Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: screening
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Between 6 May 2008 and 20 November 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)) Date of inspection 19/08/2008. Date of signature 04/03/2009.
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Wistar Han™:HsdRccHan™:WIST strain rats from Harlan UK, Ltd, Oxon, UK
- Age at study initiation: Approximately 12 weeks old
- Weight at study initiation: 264 to 315 g (male); 158 to 219 g (female)
- Fasting period before study: Not applicable
- Housing:Initially, all animals were housed in groups of five in solid floor polypropylene cages with
stainless steel mesh lids and softwood flake bedding (Harlan UK Ltd). During the mating
phase, animals were transferred to polypropylene grid floor cages suspended over trays
lined with absorbent paper on a one male: one female basis within each dose group.
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.
- Use of restrainers for preventing ingestion (if dermal): Not applicable
- Diet: The animals were allowed free access to food. A pelleted diet Rodent 2018C
Teklad Global Certified Diet Harlan UK Ltd, Oxon, UK was used throughout the study
period. The diet was considered not to contain any contaminant at a level that might
have affected the purpose or integrity of the study.
- Water:The animals were allowed free access to water. Mains drinking water was supplied from polycarbonate bottles attached to the cage. The drinking water was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Acclimation period: For 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness

IN-LIFE DATES: From: Day 1 To: Day 43 (males); Day 1 To: Day 5 post partum (females).
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: Not applicable
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: For the purpose of the study, the test material was prepared at the appropriate
concentrations as a suspension in distilled water. The stability and homogeneity of the
test material formulations were determined by Harlan Laboratories Ltd. Formulations were prepared daily and analysed weekly.
Samples were taken of each test material formulation and were analysed for
concentration of Cellcore QX at Harlan Laboratories Ltd. The results indicate
that the prepared formulations were within ± 10% of the nominal concentration.

DIET PREPARATION
A pelleted diet Rodent 2018C Teklad Global Certified Diet Harlan UK Ltd, Oxon, UK was used throughout the study period.
- Rate of preparation of diet (frequency): Daily
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
Distilled water
- Justification for use and choice of vehicle (if other than water): Not applicable
- Concentration in vehicle: 100, 30 and 10 mg/ml
- Amount of vehicle (if gavage): 5 mg/kg
- Lot/batch no. (if required): Not applicable
Details on mating procedure:
- M/F ratio per cage: 1/1 (Animals were paired on a 1 male: 1 female basis within each dose group)
- Length of cohabitation: Up to 14 days
- Proof of pregnancy: 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 the oestrous cycle 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)
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.: No data
- Further matings after two unsuccessful attempts: No data
- After successful mating each pregnant female was caged: Mated females were housed individually during the period of gestation and lactation.
- Any other deviations from standard protocol: Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of Lithium Nickel Cobalt Aluminium Oxide in the test material formulations was determined using a gravimetric technique. The test material formulations were weighed into tared glass sintered crucibles and then
rinsed with acetone to leave a test material residue. The samples were then dried in an
oven at approximately 105oC before allowing to cool over silica gel in a desiccator and
re-weighed.
The test material formulations were mixed thoroughly and samples were taken from the
top, middle and bottom of the container, shaking between sampling. Sampling was
performed in triplicate.
The test material formulations were sampled and analysed within three days of
preparation.
The analytical method has been satisfactorily validated in terms of specificity and
accuracy for the purposes of the study.
Duration of treatment / exposure:
Day 1 To: Day 42 (males); Day 1 To: Day 4 of lpost partum (females).
Frequency of treatment:
Daily
Details on study schedule:
i) Groups of ten male and ten 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) Prior to the start of treatment and once weekly thereafter, all animals were
observed for signs of functional/behavioural toxicity.
iii) One day prior to pairing (Day 14), blood samples were taken from five males and
five females, randomly selected from each dose group and analysed for
haematological and blood chemical assessment.
iv) On Day 15, animals were paired on a 1 male: 1 female basis within each dose
group for a maximum of four days.
v) 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.
vi) On completion of mating (during Week 6), five selected males per dose group
were evaluated for functional/sensory responses to various stimuli was performed.
vii) Pregnant females were allowed to give birth and maintain their offspring until Day
5 post partum. Evaluation of each litter size, litter weight, mean offspring weight
by sex, clinical observations and landmark developmental signs were also
performed during this period.
viii) At Day 4 post partum, five selected females per dose group were evaluated for
functional/sensory responses to various stimuli.
ix) Additional blood samples were taken from five males from each dose group for
haematological and blood chemical assessments on Day 42. Following
completion of the female gestation and lactation phases, the male dose groups
were killed and examined macroscopically.
x) Additional blood samples were taken from five randomly selected females from
each dose group at termination for haematological and blood chemical
assessment on Day 4 post partum. At Day 5 post partum, all surviving females
and surviving offspring were killed and examined macroscopically.
xi) The surviving high dose treatment animals were terminated early due to excessive
toxicity. Additional blood samples were taken at termination for haematological
and blood chemical assessments.
Remarks:
Doses / Concentrations:
50 mg/kg/day (10 mg/mk)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg/day (30 mg/ml)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg/day (100 mg/ml)
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose (including control).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of a preliminary range-finder performed as part of the study.
- Rationale for animal assignment (if not random): The rat was selected for this study as it is a readily available rodent species historically
- Other: Not applicable
Positive control:
Not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, up to thirty minutes after dosing, and one and five hours after
dosing, during the working week. Animals were observed immediately before dosing,
soon after dosing, and one hour after dosing at weekends and public holidays (except for
females during parturition where applicable).
- Cage side observations checked: Overt signs of toxicity, ill-health and behavioural change.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, up to thirty minutes after dosing, and one and five hours after
dosing, during the working week. Animals were observed immediately before dosing,
soon after dosing, and one hour after dosing at weekends and public holidays (except for
females during parturition where applicable).

BODY WEIGHT: Yes
- Time schedule for examinations:Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for
males until termination and weekly for females until mating was evident. Bodyweights
were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and
4 post partum.

FOOD CONSUMPTION: Yes
- During the maturation period, weekly food consumption was recorded for each cage of
adults. This was continued for males after the mating phase. For females showing
evidence of mating, food consumption was recorded for the periods covering Days 0-7,
7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1
and 4 post partum. Weekly food consumptions were performed weekly for each cage of
adults throughout the study period.

FOOD EFFICIENCY: Yes
- Food efficiency (the ratio of bodyweight change/dietary intake) was calculated
retrospectively for males and females throughout the study period.

WATER CONSUMPTION: Yes
- Time schedule for examinations:Water intake was observed daily by visual inspection of water bottles for any overt
changes.
A possible treatment-related effect was detected on the range finder animals, therefore
gravimetric measurements were initiated from Day 1 throughout to study termination.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group on Day 14 (day prior to pairing) and five males and five females selected from each surviving treatment group on Day 42. The surviving high dose treatment animals were killed on Day 41. Blood samples were obtained from the lateral tail vein at Day 14 and by cardiac puncture at Days 41 and 42. Animals were not fasted prior to sampling.
- Anaesthetic used for blood collection: Not stated in the report
- Animals fasted: No
- How many animals:
Five males and 5 females selected from each test and control group on Day 14.
Five males and 5 females selected from each surviving treatment group on Day 42
- Parameters checked in tables 18 -21 which are attached were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
(Behavioural assessment)
Prior to the start of treatment and at weekly intervals thereafter
(Functional Performance Tests)
Prior to termination
(Sensory Reactivity)
Prior to termination

- Dose groups that were examined:
(Behavioural assessment)
All animals
(Functional Performance Tests)
Five selected males and females per dose level
(Sensory Reactivity)
Five selected males and females per dose level

- Parameters examined:
(Behavioural assessment)
Detailed individual clinical observations were performed for each animal using a purpose-built arena.
The following parameters were observed:
Gait, hyper/hypothermia, tremors, skin colour, twitches, respiration, convulsions, palpebral closure, bizarre/abnormal/stereotypic behaviour, urination, salivation, defecation, pilo-erection, transfer arousal, exophthalmia, tail elevation and lachrymation
(Functional Performance Tests)
Motor Activity.
Purpose-built 44 infra-red beam automated activity monitors were used
to assess motor activity. Animals were randomly allocated to the activity monitors. The
tests were performed at approximately the same time each day, under similar laboratory
conditions. The evaluation period was thirty minutes for each animal. The percentage of
time each animal was active and mobile was recorded for the overall thirty minute period
and also during the final 20% of the period (considered to be the asymptotic period).
Forelimb/Hindlimb Grip Strength.
An automated meter was used. Each animal was
allowed to grip the proximal metal bar of the meter with its forepaws. The animal was
pulled by the base of the tail until its grip was broken. The animal was drawn along the
trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal
was pulled by the base of the tail until its grip was broken. A record of the force required
to break the grip for each animal was made. Three consecutive trials were performed for
each animal.
(Sensory Reactivity)
Animals were assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed:
Grasp response, touch escape, vocalisation, pupil reflex, toe pinch, startle reflex, tail pinch, blink reflex and finger approach


OTHER:
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 the
oestrous cycle 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 mating
ii) Date and time of observed start of parturition
iii) Date and time of observed completion of parturition
iv) Duration of gestation
Oestrous cyclicity (parental animals):
A vaginal smear was prepared for each female and the stage of the oestrous cycle was recorded.
Sperm parameters (parental animals):
Parameters examined in all male parental generations:testis During histopathology, the male epididymides were examined for spermatocoel granuloma formation.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in offspring: Number of offspring born, number and sex of offspring alive recorded daily and reported on Day 1 and 4 post partum, clinical condition of offspring from birth to Day 5 post partum, individual offspring and litter weights on Day 1 and 4 post partum, physical Development and pathology.

