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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jun 2012 - May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted with no exceptions

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Lot number USRC905, Expiry 31 May 2013

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Strain/Species Crl:CD(SD) rat.
Supplier Charles River (UK) Ltd
Number of animals 128 males and 128 females.
Animals were identified littermates of four of one sex per
litter. 32 litters of 4 males and 32 litters of 4 females were
received and males were not related to females.
Duration of acclimatisation
for the F0 animals
12 days before commencement of treatment.
Age of the F0 animals at start
of treatment
34 to 40 days old.
Weight range of the F0
animals at the start of
treatment
Males: 131 g to 199 g
Females: 116 g to 168 g

Environmental control
Rodent facility Full barrier to minimise entry of external biological and
chemical agents and to minimise the transference of such
agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not
recirculated.
Temperature and relative
humidity
Monitored and maintained within the range of 19-23ºC and
40-70%.
Although conditions were occasionally outside the indicated
ranges, these deviations were minor and of short duration and
were not considered to have influenced the health of the
animals or the outcome of the study (see Deviations from
protocol section).
Lighting Artificial lighting, 12 hours light : 12 hours dark.

Animal accommodation and bedding
Cages Cages comprised of a polycarbonate body with a stainless
steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used throughout the
study except during pairing.
Grid bottomed cages (polypropylene) were used during
pairing. These were suspended above absorbent paper which
was changed daily during pairing.
Cage distribution The cages constituting each group were dispersed in batteries
so that possible environmental influences arising from their
spatial distribution were equilibrated, as far as was
practicable.
Bedding Solid bottom cages contained wood based bedding, which was
changed at appropriate intervals each week.

Number of animals per cage Pre-pairing (acclimatisation
and after selection)
up to 4 animals
Pairing one male and one female animal
Males to termination up to 4 animals
Females after mating (from
Day 0 after mating)
one animal
Females during littering
(from Day 20 after mating)
one animal + litter
Females to termination
(after weaning)
up to 4 animals
Offspring maturation (from
weaning until selection)
litter
Environmental enrichment
Aspen chew block Provided to each cage throughout the study (except during
pairing and lactation) and replaced when necessary.
Polycarbonate shelter Provided to each cage throughout the study (except during
pairing and lactation) and replaced at the same time as the
cages.

