indicated any adverse effect of maternal

treatment.

Pathology

Necropsy

Macroscopic necropsy findings for offspring on the study were typical for the age observed and

neither the incidence or distribution of these observations indicated any adverse effect of

maternal treatment on offspring development at 100, 300 or 1000 mg/kg bw/day.

Organ Weights

The majority of animals treated with 1000 mg/kg bw/day and a proportion of animals treated

with 300 mg/kg bw/day had colored contents throughout most of the digestive tract.

Discoloration or dark tissue was apparent in the mammary gland, liver, spleen, kidney and

stomach of the majority of animals treated with 1000 mg/kg bw/day. Males at this dose level

also had discolored testes and epididymides, whilst the majority females had discolored uterus,

cervix and vagina. Discoloration of the kidneys extended to some animals treated with

300 mg/kg bw/day.

Histopathology

Testes: Pigmented macrophages, particularly notable with PAS staining were present in

increased amounts in Group 4 animals. Testes were not examined from animals in Groups 2

and 3.

Kidneys: At 1000 mg/kg bw/day the following changes were apparent in the kidneys. Tubular

degeneration was present in 12/12 males and 5/12 females. Fine brown pigment within the

tubular cells (probably lipofuscin) was present in 12/12 males and 11/12 females. Other

pigment, likely to be test item or a metabolite was present in 10/12 females. There was an

increase in incidence and severity of hyaline droplets in males. There were no changes related to

treatment in the kidneys from animals in Groups 2 and 3.

Conclusion

The oral administration of FAT 20341/A TE to male rats by gavage for a period of 42

consecutive days and to females by gavage up to and including Day 4 post partum (including a

two week pre-pairing phase, pairing, gestation and early lactation) at dose levels of 100, 300 and

1000 mg/kg bw/day resulted in treatment-related effects in animals of either sex treated with

1000 mg/kg bw/day.

Degenerative changes in the kidneys of animals of either sex treated with 1000 mg/kg bw/day

were considered to be treatment-related and adverse. Therefore, the No Observable Adverse

Effect Level (NOAEL) for systemic toxicity for adult animals of either sex was considered to be

300 mg/kg bw/day.

Increased pre-implantation loss, a lower number of implantations and a smaller litter size at birth

and a subsequent decline to Day 4 post partum were considered to preclude the 1000 mg/kg

bw/day dose level from consideration as the No Observed Effect Level (NOEL) for reproductive

toxicity.

Therefore the clear `No Observed Effect Level' (NOEL) for reproductive toxicity for animals of

either sex and survival, growth and development of offspring was considered to be 300 mg/kg

bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Species:
rat
Quality of whole database:
Study 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
Additional information
Justification for selection of Effect on developmental toxicity: via oral route:
Only this study is available

Toxicity to reproduction: other studies

Additional information

The available report (project no. 41304631) was conducted according to OECD 421 under GLP condition. FAT 20341/A TE was administred by daily oral gavage to male and female at dose levels of 100, 300 and 1000 mg/kg. Males were treated for 42 consecutive days and females were treated up to and including Day 4 post partum (including a two week prepairing phase, pairing, gestation and early lactation for females).

Degenerative changes in the kidneys of animals of either sex treated with 1000 mg/kg bw/day were considered to be treatment-related and adverse. Therefore, the No Observable Adverse Effect Level (NOAEL) for systemic toxicity for adult animals of either sex was considered to be300 mg/kg bw/day.

Increased pre-implantation loss, a lower number of implantations and a smaller litter size at birth and a subsequent decline to Day 4 post partum were considered to preclude the 1000 mg/kg bw/day dose level from consideration as the No Observed Effect Level (NOEL) for reproductive toxicity.

Therefore the clear `No Observed Effect Level' (NOEL) for reproductive toxicity for adult animals of either sex and survival, growth and development of male and female offspring was considered to be 300 mg/kg bw/day.

-Maternal toxicity: NOAEL is 300 mg/kg bw and effect for the systemeic toxicity were obeserved at 1000 mg/kg bw.

- Reproductive toxicity: NOEL is 300 mg/kg bw and at 1000 mg/kg effects in the reproduction and the fertility were observed.

- Developmental toxicity: NOEL is 300 mg/kg bw and at 1000 mg/kg developmental effects were observed.

Justification for classification or non-classification

Based on the available data, the test substance caused treatment-related effects at 1000 mg/kg bw/d, on fertility and development. Therefore, this substance shall be classified as category 2 for reproductive toxicity (fertility and developmental effects) according to CLP regulation (EC No. 1272/2008) and H360: May damage fertility on the unborn child. ( State specific effect is known) (State route of exposure if it is conclusively proven that no other routes of exposure cause the hazard).