GROSS EXAMINATION OF DEAD PUPS: Dying offspring during the study were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Adult surviving males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43.
- Maternal animals: Adult surviving females were killed by
intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Any females that failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.

GROSS NECROPSY / ORGAN WEIGHTS

For all females the uterus was examined for signs of implantation and the number of
uterine implantations in each born was recorded. This procedure was enhanced; as
necessary, by staining the uteri with a 1% ammonium polysulphide solution. In addition, the corpora lutea of all ovaries from pregnant females were counted at necropsy. All adult animals, including those dying during the study, were subjected to
a full external and internal examination, and any macroscopic abnormalities were
recorded.

HISTOPATHOLOGY

The following organs, removed from the five selected males and parental females from
each group that were killed at the end of the study, were dissected free from fat and
weighed before fixation.
Adrenals, ovaries, brain, spleen, epididymides, testes, heart, thymus, kidneys, thyroid and liver.

The following reproductive organs were weighed from all animals that were killed at the end of the study: ovaries, epididymides and testes.

Samples of the following tissues were preserved from five males and five females from
each dose group, in buffered 10% formalin except where indicated.
Adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, coagulating gland, colon, duodenum, epididymides (preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) up to 48 hours later), eyes (fixed in Davidson’s fluid), gross lesions, heart, ileum, jejunum, kidneys, liver, lungs (with bronchi)(lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative), lymph nodes (cervical and mesenteric), mammary gland, muscle (skeletal), ovaries, pancreas, pituitary, prostate, oesophagus, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical), mid thoracic and lumbar, spleen, stomach, thyroid, trachea, testes (preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) up to 48 hours later), thymus, urinary bladder, uterus/cervix and vagina.

The following tissues were also removed from the remaining animals:coagulating gland, epididymides, ovaries, pituitary, prostate, seminal vesicles, testes and uterus/cervix.

All tissues were despatched to Harlan Laboratories Ltd, Switzerland (Principal
Investigator: K Weber). The tissues from five selected control, 150 and 500 mg/kg/day
dose group animals and those animals dying during the study, were prepared as paraffin
blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin
for subsequent microscopic examination. The tissues from oagulating gland, epididymides, ovaries, pituitary, prostate, seminal vesicles, testes and uterus/cervix from the remaining
control, 150 and 500 mg/kg/day were also processed.
Since there were indications of treatment-related changes, examination was
subsequently extended to include similarly prepared sections of kidney and spleen from
five animals per sex from the low dose groups.
Microscopic examination was conducted by the Study Pathologist. All findings were
entered into the ROELEE Pathology computerisation system for tabulation and report
production.
Postmortem examinations (offspring):
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Necropsy findings checked in table 28 were included. All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
The following parameters were subjected to statistical analysis:
Quantitative functional performance data
Bodyweight and bodyweight change
Food consumption during gestation and lactation
Haematology, blood chemistry, absolute and bodyweight relative organ weights
Litter data
Sex ratio
Implantation losses and viability indices
Offspring bodyweight and bodyweight change
Offspring surface righting
Adult absolute and bodyweight relative organ weights
The following statistical procedures were used:
Data were assessed for dose response relationships by linear regression analysis,
followed by one way analysis of variance (ANOVA) incorporating Levene’s test for
homogeneity of variance. Where variances were shown to be homogenous, pairwise
comparisons were conducted using Dunnett’s test. Where Levene’s test showed
unequal variances the data were analysed using non-parametric methods: Kruskal-
Wallis ANOVA and Mann-Whitney ‘U’ test.
Non-parametric methods were used to analyse implantation loss, offspring sex ratio and
landmark developmental markers.
Probability values (p) are presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
In addition, histopathological findings were analysed using the following methods:
Histopathology data were analysed using the following methods to determine significant
differences between control and treatment groups for the individual sexes:
1. Chi-squared analysis for differences in the incidence of lesions occurring with an
overall frequency of 1 or greater.
2. Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of
severity grades for the more frequently observed graded conditions.
Probability values (p) were calculated as follows:
p<0.001 +++ --- ***
p<0.01 ++ -- **
p<0.05 + - *
p<0.1 (+) (-) (*)
p≥0.01 N.S. (not significant)
Plus (+) signs indicate positive differences from the control group and minus (-) signs
indicate negative differences.

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 for 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:
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)] x 100

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 1 (%) = (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 Day 1 and 4post partum, using the following formula:

(Number of male offspring ÷ Total number of offspring) x 100
Clinical signs:
effects observed, treatment-related
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:
effects observed, treatment-related
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
(Clinical signs)
A summary incidence of daily clinical observations is given in Table 2 and Table 3 attached.
Prior to being found dead one male treated with 500 mg/kg/day showed staining of the mouth on Day 16 with diarrhoea and stained ano-genital region seen on Day 19. Whilst one female, treated with 500 mg/kg/day, displayed, pilo-erection and pallor of the extremities on Day 40.
Episodes of staining of the snout, hunched posture, pilo-erection, hypothermia, pallor of the extremities, dehydration, staining of the mouth and tiptoe gait were observed in two 500 mg/kg/day females killed in extremis. Reduced respiration was also evident in one of these animals.
One female treated with 150 mg/kg/day showed episodes of hunched posture, pilo-erection, dehydration, diarrhoea, fur loss, tiptoe gait, diuresis, emaciation and lethargy prior to being killed in extremis.
Dark faeces were noted in the cages of animals of either sex treated with 500 mg/kg/day from Day 16 onwards.
Incidental findings of increased salivation was detected immediately prior to dosing in one 500 mg/kg/day male, on Day 39, whilst an isolated episode of red/brown staining of the mouth was noted in another male from this treatment group on Day 22. Increased salivation and staining around the mouth is often reported following oral administration of an unpalatable or slightly irritant test material formulation and considered not to be an indication of systemic toxicity. In addition, isolated episodes of noisy respiration were also detected in two males from this treatment group on Days 21 and 39 respectively. An incidence of fur stained black by the test material was seen in one 50 mg/kg/day male on Day 42, whilst one female from this treatment group showed fur loss between Days 37 and 39. One control female showed episodes of fur loss between Days 41 and 42. In isolation these findings are considered incidental and of no toxicological importance.
(Mortality)
Two males treated with 500 mg/kg/day were found dead, one on Day 20 and one on Day 29. Two females from this dose group were also found dead, one on study Day 12 and one on Day 41.
On Day 39, two 500 mg/kg/day females were killed in extremis whilst one 150 mg/kg/day female was killed in extremis on Day 42.
There were no further unscheduled deaths.
The 500 mg/kg/day treatment group was considered to be excessive and terminated on Day 41.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
(Body weight)
No adverse effect on bodyweight development was detected for treatment animals, in comparison to controls.
(Food consumption)
No adverse effect on dietary intake was detected for treated animals, in comparison to controls.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Not examined.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no intergroup differences in mating performance including pre-coital interval.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No treatment-related changes were seen. Spermatocoel
granuloma formation is observed occasionally as a spontaneous condition in the
epididymis of male rats.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
(Mating)
There were no intergroup differences in mating performance or gestation lengths.
Ten out of ten control, 50 and 150 mg/kg/day females together with nine out of nine 500 mg/kg/day females showed signs of positive mating.
(Fertility)
No adverse effect on fertility was evident for treatment animals in comparison to controls. Pregnancy was achieved for eight out of ten control females. Nine out of ten females treated with 50 and 150 mg/kg/day and seven out of nine females treated with 500 mg/kg/day.
(gestation length)
There was no adverse effect on gestation lengths between treated and control females. Females from all treatment groups went on to give birth following 22 to 24 days of gestation.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No abnormalities were detected for treatment animals, in comparison to controls.