Diet supply
Diet SDS VRF1 Certified powdered diet.
The diet contained no added antibiotic or other
chemotherapeutic or prophylactic agent.
Availability Non-restricted.
Water supply
Supply Potable water from the public supply via polycarbonate
bottles with sipper tubes. Bottles were changed at
appropriate intervals.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: Powdered laboratory animal diet (SDS VFR1 Certified).
Details on exposure:
2.2.3 Formulation
The test substance was incorporated into the basal diet to provide the required concentrations
by dilution of an appropriate premix.
The required amount of [test substance] was added to an approximately equal amount of fine
sieved diet (355μm) by gentle stirring, using a spoon. An amount of sieved diet (355μm)
approximately equal to the weight of the mixture was then added and the mixture stirred until
it appeared visibly homogeneous. The doubling-up process was repeated until approximately
half the pre-mix weight was achieved. This mix was then ground using a mechanical grinder
and made up to the required weight using the remaining coarse diet from the sieving process.
At this stage, the mixture was stirred then mixed in a Turbula mixer for 100 cycles at 16 rpm
to ensure all the test material was dispersed in the diet.
Aliquots of the appropriate premix were then diluted with further diet to produce the required
dietary concentrations for feeding to the animals; each batch of treated diet was mixed for a
further 100 revolutions at 16 rpm in the Turbula mixer.
The homogeneity and stability of formulations during storage were confirmed as part of
another study (Huntingdon Life Sciences Study Number: HJL0157). On that study,
homogeneity and stability were confirmed following storage at ambient temperature
(nominally 21 °C) for 22 days.
All diets were prepared weekly at the Huntingdon Research Centre and transported to the Eye
Research Centre for feeding to the animals.
Detailed records of compound usage were maintained. The amount of test substance
necessary to prepare the formulated diets and the amount actually used were determined on
each occasion of diet preparation. The difference between these amounts was checked before
the diets were dispensed.
Details on mating procedure:
F0 pairing commenced After 10 weeks of treatment.
F1 pairing commenced 10 weeks after selection (formal commencement of F1).
Male/female ratio 1:1 from within the same treatment groups (sibling pairing
was not permitted).
Duration of pairing Up to two weeks.
mating
Presence of ejected copulation plugs.
Vaginal smear - examined for the presence of spermatozoa
and the stage of the oestrous cycle.
Day 0 of gestation When positive evidence of mating was detected.
Male/female separation Day when mating evidence was detected.
Pre-coital interval Calculated for each female as the time between first pairing
and evidence of mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
2.2.4 Formulation analysis
The stability of a homogenous preparation of the test substance in the diet, at nominal
concentrations of 500 ppm and 15000 ppm, was demonstrated over a period of up to 22 days
at ambient temperature in Study number HJL0157. Stability was also confirmed following
storage at nominally -20°C for 22 days.
In this study, at specified intervals during treatment, the test formulations were analysed for
achieved concentration of the test substance (see Deviations from protocol section). On five
occasions, approximately 10 weeks apart, four samples (nominally 200 g) were taken from all
groups; two assays from each sample. Stored samples were retained as contingency for
analysis should any result require confirmation.
The method of analysis was an adaptation of a method supplied by the Sponsor; further
details on test procedures and results are presented in the Formulation Analysis Report
(Annex 2).
Achieved dosage for the F1 generation males Level (ppm) Achieved dosage (mg/kg/day) Mean Range 1200 105 68-171 3500 307 199-490 12000 1189 769-1686 Achieved dosage for the F1 generation females Level (ppm) Achieved dosage (mg/kg/day) Pre-pairing Gestation Lactation Mean Range Range Range 1200 111 83-169 89-99 129-209 3500 330 251-505 252-277 379-634 12000 1162 836-1739 873-951 1296-2194
Duration of treatment / exposure:
F0: 10 weeks of treatment before pairing commenced. Males: 19 weeks. Females until d28 post-partum
F1: lifetime (10 weeks beginning after selection on PND 28, 10 weeks of treatment before pairing commenced. Males: 19 weeks. Females until d28 post-partum)
F2: until d28 post-partum
Frequency of treatment:
daily ad libitum
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1200 ppm
Basis:
nominal in diet
Refer to results section for achieved dose
Remarks:
Doses / Concentrations:
3500
Basis:
nominal in diet
Refer to results section for achieved dose
Remarks:
Doses / Concentrations:
12000
Basis:
nominal in diet
Refer to results section for achieved dose
No. of animals per sex per dose:
F0: 28 m/f per dose
F1: 24 m/f per dose
Positive control:
none

Examinations

Parental animals: Observations and examinations:
2.5.1 Clinical observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to
treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the
occupants. Any deviation from normal was recorded at the time in respect of nature and
severity, date and time of onset, duration and progress of the observed condition, as
appropriate.
During the acclimatisation period, observations of the animals and their cages were recorded
at least once per day.
Clinical signs
A detailed weekly physical examination was performed once each week for F0 animals and
for F1 animals selected for the formal F1 generation to monitor general health. Females also
had a detailed examination on Days 0, 5, 12, 18 and 20 after mating and on days 1, 7, 14, 21
and 25 of lactation.
Mortality
A viability check was performed near the start and end of each working day. Animals were
killed for reasons of animal welfare where necessary.
A complete necropsy was performed in all cases as described in section 2.6.
2.5.2 Bodyweight
Animals were weighed as follows:
F0 Males Day that treatment commenced (Week 0).
Each week.
At necropsy.
F0 Females Day that treatment commenced (Week 0).
Each week until mating was detected.
Days 0, 7, 14 and 20 after mating.
Days 1, 4, 7, 14, 21, 25 and 28 of lactation.

2.5.3 Food consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was
recorded was recorded as follows:
F0 males and females: Weekly until paired for mating. From these records the
mean weekly consumption per animal (g/rat/week) was
calculated for each cage.
F0 females: Days 0-6, 7-13 and 14-19 after mating and Days 1-3, 4-6,
7-13 and 14-20 of lactation. From these records the mean
daily consumption (g/rat/day) was calculated for each
animal.