GROSS PATHOLOGY (PARENTAL ANIMALS)
A summary incidence of necropsy findings for adults are given in Tables 29 and 30 attached.
There were macroscopic abnormalities considered to be attributable to treatment.
One 500 mg/kg/day male showed small seminal vesicles, whilst another showed a mass attached to the mandibular lymph nodes. In addition three females from this treatment group had dark contents of the gastrointestinal tract, two of these also showed a distended stomach. Dark contents of the stomach were also seen in two of these females with enlarged adrenals also noted in one of these. One female showed a dark mass on the left horn of the uterus.
A yellow fluid filled mass on the right epididymis was evident in one 50 mg/kg/day male. Two females treated with 50 mg/kg/day showed epithelial sloughing of the glandular region of the stomach, whilst two additional females from this treatment group showed epithelial sloughing of both regions of the stomach. In addition, reddened lungs were seen in one 50 mg/kg/day female. In the absence of any histopathological finding to suggest an effect of treatment these findings are considered to be of no toxicological significance.
Epithelial sloughing of the glandular region of the stomach was seen in two control females one of which also showed both ovaries encased in a clear fluid filled sac. There was no adverse effect on implantations for this animal and histopathological examination did not reveal any treatment-related effects and as such, this finding was considered to be a congenital abnormality and unrelated to treatment. One additional control female showed a dark viscous fluid filled uterus.
One interim death male treated with 500 mg/kg/day showed a distended stomach with autolysis of the gastrointestinal tract and small seminal vesicles. Whilst another male, showed a distended stomach, with fluid filled dark viscous contents and a small caecum. In addition, one interim death female treated with 500 mg/kg/day was found partially cannibalised, with enlarged adrenals and an enlarged stomach, with dark contents. Furthermore, a distended stomach, with autolysis of the gastrointestinal tract was seen in another interim death female. Dark coloured contents of the stomach and enlarged adrenals were evident in the remaining interim death females treated with 500 mg/kg/day. One also showed dark contents of the gastrointestinal tract.
One interim death female treated with 150 mg/kg/day showed dark coloured contents of the stomach with the remaining gastrointestinal tract empty.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Summary incidences of histopathological findings are given in Tables 35 and 36 attached. Individual histopathology is presented in Appendices 26 and 27 attached.
As the high dose animals were killed before scheduled termination the intermediate dose group was taken to be the effective high dose, having received 150 mg/kg/day of the test material for the full dosing period. A comparison of this group with control animals is thus more reliable as an indicator of treatment-related changes, although the findings for high dose animals are also taken into consideration. The following treatment-related changes were observed:
SPLEEN: Generally higher grades of severity of extramedullary haemopoiesis were seen in relation to treatment for males, and possibly also for females, treated with 500 mg/kg/day which died or were killed prematurely. Animals of either sex treated with 150 mg/kg/day and surviving until scheduled termination of the study showed a similar effect compared with controls, although females demonstrated a stronger effect than males at this dose level. There was no evidence of a convincing effect of treatment on extramedullary haemopoiesis among animals of either sex treated with 50 mg/kg/day.
KIDNEY: Globular accumulations of eosinophilic material were observed in the tubular epithelium of 3/5 males treated with 500 mg/kg/day all of which died before scheduled termination; one control was similarly affected. 3/5 surviving males treated with 150 mg/kg/day also demonstrated globular accumulations of eosinophilic material in the tubular epithelium, but female treatment animals were not affected. This finding is consistent with the presence of hydrocarbon nephropathy, which results from the excessive accumulation of α2-microglobulin in renal proximal tubular epithelial cells. α2-Microglobulin is found only in the proximal tubular epithelium of adult male rats. 2/5 males treated with 50 mg/kg/day were similarly affected, one with moderate severity, and an effect of treatment at this dose level cannot be excluded even though this is a relatively low incidence of a condition which is seen occasionally among untreated rats of this age and strain.
Hypertrophy of the collecting duct epithelium was seen in 3/5 high dose females treated with 500 mg/kg/day, but all were found dead prior to scheduled termination of the study. The condition was not observed among females treated with 150 mg/kg/day and cannot be reliably attributed to treatment.
OTHER HISTOPATHOLOGY
The following animals were found dead or killed in extremis prior to scheduled termination: Female 54 (Group 3), males 63, 70 and females 71, 72, 75, 77 and 79 (Group 4). The following animals were terminated prematurely because the dose level proved to be excessive: Males 61, 62, 64-69 and females 73, 74, 76, 78 and 80 (Group 4).
No treatment-related histopathological changes were seen consistently among animals dying before scheduled termination. Among those found dead or killed in extremis marked or severe thymic lymphoid atrophy was observed for several animals, although this is a change frequently seen in moribund rodents and may not be specifically related to treatment. Hypertrophy of the collecting duct epithelium was seen in the renal papilla of three high dose females, which was similarly of doubtful toxicological significance. Haemometra was observed for three high dose females found dead or killed in extremis.

OTHER FINDINGS (PARENTAL ANIMALS)
WATER CONSUMPTION
Group mean daily water consumptions for males and females are given in Tables 16 and 17 attached.
Males treated with 500 mg/kg/day showed a statistically significant increase in water consumption (p<0.01) during Week 1 of treatment, in comparison to controls. The increase was noted throughout the remainder of the study although statistical significance was not achieved. Females treated with 500 mg/kg/day showed an increase in water consumption from Week 2 of maturation, in comparison to controls. During the gestation phase, however, statistical significance (p<0.01) was only achieved for Weeks 1 and 2, in comparison to controls.
No such effect was detected for females treated with 150 and 50 mg/kg/day during the maturation, gestation and lactation phases.

HAEMATOLOGY
Group mean values and standard deviations for test and control group animals are given in Table 18 and Table 19 attached (statistically significant differences are indicated). There were no toxicologically significant effects detected in the haematological parameters measured.
On Day 14 a statistically significant reduction in lymphocytes (p<0.05) was evident throughout the male treatment animals in comparison to controls. A statistically significant reduction in total leucocyte count (p<0.0.5) was also noted in males treated with 150 mg/kg/day in comparison to controls. In the absence of any histopathological correlates these findings are considered to be of no toxicological importance.
On Day 42 a statistically significant increase in mean corpuscular haemoglobin and mean cell volume levels (p<0.01) were evident for males treated with 150 and 50 mg/kg/day, in comparison to controls. In the absence of any histopathological correlates these findings are considered to be of no toxicological significance.
No such effect was detected in females treated with 150 and 50 mg/kg/day.

CLINICAL CHEMISTRY
Group mean values and standard deviations for test and control group animals are given in Table 20 and Table 21 attached (statistically significant differences are indicated). There were no toxicologically significant changes detected in the blood chemical parameters measured.
On Day 14 a statistically significant increase in aspartate aminotransferase (p<0.01) and reduction in glucose levels (p<0.05) was noted for females treated with 500 mg/kg/day, in comparison to controls. The majority of the individual values were within the normal ranges for rats of the strain and age used and in the absence of any other further supporting evidence, the inter-group differences were considered to be of no toxicological importance.
No such effect was detected for males treated with 500 mg/kg/day or animals of either sex treated with 150 and 50 mg/kg/day.
There were no abnormalities detected for treatment animals, in comparison to controls on Day 42.

NEUROBEHAVIOUR
(Behavioural assessment)
There were no treatment-related changes in the behavioural parameters measured.
All remaining inter and intra group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and are considered to be of no toxicological importance.
(Functional Performance Tests)
There were no treatment-related changes in the functional performance parameters measured.
(Sensory Reactivity)
There were no treatment-related changes in sensory reactivity.
Statistical analysis of the data revealed no significant inter-group differences.
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
50 mg/kg bw/day
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Remarks:
systemic toxicity
Effect level:
50 mg/kg bw/day
Sex:
female
Basis for effect level:
other: Deaths at 500 and 150 mg/kg/day and microscopic changes in the spleen and kidneys seen in females.
Dose descriptor:
NOEL
Remarks:
Reproductive and development toxicity
Effect level:
150 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: Parents and offsprings (migrated information)
Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
not examined
The following assessment of litter response is generally based on those litters reared to termination on Day 5 post partum, although data available for females showing total litter loss has also been taken into consideration, where considered appropriate.

VIABILITY (OFFSPRING)
Group mean corpora lutea and implantation counts, litter size, litter weights; implantation losses and survival indices and sex ratio are given in Tables 23 to 25 attached. There was no adverse effect on litter size for treatment animals when compared to controls and there was no obvious difference in sex ratio for litters from treated females in comparison to controls.
At 500 mg/kg/day a reduction in group mean corpora lutea with a reduction in mean implantation site number and mean litter size at birth was evident in comparison to controls. This may not be considered a treatment related effect, primarily due to the reduced group size.
At 150 mg/kg/day there was a slightly lower corpora lutea count with a concomitant reduction in mean implant number and litter size at both. These differences were not statistically significant when compared with controls and therefore are likely to reflect normal biological variation.
At 50 mg/kg/day the difference in mean corpora lutea count compared with controls was not biologically or statistically significant. There was a slight increase in pre-implantation loss with a reduction in implant numbers. This was artificially increased as a consequence of two values (females 31 and 35) which had a significantly higher value when compared to other females from this group.

CLINICAL SIGNS (OFFSPRING)
Group values for surface righting reflex and the incidence of clinical signs are given in Tables 26 and 27 attached.
Of the nine pregnancies at 150 mg/kg/day one litter showed dead offspring and the remaining offspring and dam were of poor physical health on Day 2 post partum, resulting in the termination of the dam and remaining litter. The effect on offspring were considered a consequence of maternal effect. One litter had a pup with a swollen tail on Day 4 post partum which progressed to an open wound on Day 5 whilst another litter was observed with one small pup from Pre Day 1 to Day 5 post partum. In addition, three litters had one pup missing, one on Day 2 and two on Day 3 post partum. Prior to one of the pups missing on Day 3 it was seen to be weak and cold on Day 2. These effects seen are considered incidental and unrelated to treatment.
The litter responses of the 50 mg/kg/day treatment animals were considered to be of no toxicological significance.
Of the nine pregnancies at 50 mg/kg/day one litter showed one dead and one missing offspring together with two cold pups one of which was weak on Day 2 post partum. By Day 3 post partum the cold and weak pup was missing and by Day 4 post partum the previously observed cold pup was also small. This pup was subsequently missing on Day 5 post partum whilst a further pup was observed with a swollen left hindlimb. From Pre Day 1 to Day 5 post partum another litter showed two small offspring together with two dead pups on Day 2 post partum. An additional litter was observed with one small offspring between Day 4 and Day 5 post partum only. Furthermore, another litter showed one cold pup on Day 2 post partum only.
Of the eight litters produced in the controls one litter showed five dead offspring Pre Day 1, whilst another showed one small offspring between Pre Day 1 and Day 5 post partum. Another litter showed one pup with a swollen right hind paw between Days 4 and 5 post partum. In addition two animals from a further litter were observed as cold on Day 4 post partum one was also small. Furthermore, one litter showed one missing offspring on Day 2 post partum and two cold offspring on Day 5 post partum.
A statistically significant reduction in reflexological response (p<0.05) was evident in the 500 mg/kg/day offspring, in comparison to controls. The effect on offspring were considered a consequence of maternal effect.
No such effect was detected for the 150 and 50 mg/kg/day offspring.

BODY WEIGHT (OFFSPRING)
There was no significant intergroup difference in mean litter weights or offspring bodyweights for treated animals in comparison to controls.
Of the four pregnancies at 500 mg/kg/day, one litter lost 7/9 pups on Day 2 post partum after the whole litter was weak, cold with no milk in the stomach. Two litters showed one dead pup Pre Day 1, two also showed instances of cold pups, one also showed weak pups. A reduction in offspring bodyweight and bodyweight change was also seen in this treatment group, in comparison to controls. However, due to the number of surviving litters no conclusive relationship to treatment could be established. The dose group was terminated early due to toxicity to adults at this dose level.

SEXUAL MATURATION (OFFSPRING)
Not applicable.

ORGAN WEIGHTS (OFFSPRING)
Not applicable.

GROSS PATHOLOGY (OFFSPRING)
A summary incidence of necropsy findings for offspring are given in Table 28 attached.
Two females treated with 500 mg/kg/day produced a litter with one death at birth. One pup showed autolytic changes and one pup had no milk in the stomach. On Day 2 post partum three pups in one 150 mg/kg/day and two pups in one 50 mg/kg/day litter were found dead. These pups displayed autolytic changes. In addition, on Day 2 post partum one up from a 50 mg/kg/day was found dead, however, no abnormalities were detected. Furthermore, one control female produced a litter with five deaths at birth. Two animals showed autolytic changes, two had no milk in their stomach and the remaining pup was seen to have intestines protruding into the thoracic cavity.

HISTOPATHOLOGY (OFFSPRING)
Not examined.

OTHER FINDINGS (OFFSPRING)
Not applicable.
Reproductive effects observed:
not specified
Conclusions:
The oral administration of Cellcore QX to rats for a period of up to forty-one consecutive days for the 500 mg/kg/day dose group and up to forty-two consecutive days for the 150 and 50 mg/kg/day dose groups, resulted in treatment-related effects at 500 and 150 mg/kg/day for both sexes and males only at 50 mg/kg/day. These included deaths at 500 and 150 mg/kg/day and microscopic changes in the spleen and kidneys seen for animals of either sex at 500 and 150 mg/kg/day and males only at 50 mg/kg/day. The changes identified in the kidneys of male rats are specific for male rats only and are considered not to represent “serious damage” to health. Therefore the “No Observed Adverse Effect Level” (NOAEL) for systemic toxicity for males was considered to be 50 mg/kg/day and the “No Observed Effect Level” (NOEL) for systemic toxicity for females was considered to be 50 mg/kg/day.
As the 500 mg/kg/day treatment group was terminated early due to excessive dose level no definitive reproductive effect could be established. However there was no effect on pre-natal development in comparison to controls.
No effect of treatment was detected on reproduction or offspring development, at a treatment level up to 150 mg/kg/day therefore the ‘No Observed Effect Level’ (NOEL) for reproductive and development toxicity was considered to be 150 mg/kg/day.

Executive summary:

Introduction.The study was designed to investigate the systemic toxicity and potential adverse effects of the test material on reproduction (including offspring development) and complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

This study was designed to comply with Commission Regulation (EC) No 440/2008 of 30 May 2008, test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Regulation, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods. The test material was administered by gavage to three groups each of ten male and ten femaleWistar Han™:HsdRccHan™:WIST strain rats, for up to forty-one consecutive days for the 500 mg/kg/day dose group and up to forty-two consecutive days for the 150 and 50 mg/kg/day dose groups (including a two week maturation phase, pairing, gestation and early lactation for females). A control group of ten males and ten females was dosed with vehicle alone (distilled water). 

Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating and at termination on five selected males and females from each dose group. 

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.

Extensive functional observations were performed on five selected males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4post partum.

Surviving males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Adult Responses:

Mortality.Two males treated with 500 mg/kg/day were found dead, one on Day 20 and one on Day 29. Two females from this dose group were also found dead, one on study Day 12 and one on Day 41. 

On Day 39, two 500 mg/kg/day females were killed in extremis whilst one 150 mg/kg/day female was killed in extremis on Day 42.

There were no further unscheduled deaths.

The 500 mg/kg/day treatment group was considered to be excessive and terminated on Day 41.

Clinical Signs.Prior to being found dead one male treated with 500 mg/kg/day showed staining of the mouth plus diarrhoea and stained ano-genital region. Whilst one female, treated with 500 mg/kg/day, displayed, pilo-erection and pallor of the extremities.

Episodes of staining of the snout, hunched posture, pilo-erection, hypothermia, pallor of the extremities, dehydration, staining of the mouth and tiptoe gait were observed in two 500 mg/kg/day females killed in extremis. Reduced respiration was also evident in one of these animals.

One female treated with 150 mg/kg/day showed episodes of hunched posture, pilo-erection, dehydration, diarrhoea, fur loss, tiptoe gait, diuresis, emaciation and lethargy prior to being killed in extremis.

Dark faeces were noted in the cages of animals of either sex treated with 500 mg/kg/day from Day 16 onwards.

Behavioural Assessment.There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests.There were no treatment-related changes in the functional performance parameters measured.

Sensory Reactivity Assessments.There were no treatment-related changes in sensory reactivity.

Bodyweights.No adverse effect on bodyweight development was detected for treated animals, in comparison to controls.

Food Consumption and Food Efficiency.No adverse effect on dietary intake was detected for treated animals, in comparison to controls.