2.5.6 Parturition observations and gestation length
Duration of gestation Time that elapsed between mating and commencement of
parturition.
Parturition observations From Day 20 after mating animals were checked three times
daily for evidence of parturition. The progress and
completion of parturition was monitored; numbers of live
and dead offspring were recorded and any difficulties
observed were noted.
Oestrous cyclicity (parental animals):
2.5.4 Oestrous cycles
Dry smears Smears were taken daily for 22 days before pairing, using
cotton swabs moistened with saline. The smears were
subsequently examined to establish the duration and
regularity of the oestrous cycle.
Wet smears After pairing with the male, daily smearing was continued
using pipette lavage, until evidence of mating was observed.
For four days before scheduled termination (nominally
Days 25 to 28 post partum) daily vaginal smears were taken
and used to determine the stage of the oestrous cycle at
termination.
For females whose litters had previously died, smears were taken on a theoretical
Days 25-28. Females that failed to litter or mate were retained and smeared for four days
starting on the day on which the first batch of ‘true’ Day 25 females started smearing, then
killed with that first batch of females.
Sperm parameters (parental animals):
2.6.1 Sperm analysis
Immediately after scheduled sacrifice of each F0 and selected F1 male, the left vas deferens,
epididymis and testis was removed and the epididymis and testis were weighed. Samples
were obtained from the right side for male 49.
The following tests were performed:
Sperm motility - all groups A sample of sperm was expressed from the vas deferens into
pre-warmed (37C) medium M199, which contained 0.5%
w/v bovine serum albumin (BSA Fraction V). A sample for
assessment was taken into a 100μm depth cannula by
capillary action and at least 200 sperm per animal analysed
using the Hamilton Thorne IVOS Computer Assisted Sperm
Analyser (CASA) version 12.3d. The percentages of motile
and progressively motile sperm were reported.
Sperm morphology - Groups 1
and 4
A 200L aliquot of the sperm/medium mixture (described
above) was diluted with 800L of 10% neutral buffered
formalin. After staining with nigrosine and eosin an
air-dried smear was prepared and examined by light
microscopy for the assessment of sperm morphology. At
least 200 sperm were assessed for each male of groups 1 and
4, where possible. The percentages of normal sperm and
abnormal sperm were reported
Sperm count - Groups 1 and 4 The left cauda epididymis of each male was weighed (right
cauda epididymis for male 49). The left cauda epididymis
for groups 2 and 3 were then frozen (except for group 2
male No. 328; see Deviations from protocol section). For
groups 1 and 4 and male 328, 10 mL of a mixture of 0.9%
saline, 0.01% merthiolate and 0.05% Triton X-100 (SMT)
was added and the cauda epididymis was homogenised for
at least one minute. An aliquot of this mixture was added to
a pre-prepared IDENT stain tube before being assessed for
sperm count using CASA. The concentration (Million/g)
and total number of sperm were reported. The data for male
328 was retained but has not been reported.
Homogenisation-resistant
spermatids count - Groups 1
and 4
The left testis of each male of groups 1 and 4 and male
2M 328 was homogenised for at least two minutes in 25 mL
of SMT. An aliquot of this mixture was added to a preprepared
IDENT stain tube before being assessed for
homogenisation-resistant spermatid count using CASA.
The concentration (Million/g) and total number of
spermatids were reported. Testes for groups 2 and 3 (except
male 328) were frozen. The data for male 328 was retained
but has not been reported.
Litter observations:
2.5.7 Records made during littering phase (F0 and F1 generation)
The records maintained were as follows:
Clinical signs All litters were examined at approximately 24 hours after
birth (Day 1 of age) and then daily thereafter.
Offspring identification On Day 1 of age each offspring was numbered individually
within each litter using a toe tattoo.
Litter size Daily records were maintained of mortality and consequent
changes in litter size from Days 1-21 of age.
On Day 4 of age, litters containing more than ten offspring
were reduced to ten by random culling, leaving, whenever
possible, five male and five female offspring in each litter.
Sex ratio of each litter Recorded on Days 1, 4 (before and after culling) and on
Day 21 of age.
Individual offspring
bodyweights
Recorded on Days 1, 4 (before culling), 7, 14 and 21 of age.
Weaning of offspring The dam was removed from the litter cage and offspring
were weaned on Day 21 of age.
Selection of offspring (F1
generation)
The selection of offspring to form the F1 generation was
made on Day 18 of age.
Postmortem examinations (parental animals):
The organs weighed, tissue samples fixed and sections examined microscopically are detailed
as follows:
Necropsy Histology Pathology
Tissue and regions to be examined Weigh Fix Light
microscopy
ADULT ANIMALS
Abnormalities * * *
Adrenals * * * *
Brain (cerebellum, cerebrum, midbrain) * * # #
Epididymides (caput, corpus and cauda) L+R † † †
Kidneys * * # #
Liver (section from all main lobes) * * # #
Mammary area – caudal b) # #
Ovaries d) L+R * * *
Pituitary * * * *
Prostate * * * *
Seminal vesicles (with coagulation gland) * * * *
Spleen * * # #
Testes L+R † † †
Thyroid with parathyroids a) * # #
Uterus with cervix and oviducts (separate section of each) * * * *
Vagina e) * * *
Target organs, if possible  * * * *
OFFSPRING c)
Abnormalities * $ $
Brain * * # #
Epididymides * # #
Ovaries * # #
Prostate * # #
Seminal vesicles * # #
Spleen * * # #
Testes * # #
Thymus * * # #
Uterus with cervix and oviducts * # #
Vagina * # #
L+R Bilateral organs weighed individually.
* Organs weighed, samples fixed or sections examined microscopically.
# Examined if effects suspected during the study.
† Only one examined - left testis and epididymis reserved for seminology.
$ Consideration was given to the offspring abnormalities recorded; only those deemed appropriate will be examined.
a) Weighed after partial fixation.
b) Females with total litter loss only
c) Procedures for only one male and one female offspring per litter (but abnormalities fixed from all offspring in
litter).
d) 5 sections cut at ca 100 micron intervals from the inner third of each ovary.
e) Section ca 5 mm from vulva.
The retained tissues were checked before disposal of the carcass.