Water Consumptions.An increase in water consumption was evident in the 500 mg/kg/day males from Week 1 onwards, whilst the females treated with 500 mg/kg/day showed an increase in water consumption from Week 2 (maturation) onwards.

No such effect was detected for animals of either sex treated with 150 and 50 mg/kg/day.

Haematology.There were no toxicologically significant changes detected in the haematological parameters measured.

Blood Chemistry.There were no toxicologically significant changes detected in the blood chemical parameters measured.

Reproductive Performance:

Mating, Gestation and Fertility.There were no intergroup differences in mating performance or gestation lengths.

Pregnancy was achieved for eight out of ten control females, nine out of ten 50 and 150 mg/kg/day females. However, only seven out of ten females treated with 500 mg/kg/day achieved pregnancy.

Litter Responses.

Offspring Litter Size and Viability.There was no adverse effect on litter size for treated animals when compared to controls and there was no obvious difference in sex ratio for litters from treated females in comparison to controls.

Offspring Growth and Development.There was no significant intergroup difference in mean litter weights and offspring bodyweights for treated animals in comparison to controls.

A reduction in surface righting was evident in the 500 mg/kg/day offspring in comparison to controls. This was considered to be attributed to maternal nurturing.

No such effect was detected at 150 and 50 mg/kg/day.

Offspring Observations. Of the four litters produced at 500 mg/kg/day, one litter had one dead offspring. No abnormalities were detected in the remaining animals. Another litter had one dead offspring and four of the remaining offspring were cold and weak. All the animals in a further litter were cold. The whole of the remaining litter were cold with no milk in the stomach, they were also weak and two pups were found dead and five were missing.

Of the nine pregnancies at 150 mg/kg/day one litter had a number of dead offspring andthe remaining offspring and dam were of poor physical health resulting in their termination.

The incidental findings detected in the control, 50 and remaining 150 mg/kg/day dose group were considered to be incidental.

Pathology.

Necropsy.

Offspring:Two females treated with 500 mg/kg/day produced a litter with one death at birth. One pup displayed autolytic changes and one pup had no milk in the stomach. On Day 2 post partum three pups in one 150 mg/kg/day and two pups in one 50 mg/kg/day litter were found dead. These pups displayed autolytic changes.

The incidental signs detected in litters from the remaining dose groups were of no toxicological importance.

Adults:One 500 mg/kg/day male showed small seminal vesicles, whilst another, showed a mass attached to the mandibular lymph nodes. In addition three females from this treatment group had dark contents of the gastrointestinal tract, two of these also showed a distended stomach. A dark content of the stomach was seen in two of the females with enlarged adrenals also noted in one of these. One female showed a dark mass on the left horn of the uterus.

A yellow fluid filled mass on the right epididymis was evident in one 50 mg/kg/day male. Two females treated with 50 mg/kg/day showed epithelial sloughing of the glandular region of the stomach, whilst two additional females showed epithelial sloughing of both regions of the stomach. In addition, reddened lungs were seen in one 50 mg/kg/day female.

Epithelial sloughing of the glandular region of the stomach was seen in two control females one of which also showed both ovaries encased in a clear fluid filled sac. One additional control female displayed dark viscous fluid filled uterus.

One interim death male treated with 500 mg/kg/day showed a distended stomach with autolysis of the gastrointestinal tract and small seminal vesicles. One male showed distended stomach with fluid filled dark viscous contents and a small caecum. In addition, one interim death female treated with 500 mg/kg/day was found partially cannibalised, with enlarged adrenals and an enlarged stomach, with dark contents. A distended stomach, with autolysis of the gastrointestinal tract was seen in another interim death female. Dark coloured contents of the stomach and enlarged adrenals were evident in the remaining interim death females treated with 500 mg/kg/day. One also showed dark contents of the gastrointestinal tract.

One interim death female treated with 150 mg/kg/day showed dark coloured contents of the stomach with the remaining gastrointestinal tract empty.

Uterine Examination.From evaluation of the corpora lutea and implantation data, two females treated with 500 mg/kg/day, one female treated with 150 mg/kg/day and one female treated with 50 mg/kg/day showed no corpora lutea or implantation sites, therefore were never pregnant. Two control females that did not produce litters displayed corpora lutea and implantation sites, suggesting post-implantation losses.

Organ Weights.No abnormalities were detected for treatment animals, in comparison to controls.

Histopathology.The following treatment-related changes were observed:

The following treatment-related changes were observed:

SPLEEN:Generally higher grades of severity of extramedullary haemopoiesis were seen in relation to treatment for males rats, and possibly also for females treated with 500 mg/kg/day which died or were killed prematurely. Animals of either sex treated with 150 mg/kg/day and surviving until scheduled termination of the study showed a similar effect compared with controls, although females demonstrated a stronger effect than males at this dose level. There was no evidence of a convincing effect of treatment on extramedullary haemopoiesis among animals of either sex treated with 50 mg/kg/day. 

KIDNEY:Globular accumulations of eosinophilic material were observed in the tubular epithelium of 3/5 males treated with 500 mg/kg/day all of which died before scheduled termination; one control was similarly affected. 3/5 surviving males treated with 150 mg/kg/day also demonstrated globular accumulations of eosinophilic material in the tubular epithelium, but female treatment animals were not affected. 2/5 males treated with 50 mg/kg/day were similarly affected, one with moderate severity.

Hypertrophy of the collecting duct epithelium was seen in 3/5 high dose females treated with 500 mg/kg/day, but all were found dead prior to scheduled termination of the study. The condition was not observed among females treated with 150 mg/kg/day. 

There were no treatment-related changes in the reproductive organs examined.

Conclusion.The oral administration of NCA to rats for a period of up to forty-one consecutive days for the 500 mg/kg/day dose group and up to forty-two consecutive days for the 150 and 50 mg/kg/day dose groups,resulted in treatment-related effects at 500 and 150 mg/kg/day for both sexes and males only at 50 mg/kg/day. These included deaths at 500 and 150 mg/kg/day and microscopic changes in the spleen and kidneys seen for animals of either sex at 500 and 150 mg/kg/day and males only at 50 mg/kg/day. The changes identified in the kidneys of male rats are specific for male rats only and are considered not to represent “serious damage” to health. Therefore the “No Observed Adverse Effect Level” (NOAEL) for systemic toxicity for males was considered to be 50 mg/kg/day and the “No Observed Effect Level” (NOEL) for systemic toxicity for females was considered to be 50 mg/kg/day.

As the 500 mg/kg/day treatment group was terminated early due to excessive toxicity no definitive reproductive effect could be established. However there was no effect on pre-natal development in comparison to controls.

No effect of treatment was detected on reproduction or offspring development, at a treatment level up to 150 mg/kg/day therefore the ‘No Observed Effect Level’ (NOEL) for reproductive and development toxicity was considered to be 150 mg/kg/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The key study was conducted in accordance with the standardised guideline OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).It complies with GLP and was awarded a reliability score of 1 in accordance with the principles laid out by Klimisch (1997).
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 systemic toxicity and potential adverse effects of the test material on reproduction (including offspring development) was investigated in accordance with the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

 

The test material was administered by gavage to three groups each of ten male and ten female Wistar strain rats, for up to forty-one consecutive days at 500 mg/kg/day and up to forty-two consecutive days at 150 and 50 mg/kg/day (including a two week maturation phase, pairing, gestation and early lactation for females). A control group of ten males and ten females was dosed with vehicle alone (distilled water). 

Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating and at termination on five selected males and females from each dose group. 

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.

Extensive functional observations were performed on five selected males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4post partum.

Surviving males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Mortality

Two males treated with 500 mg/kg/day were found dead, one on Day 20 and one on Day 29. Two females from this dose group were also found dead, one on study Day 12 and one on Day 41. 

On Day 39, two 500 mg/kg/day females were killed in extremis whilst one 150 mg/kg/day female was killed in extremis on Day 42.

There were no further unscheduled deaths.

The 500 mg/kg/day treatment group was considered to be excessive and terminated on Day 41.

 

Reproductive Performance

Mating, Gestation and Fertility

There were no intergroup differences in mating performance or gestation lengths.

Pregnancy was achieved for eight out of ten control females, nine out of ten 50 and 150 mg/kg/day females. However, only seven out of ten females treated with 500 mg/kg/day achieved pregnancy.

 

Pathology

One 500 mg/kg/day male showed small seminal vesicles; in addition, one female showed a dark mass on the left horn of the uterus. A yellow fluid filled mass on the right epididymis was evident in one 50 mg/kg/day male. These findings were considered to be incidental.