2.6.2 Organ weights
Requisite organs were weighed for all F0 and F1 adult animals killed at scheduled intervals.
For F1 unselected and F2 offspring, one male and one female were selected at random from
each litter for organ weights.
Bilateral organs were weighed individually.
Statistics:
For bodyweight, food consumption, litter
size and survival indices, sexual maturation, organ weight and primordial ovarian follicle
counts data:
F1 approximate test used to determine if data is normally distributed (Bartlett's test for variance homogeneity was not significant at 1%).
If the F1 approximate test for monotonicity of dose-response was not
significant at the 1% level, Williams' test for a monotonic trend was applied.
F1 approximate test was significant, suggesting that the dose-response was not
monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead. Where there
were only two groups, comparisons were made using t-tests.

For litter size and survival indices and sexual maturation data: Fisher's Exact test (pairwise with each group).

Sex ratio by Wald Chi-quare test.

Gestation length, sperm count, copulation plug: exact 2-tailed linear-by-linear test. If significant, higheest dose group excluded and test reapplied (step-down repeated until significance was lost).

Estrous cycles: 1-tailed linear-by-linear test, using step down approach when significant.
Reproductive indices:
Mating performance and fertility
Individual data was tabulated. Group values were calculated for males and females
separately for the following:
Percentage mating = Number animals mating Animals paired x 100
Conception rate (%) = Number aniAmnailms aalcsh mieavtiendg pregnancy x 100
Fertility index (%) = Number anAimnaimls aalcs hpiaeivriinngg pregnancy x 100
Huntingdon Life Sciences
HJL0156
39
Gestation length
Gestation length was calculated as the number of gestation days up to and including the day
on which offspring were first observed, with Day 1 = day of mating for calculation purposes.
Where parturition had started overnight, this value was adjusted by subtracting half of one
day. Gestation index was calculated for each group as:
Gestation index (%) = Number of live litters born Number pregnant x 100
Offspring viability indices:
Survival indices
The following were calculated for each litter:
Post-implantation survival index (%) =
Total number of offspring born
Total number of uterine implantation sites x 100
Post-implantation survival index was expressed as 100% where the number of offspring
exceeded the number of implantation sites recorded.
Live birth index (%) = Number of live offspring on Day 1 after littering Total number of offspring born x 100
Viability index (%) = Number of live offspring on Day 4 before culling Number live offspring on Day 1 after littering x 100
Lactation index (%) = Number of live offspring on Day 21 after littering Number live offspring on Day 4 (after culling) x 100
Group mean values were calculated from individual litter values.
Sex ratio
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring
on Days 1, 4 (before culling) and 21 of age.
Percentage males =
Number of males in litter
Total number of offspring in litter x 100
Group mean values were calculated from individual litter values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

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

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
12 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no consistent effect of treatment on mean bodyweight gain but at 12000 ppm for
F1 females, mean bodyweight gain during Days 0-20 of gestation was marginally but
statistically significantly lower than in Controls; mean bodyweights were significantly lower
than Controls from Day 14 of gestation until Day 14 of lactation. Bodyweight gain during
Days 1-14 of lactation was similar to Controls. Between Days 14 and 28 of lactation, dams
are expected to show mean bodyweight loss so that overall mean weight loss is apparent
during lactation; the extent of the overall mean weight loss in the 3500 or 12000 ppm groups
was significantly lower than in Controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During Days 1-13 of lactation mean food
consumption of F1 females receiving 12000 ppm was marginally but statistically
significantly lower than in Controls.
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute and bodyweight relative pituitary weights for F1 adult males receiving 12000 ppm
were marginally but statistically significantly lower than in Controls. Bodyweight relative
kidney weights for F1 males and females receiving 12000 ppm were marginally but
significantly higher than in Controls.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

Bodyweight
Bodyweight and bodyweight gain prior to pairing for F1 males and females was not adversely
affected by treatment with [test substance] when compared with Controls.
Among males receiving 12000 ppm mean bodyweight gain during Week 4-5, 7-8 and 9-10
and during Week 7-8 for males receiving 3500 ppm was marginally but statistically
significantly low when compare with Controls.
Bodyweight gain and overall group mean bodyweight gain for F1 females prior to pairing
was unaffected by treatment. However, bodyweight gain during Weeks 9-10 prior to pairing
was statistically significantly lower for F1 females receiving MCP2484, when compared to
Controls.
At 12000 ppm, mean bodyweight gain during Days 0-20 of gestation was marginally but
statistically significantly lower than in Controls; mean bodyweights were significantly lower
than Controls from Day 14 of gestation until Day 14 of lactation. Bodyweight gain during
Days 1-14 of lactation was similar to Controls. Between Days 14 and 28 of lactation, dams
are expected to show mean bodyweight loss so that overall mean weight loss is apparent
during lactation; the extent of the overall mean weight loss in the 3500 or 12000 ppm groups
was significantly lower than in Controls.


The mean ovarian primordial follicle count in the 12000 ppm group was marginally but
statistically significantly lower than in Controls. However, no effect of treatment was
inferred since mean counts were highly variable in each group and review of the individual
values did not reveal any evidence for a treatment related trend to lower follicle counts; two
control females had atypically high mean follicle counts in excess of 32.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
12 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
mortality
other: Reproductive Perfomance

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 12000 ppm, body weight gains of F2 male and female offspring between Days 1 and 21 of
age were marginally, but statistically significantly lower than in Controls.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
12 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Executive summary:

There were no clinical signs observed during the study that were related to treatment with

Dietary administration of [test substance], a base oil, to adult male and female F0 and

F1 Crl:CD (SD) rats at dietary levels up to 12000 ppm was not associated with any overt

signs of systemic toxicity, or any effects on reproductive performance.

There were no clinical signs observed during the study that were related to treatment with

[test substance].

One F0 female receiving 12000 ppm appeared to complete parturition but showed poor

clinical condition and was killed for welfare reason on Day 1 of lactation. Microscopic

examination of the uterus revealed lesions which were considered to have contributed to the

poor condition of this female and macroscopic examination revealed that parturition had been

incomplete and 5 fetuses were present in utero. Since this was a single incidence of

incomplete parturition among F0 females which was not replicated among F1 females

receiving 12000 ppm this isolated finding was considered to be unrelated to treatment.

There were no effects of treatment on oestrous cycles, pre-coital interval, mating

performance, fertility, gestation lengths and gestation index, offspring organ weights, sperm

motility, concentrations or morphology, for either generation at dietary concentrations up to

12000 ppm of [test substance]. Two F0 females receiving 12000 ppm were observed with total

litter loss, which subsequently resulted in the viability index being marginally lower at this

dose level. This was not considered to be treatment related as it was not replicated in the

F1-F2 generation; where F1 animals have greater exposure to [test substance] compared to that of

the F0 generation. F1 parental animals eat treated diet from a younger age and thus through

more stages of life cycle than F0 parent animals; so if this finding was treatment related, total

litter loss should also have been apparent in F2 litters. There was no effect of treatment on

the ability of the F0 or F1 females to successfully litter and rear their offspring to weaning.

Litter size, survival of the offspring and the offspring sex ratio were not affected by treatment

of either generation.