Uterine Examination

From evaluation of the corpora lutea and implantation data, two females treated with 500 mg/kg/day, one female treated with 150 mg/kg/day and one female treated with 50 mg/kg/day showed no corpora lutea or implantation sites, therefore were never pregnant. Two control females that did not produce litters displayed corpora lutea and implantation sites, suggesting post-implantation losses.

 

There were no treatment-related changes in the reproductive organs examined.

 

As the 500 mg/kg/day treatment group was terminated early due to excessive toxicity no definitive reproductive effect could be established.

No effect of treatment was detected on reproduction at a treatment level up to 150 mg/kg/day therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 150 mg/kg/day.


Short description of key information:
The 500 mg/kg/day treatment group was terminated early due to excessive toxicity; therefore no definitive reproductive effect could be established.
No effect of treatment was detected on reproduction at a treatment level up to 150 mg/kg/day therefore the ‘No Observed Effect Level’ (NOEL) for reproductive and development toxicity was considered to be 150 mg/kg/day.

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

Effects on developmental toxicity

Description of key information
The 500 mg/kg/day treatment group was terminated early due to excessive toxicity; however there was no effect on pre-natal development in comparison to controls.
No effect of treatment was detected for offspring development, at a treatment level up to 150 mg/kg/day therefore the ‘No Observed Effect Level’ (NOEL) for developmental toxicity was considered to be 150 mg/kg/day.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Between 6 May 2008 and 20 November 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test”
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). Date of inspection 19/08/2008. Date of signature 04/03/2009.
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Wistar Han™:HsdRccHan™:WIST strain rats from Harlan UK, Ltd, Oxon, UK
- Age at study initiation: Approximately 12 weeks old
- Weight at study initiation: 264 to 315 g (male); 158 to 219 g (female)
- Fasting period before study: Not applicable
- Housing:Initially, all animals were housed in groups of five in solid floor polypropylene cages with
stainless steel mesh lids and softwood flake bedding (Harlan UK Ltd). During the mating
phase, animals were transferred to polypropylene grid floor cages suspended over trays
lined with absorbent paper on a one male: one female basis within each dose group.
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.
- Diet: The animals were allowed free access to food. A pelleted diet Rodent 2018C
Teklad Global Certified Diet Harlan UK Ltd, Oxon, UK was used throughout the study
period. The diet was considered not to contain any contaminant at a level that might
have affected the purpose or integrity of the study.
- Water:The animals were allowed free access to water. Mains drinking water was supplied from polycarbonate bottles attached to the cage. The drinking water was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Acclimation period: For 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness

IN-LIFE DATES: Day 1 To: Day 43 (males); Day 1 To: Day 5 post partum (females).
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: Not applicable
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: For the purpose of the study, the test material was prepared at the appropriate
concentrations as a suspension in distilled water. The stability and homogeneity of the
test material formulations were determined by Harlan Laboratories Ltd. Formulations were prepared daily and analysed weekly.
Samples were taken of each test material formulation and were analysed for
concentration of Cellcore QX at Harlan Laboratories Ltd. The results indicate
that the prepared formulations were within ± 10% of the nominal concentration.

DIET PREPARATION
A pelleted diet Rodent 2018C Teklad Global Certified Diet Harlan UK Ltd, Oxon, UK was used throughout the study period.
- Rate of preparation of diet (frequency): Daily
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
Distilled water
- Justification for use and choice of vehicle (if other than water): Not applicable
- Concentration in vehicle: 100, 30 and 10 mg/ml
- Amount of vehicle (if gavage): 5 mg/kg
- Lot/batch no. (if required): Not applicable
- Purity: Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of Lithium Nickel Cobalt Aluminium Oxide in the test material formulations was determined using a gravimetric technique. The test material formulations were weighed into tared glass sintered crucibles and then
rinsed with acetone to leave a test material residue. The samples were then dried in an
oven at approximately 105oC before allowing to cool over silica gel in a desiccator and
re-weighed.
The test material formulations were mixed thoroughly and samples were taken from the
top, middle and bottom of the container, shaking between sampling. Sampling was
performed in triplicate.
The test material formulations were sampled and analysed within three days of
preparation.
The analytical method has been satisfactorily validated in terms of specificity and
accuracy for the purposes of the study.

Details on mating procedure:
- M/F ratio per cage: 1/1 (Animals were paired on a 1 male: 1 female basis within each dose group)
- Length of cohabitation: Up to 14 days
- Proof of pregnancy: 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 the oestrous cycle 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)
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.: No data
- Further matings after two unsuccessful attempts: No data
- After successful mating each pregnant female was caged: Mated females were housed individually during the period of gestation and lactation.
- Any other deviations from standard protocol: Not applicable
Duration of treatment / exposure:
From Day 1 To: Day 42 (males); Day 1 To: Day 4 of post partum (females).
Frequency of treatment:
Daily
Duration of test:
Day 1 To: Day 43 (males); Day 1 To: Day 5 post partum (females).
Remarks:
Doses / Concentrations:
50 mg/kg/day (10 mg/ml)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg/day (30 mg/ml)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg/day (100 mg/ml)
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose (including control).
Control animals:
yes, concurrent vehicle
Details on study design:
Details on study schedule
i) Groups of ten male and ten 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) Prior to the start of treatment and once weekly thereafter, all animals were
observed for signs of functional/behavioural toxicity.
iii) One day prior to pairing (Day 14), blood samples were taken from five males and
five females, randomly selected from each dose group and analysed for
haematological and blood chemical assessment.
iv) On Day 15, animals were paired on a 1 male: 1 female basis within each dose
group for a maximum of four days.
v) 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.
vi) On completion of mating (during Week 6), five selected males per dose group
were evaluated for functional/sensory responses to various stimuli was performed.
vii) Pregnant females were allowed to give birth and maintain their offspring until Day
5 post partum. Evaluation of each litter size, litter weight, mean offspring weight
by sex, clinical observations and landmark developmental signs were also
performed during this period.
viii) At Day 4 post partum, five selected females per dose group were evaluated for
functional/sensory responses to various stimuli.
ix) Additional blood samples were taken from five males from each dose group for
haematological and blood chemical assessments on Day 42. Following
completion of the female gestation and lactation phases, the male dose groups
were killed and examined macroscopically.
x) Additional blood samples were taken from five randomly selected females from
each dose group at termination for haematological and blood chemical
assessment on Day 4 post partum. At Day 5 post partum, all surviving females
and surviving offspring were killed and examined macroscopically.
xi) The surviving high dose treatment animals were terminated early due to excessive
toxicity. Additional blood samples were taken at termination for haematological
and blood chemical assessments.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, up to thirty minutes after dosing, and one and five hours after
dosing, during the working week. Animals were observed immediately before dosing,
soon after dosing, and one hour after dosing at weekends and public holidays (except for
females during parturition where applicable).
- Cage side observations checked: Overt signs of toxicity, ill-health and behavioural change.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, up to thirty minutes after dosing, and one and five hours after
dosing, during the working week. Animals were observed immediately before dosing,
soon after dosing, and one hour after dosing at weekends and public holidays (except for
females during parturition where applicable).

BODY WEIGHT: Yes
- Time schedule for examinations:Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for
males until termination and weekly for females until mating was evident. Bodyweights
were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and
4 post partum.

FOOD CONSUMPTION: Yes
- During the maturation period, weekly food consumption was recorded for each cage of
adults. This was continued for males after the mating phase. For females showing
evidence of mating, food consumption was recorded for the periods covering Days 0-7,
7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1
and 4 post partum. Weekly food consumptions were performed weekly for each cage of
adults throughout the study period.

FOOD EFFICIENCY: Yes
- Food efficiency (the ratio of bodyweight change/dietary intake) was calculated
retrospectively for males and females throughout the study period.

WATER CONSUMPTION: Yes
- Time schedule for examinations:Water intake was observed daily by visual inspection of water bottles for any overt
changes.
A possible treatment-related effect was detected on the range finder animals, therefore
gravimetric measurements were initiated from Day 1 throughout to study termination.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice
Male animals: Adult surviving males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43.
Maternal animals: Adult surviving females were killed by
intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Any females that failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.
- Organs examined:
Gross pathology: For all females the uterus was examined for signs of implantation and the number of
uterine implantations in each born was recorded. This procedure was enhanced; as
necessary, by staining the uteri with a 1% ammonium polysulphide solution. In addition, the corpora lutea of all ovaries from pregnant females were counted at necropsy. All adult animals, including those dying during the study, were subjected to
a full external and internal examination, and any macroscopic abnormalities were
recorded.
Histopathology/organ weights:
The following organs, removed from the five selected males and parental females from
each group that were killed at the end of the study, were dissected free from fat and
weighed before fixation.
Adrenals, ovaries, brain, spleen, epididymides, testes, heart, thymus, kidneys, thyroid and liver.

The following reproductive organs were weighed from all animals that were killed at the end of the study: ovaries, epididymides and testes.


Samples of the following tissues were preserved from five males and five females from
each dose group, in buffered 10% formalin except where indicated.
Adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, coagulating gland, colon, duodenum, epididymides (preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) up to 48 hours later), eyes (fixed in Davidson’s fluid), gross lesions, heart, ileum, jejunum, kidneys, liver, lungs (with bronchi)(lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative), lymph nodes (cervical and mesenteric), mammary gland, muscle (skeletal), ovaries, pancreas, pituitary, prostate, oesophagus, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical), mid thoracic and lumbar, spleen, stomach, thyroid, trachea, testes (preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) up to 48 hours later), thymus, urinary bladder, uterus/cervix and vagina.

The following tissues were also removed from the remaining animals:coagulating gland, epididymides, ovaries, pituitary, prostate, seminal vesicles, testes and uterus/cervix.

All tissues were despatched to Harlan Laboratories Ltd, Switzerland (Principal
Investigator: K Weber). The tissues from five selected control, 150 and 500 mg/kg/day
dose group animals and those animals dying during the study, were prepared as paraffin
blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin
for subsequent microscopic examination. The tissues shown from oagulating gland, epididymides, ovaries, pituitary, prostate, seminal vesicles, testes and uterus/cervix from the remaining
control, 150 and 500 mg/kg/day were also processed.
Since there were indications of treatment-related changes, examination was
subsequently extended to include similarly prepared sections of kidney and spleen from
five animals per sex from the low dose groups.
Microscopic examination was conducted by the Study Pathologist. All findings were
entered into the ROELEE Pathology computerisation system for tabulation and report
production.

OTHER:
HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Haematological and blood chemical investigations were performed on five males and five
females selected from each test and control group on Day 14 (day prior to pairing) and
five males and five females selected from each surviving treatment group on Day 42.
Blood samples were obtained from the lateral tail vein at Day 14 and by cardiac puncture at Days 41 and 42.
- Anaesthetic used for blood collection: Not stated in the report
- Animals fasted: No
- How many animals:
Five males and 5 females selected from each test and control group on Day 14.
Five males and 5 females selected from each surviving treatment group on Day 42
- Parameters checked in tables 18 -21 which are attached were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
(Behavioural assessment)
Prior to the start of treatment and at weekly intervals thereafter
(Functional Performance Tests)
Prior to termination
(Sensory Reactivity)
Prior to termination

- Dose groups that were examined:
(Behavioural assessment)
All animals
(Functional Performance Tests)
Five selected males and females per dose level
(Sensory Reactivity)
Five selected males and females per dose level

- Parameters examined:
(Behavioural assessment)
Detailed individual clinical observations were performed for each animal using a purpose-built arena.
The following parameters were observed:
Gait, hyper/hypothermia, tremors, skin colour, twitches, respiration, convulsions, palpebral closure, bizarre/abnormal/stereotypic behaviour, urination, salivation, defecation, pilo-erection, transfer arousal, exophthalmia, tail elevation and lachrymation
(Functional Performance Tests)
Motor Activity.
Purpose-built 44 infra-red beam automated activity monitors were used
to assess motor activity. Animals were randomly allocated to the activity monitors. The
tests were performed at approximately the same time each day, under similar laboratory
conditions. The evaluation period was thirty minutes for each animal. The percentage of
time each animal was active and mobile was recorded for the overall thirty minute period
and also during the final 20% of the period (considered to be the asymptotic period).
Forelimb/Hindlimb Grip Strength.
An automated meter was used. Each animal was
allowed to grip the proximal metal bar of the meter with its forepaws. The animal was
pulled by the base of the tail until its grip was broken. The animal was drawn along the
trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal
was pulled by the base of the tail until its grip was broken. A record of the force required
to break the grip for each animal was made. Three consecutive trials were performed for
each animal.
(Sensory Reactivity)
Animals were assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed:
Grasp response, touch escape, vocalisation, pupil reflex, toe pinch, startle reflex, tail pinch, blink reflex and finger approach

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 the
oestrous cycle 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 mating
ii) Date and time of observed start of parturition
iii) Date and time of observed completion of parturition
iv) Duration of gestation

Estrous cyclicity (Parental)
A vaginal smear was prepared for each female and the stage of the oestrous cycle was recorded.

Sperm parameters (parental animals)
Parameters examined in all male parental generations:During histopathology, the male epididymides were examined for spermatocoel granuloma formation.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
- Other: Parameters in Table 1 were examined in this study.
Fetal examinations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in offspring: Number of offspring born, number and sex of offspring alive recorded daily and reported on Day 1 and 4 post partum, clinical condition of offspring from birth to Day 5 post partum, individual offspring and litter weights on Day 1 and 4 post partum, physical Development and pathology.

GROSS EXAMINATION OF DEAD PUPS: Dying offspring during the study were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

POST-MORTEM EXAMINATION
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Necropsy findings checked in table 28 were included. All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:
The following parameters were subjected to statistical analysis:
Quantitative functional performance data
Bodyweight and bodyweight change
Food consumption during gestation and lactation
Haematology, blood chemistry, absolute and bodyweight relative organ weights
Litter data
Sex ratio
Implantation losses and viability indices
Offspring bodyweight and bodyweight change
Offspring surface righting
Adult absolute and bodyweight relative organ weights
The following statistical procedures were used:
Data were assessed for dose response relationships by linear regression analysis,
followed by one way analysis of variance (ANOVA) incorporating Levene’s test for
homogeneity of variance. Where variances were shown to be homogenous, pairwise
comparisons were conducted using Dunnett’s test. Where Levene’s test showed
unequal variances the data were analysed using non-parametric methods: Kruskal-
Wallis ANOVA and Mann-Whitney ‘U’ test.
Non-parametric methods were used to analyse implantation loss, offspring sex ratio and
landmark developmental markers.
Probability values (p) are presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
In addition, histopathological findings were analysed using the following methods:
Histopathology data were analysed using the following methods to determine significant
differences between control and treatment groups for the individual sexes:
1. Chi-squared analysis for differences in the incidence of lesions occurring with an
overall frequency of 1 or greater.
2. Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of
severity grades for the more frequently observed graded conditions.
Probability values (p) were calculated as follows:
p<0.001 +++ --- ***
p<0.01 ++ -- **
p<0.05 + - *
p<0.1 (+) (-) (*)
p≥0.01 N.S. (not significant)
Plus (+) signs indicate positive differences from the control group and minus (-) signs
indicate negative differences.

Indices:
(Parental animals)
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 for 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
Historical control data:
Not applicable.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
(Clinical signs)
A summary incidence of daily clinical observations is given in Table 2 and Table 3 attached.
Prior to being found dead one male treated with 500 mg/kg/day showed staining of the mouth on Day 16 with diarrhoea and stained ano-genital region seen on Day 19. Whilst one female, treated with 500 mg/kg/day, displayed, pilo-erection and pallor of the extremities on Day 40.
Episodes of staining of the snout, hunched posture, pilo-erection, hypothermia, pallor of the extremities, dehydration, staining of the mouth and tiptoe gait were observed in two 500 mg/kg/day females killed in extremis. Reduced respiration was also evident in one of these animals.
One female treated with 150 mg/kg/day showed episodes of hunched posture, pilo-erection, dehydration, diarrhoea, fur loss, tiptoe gait, diuresis, emaciation and lethargy prior to being killed in extremis.
Dark faeces were noted in the cages of animals of either sex treated with 500 mg/kg/day from Day 16 onwards.
Incidental findings of increased salivation was detected immediately prior to dosing in one 500 mg/kg/day male, on Day 39, whilst an isolated episode of red/brown staining of the mouth was noted in another male from this treatment group on Day 22. Increased salivation and staining around the mouth is often reported following oral administration of an unpalatable or slightly irritant test material formulation and considered not to be an indication of systemic toxicity. In addition, isolated episodes of noisy respiration were also detected in two males from this treatment group on Days 21 and 39 respectively. An incidence of fur stained black by the test material was seen in one 50 mg/kg/day male on Day 42, whilst one female from this treatment group showed fur loss between Days 37 and 39. One control female showed episodes of fur loss between Days 41 and 42. In isolation these findings are considered incidental and of no toxicological importance.