Bodyweight, bodyweight gain and food consumption for F0 adults were unaffected by

treatment with [test substance]. Among F1males receiving 12000 ppm mean bodyweight gain

during Week 4-5, 7-8 and 9-10 and during Week 7-8 for males receiving 3500 ppm was

marginally but statistically significantly low when compare with Controls. Bodyweight gain

during Weeks 9-10 prior to pairing was statistically significantly lower for F1 females

receiving [test substance], when compared to Controls. However, this had no impact on overall

group mean bodyweight gain prior to pairing or group mean bodyweights. At 12000 ppm,

mean bodyweight gain during Days 0-20 of gestation was marginally but statistically

significantly lower than in Controls; mean bodyweights were significantly lower than

Controls from Day 14 of gestation until Day 14 of lactation, which correlates with the

reduced food consumption at this period; the extent of the overall mean weight loss in the

3500 or 12000 ppm groups was significantly lower than in Controls. These effects on

bodyweight and food consumption were not consistent and not considered adverse as they

had no overall effect on clinical condition, survival or reproductive performance..

One F0 female receiving 12000 ppm appeared to complete parturition but showed poor

clinical condition and was killed for welfare reason on Day 1 of lactation. Microscopic

examination of the uterus revealed lesions which were considered to have contributed to the

poor condition of this female and macroscopic examination revealed that parturition had been

incomplete and 5 fetuses were present in utero. Since this was a single incidence of

incomplete parturition among F0 females which was not replicated among F1 females

receiving 12000 ppm this isolated finding was considered to be unrelated to treatment.

There were no effects of treatment on oestrous cycles, pre-coital interval, mating

performance, fertility, gestation lengths and gestation index, offspring organ weights, sperm

motility, concentrations or morphology, for either generation at dietary concentrations up to

12000 ppm of [test substance]. Two F0 females receiving 12000 ppm were observed with total

litter loss, which subsequently resulted in the viability index being marginally lower at this

dose level. This was not considered to be treatment related as it was not replicated in the

F1-F2 generation; where F1 animals have greater exposure to [test substance] compared to that of

the F0 generation. F1 parental animals eat treated diet from a younger age and thus through

more stages of life cycle than F0 parent animals; so if this finding was treatment related, total

litter loss should also have been apparent in F2 litters. There was no effect of treatment on

the ability of the F0 or F1 females to successfully litter and rear their offspring to weaning.

Litter size, survival of the offspring and the offspring sex ratio were not affected by treatment

of either generation.

Bodyweight, bodyweight gain and food consumption for F0 adults were unaffected by

treatment with [test substance]. Among F1males receiving 12000 ppm mean bodyweight gain

during Week 4-5, 7-8 and 9-10 and during Week 7-8 for males receiving 3500 ppm was

marginally but statistically significantly low when compare with Controls. Bodyweight gain

during Weeks 9-10 prior to pairing was statistically significantly lower for F1 females

receiving [test substance], when compared to Controls. However, this had no impact on overall

group mean bodyweight gain prior to pairing or group mean bodyweights. At 12000 ppm,

mean bodyweight gain during Days 0-20 of gestation was marginally but statistically

significantly lower than in Controls; mean bodyweights were significantly lower than

Controls from Day 14 of gestation until Day 14 of lactation, which correlates with the

reduced food consumption at this period; the extent of the overall mean weight loss in the

3500 or 12000 ppm groups was significantly lower than in Controls. These effects on

bodyweight and food consumption were not consistent and not considered adverse as they

had no overall effect on clinical condition, survival or reproductive performance.

Among F2 offspring in the 12000 ppm group, mean body weight gains between Days 1 and

21 were marginally (less than 10%) lower than in Controls. However, this minor effect of

treatment on the growth of the offspring is considered not to be adverse since there was no

effect on the survival or general condition of the offspring.

Absolute and bodyweight relative pituitary weights for F1 adult males receiving 12000 ppm

were marginally but statistically significantly lower than in Controls and bodyweight relative

kidney weights for F1 adult males and females receiving 12000 ppm were marginally but

significantly higher than in Controls. These findings were considered not to be of any

toxicological significance as there were no corresponding macroscopic or histopathological

findings.

Macroscopic and histopathological examination of the F0 and F1 adult males and females did

not reveal any findings that were considered to be related to treatment with [test substance].

Macroscopic examination of any abnormal tissues of F1 and F2 offspring did not reveal any

findings that were related to treatment with [test substance] at dietary levels up to 12000 ppm.

The mean ovarian primordial follicle count in the 12000 ppm group was marginally but

statistically significantly lower than in Controls. However, no effect of treatment was

inferred since mean counts were highly variable in each group and review of the individual

values did not reveal any evidence for a treatment related trend to lower follicle counts;

2 control females had atypically high mean follicle counts in excess of 32. The mean number

of implantation in these females was identical to Controls so even if the marginally lower

primordial follicle counts were viewed as treatment related it would not be viewed as an

adverse finding within the context of this study as it did not affect reproductive performance.