(Mortality)
Two males treated with 500 mg/kg/day were found dead, one on Day 20 and one on Day 29. Two females from this dose group were also found dead, one on study Day 12 and one on Day 41.
On Day 39, two 500 mg/kg/day females were killed in extremis whilst one 150 mg/kg/day female was killed in extremis on Day 42.
There were no further unscheduled deaths.
The 500 mg/kg/day treatment group was considered to be excessive and terminated on Day 41.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
(Body weight)
No adverse effect on bodyweight development was detected for treatment animals, in comparison to controls.

(Food consumption)
No adverse effect on dietary intake was detected for treated animals, in comparison to controls.


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No treatment-related changes were seen. Spermatocoel
granuloma formation is observed occasionally as a spontaneous condition in the
epididymis of male rats.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
(Mating)
There were no intergroup differences in mating performance or gestation lengths.
Ten out of ten control, 50 and 150 mg/kg/day females together with nine out of nine 500 mg/kg/day females showed signs of positive mating.

(Fertility)
No adverse effect on fertility was evident for treatment animals in comparison to controls. Pregnancy was achieved for eight out of ten control females. Nine out of ten females treated with 50 and 150 mg/kg/day and seven out of nine females treated with 500 mg/kg/day.

(gestation length)
There was no adverse effect on gestation lengths between treated and control females. Females from all treatment groups went on to give birth following 22 to 24 days of gestation.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No abnormalities were detected for treatment animals, in comparison to controls.

GROSS PATHOLOGY (PARENTAL ANIMALS)
A summary incidence of necropsy findings for adults are given in Tables 29 and 30 attached.
There were macroscopic abnormalities considered to be attributable to treatment.
One 500 mg/kg/day male showed small seminal vesicles, whilst another showed a mass attached to the mandibular lymph nodes. In addition three females from this treatment group had dark contents of the gastrointestinal tract, two of these also showed a distended stomach. Dark contents of the stomach were also seen in two of these females with enlarged adrenals also noted in one of these. One female showed a dark mass on the left horn of the uterus.
A yellow fluid filled mass on the right epididymis was evident in one 50 mg/kg/day male. Two females treated with 50 mg/kg/day showed epithelial sloughing of the glandular region of the stomach, whilst two additional females from this treatment group showed epithelial sloughing of both regions of the stomach. In addition, reddened lungs were seen in one 50 mg/kg/day female. In the absence of any histopathological finding to suggest an effect of treatment these findings are considered to be of no toxicological significance.
Epithelial sloughing of the glandular region of the stomach was seen in two control females one of which also showed both ovaries encased in a clear fluid filled sac. There was no adverse effect on implantations for this animal and histopathological examination did not reveal any treatment-related effects and as such, this finding was considered to be a congenital abnormality and unrelated to treatment. One additional control female showed a dark viscous fluid filled uterus.
One interim death male treated with 500 mg/kg/day showed a distended stomach with autolysis of the gastrointestinal tract and small seminal vesicles. Whilst another male, showed a distended stomach, with fluid filled dark viscous contents and a small caecum. In addition, one interim death female treated with 500 mg/kg/day was found partially cannibalised, with enlarged adrenals and an enlarged stomach, with dark contents. Furthermore, a distended stomach, with autolysis of the gastrointestinal tract was seen in another interim death female. Dark coloured contents of the stomach and enlarged adrenals were evident in the remaining interim death females treated with 500 mg/kg/day. One also showed dark contents of the gastrointestinal tract.
One interim death female treated with 150 mg/kg/day showed dark coloured contents of the stomach with the remaining gastrointestinal tract empty.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Summary incidences of histopathological findings are given in Tables 35 and 36 attached. Individual histopathology is presented in Appendices 26 and 27 attached.
As the high dose animals were killed before scheduled termination the intermediate dose group was taken to be the effective high dose, having received 150 mg/kg/day of the test material for the full dosing period. A comparison of this group with control animals is thus more reliable as an indicator of treatment-related changes, although the findings for high dose animals are also taken into consideration. The following treatment-related changes were observed:
SPLEEN: Generally higher grades of severity of extramedullary haemopoiesis were seen in relation to treatment for males, and possibly also for females, treated with 500 mg/kg/day which died or were killed prematurely. Animals of either sex treated with 150 mg/kg/day and surviving until scheduled termination of the study showed a similar effect compared with controls, although females demonstrated a stronger effect than males at this dose level. There was no evidence of a convincing effect of treatment on extramedullary haemopoiesis among animals of either sex treated with 50 mg/kg/day.
KIDNEY: Globular accumulations of eosinophilic material were observed in the tubular epithelium of 3/5 males treated with 500 mg/kg/day all of which died before scheduled termination; one control was similarly affected. 3/5 surviving males treated with 150 mg/kg/day also demonstrated globular accumulations of eosinophilic material in the tubular epithelium, but female treatment animals were not affected. This finding is consistent with the presence of hydrocarbon nephropathy, which results from the excessive accumulation of α2-microglobulin in renal proximal tubular epithelial cells. α2-Microglobulin is found only in the proximal tubular epithelium of adult male rats. 2/5 males treated with 50 mg/kg/day were similarly affected, one with moderate severity, and an effect of treatment at this dose level cannot be excluded even though this is a relatively low incidence of a condition which is seen occasionally among untreated rats of this age and strain.
Hypertrophy of the collecting duct epithelium was seen in 3/5 high dose females treated with 500 mg/kg/day, but all were found dead prior to scheduled termination of the study. The condition was not observed among females treated with 150 mg/kg/day and cannot be reliably attributed to treatment.

OTHER HISTOPATHOLOGY
The following animals were found dead or killed in extremis prior to scheduled termination: Female 54 (Group 3), males 63, 70 and females 71, 72, 75, 77 and 79 (Group 4). The following animals were terminated prematurely because the dose level proved to be excessive: Males 61, 62, 64-69 and females 73, 74, 76, 78 and 80 (Group 4).
No treatment-related histopathological changes were seen consistently among animals dying before scheduled termination. Among those found dead or killed in extremis marked or severe thymic lymphoid atrophy was observed for several animals, although this is a change frequently seen in moribund rodents and may not be specifically related to treatment. Hypertrophy of the collecting duct epithelium was seen in the renal papilla of three high dose females, which was similarly of doubtful toxicological significance. Haemometra was observed for three high dose females found dead or killed in extremis.

OTHER FINDINGS (PARENTAL ANIMALS)
WATER CONSUMPTION
Group mean daily water consumptions for males and females are given in Tables 16 and 17 attached.
Males treated with 500 mg/kg/day showed a statistically significant increase in water consumption (p<0.01) during Week 1 of treatment, in comparison to controls. The increase was noted throughout the remainder of the study although statistical significance was not achieved. Females treated with 500 mg/kg/day showed an increase in water consumption from Week 2 of maturation, in comparison to controls. During the gestation phase, however, statistical significance (p<0.01) was only achieved for Weeks 1 and 2, in comparison to controls.
No such effect was detected for females treated with 150 and 50 mg/kg/day during the maturation, gestation and lactation phases.

HAEMATOLOGY
Group mean values and standard deviations for test and control group animals are given in Table 18 and Table 19 attached (statistically significant differences are indicated). There were no toxicologically significant effects detected in the haematological parameters measured.
On Day 14 a statistically significant reduction in lymphocytes (p<0.05) was evident throughout the male treatment animals in comparison to controls. A statistically significant reduction in total leucocyte count (p<0.0.5) was also noted in males treated with 150 mg/kg/day in comparison to controls. In the absence of any histopathological correlates these findings are considered to be of no toxicological importance.
On Day 42 a statistically significant increase in mean corpuscular haemoglobin and mean cell volume levels (p<0.01) were evident for males treated with 150 and 50 mg/kg/day, in comparison to controls. In the absence of any histopathological correlates these findings are considered to be of no toxicological significance.
No such effect was detected in females treated with 150 and 50 mg/kg/day.

CLINICAL CHEMISTRY
Group mean values and standard deviations for test and control group animals are given in Table 20 and Table 21 attached (statistically significant differences are indicated). There were no toxicologically significant changes detected in the blood chemical parameters measured.
On Day 14 a statistically significant increase in aspartate aminotransferase (p<0.01) and reduction in glucose levels (p<0.05) was noted for females treated with 500 mg/kg/day, in comparison to controls. The majority of the individual values were within the normal ranges for rats of the strain and age used and in the absence of any other further supporting evidence, the inter-group differences were considered to be of no toxicological importance.
No such effect was detected for males treated with 500 mg/kg/day or animals of either sex treated with 150 and 50 mg/kg/day.
There were no abnormalities detected for treatment animals, in comparison to controls on Day 42.

NEUROBEHAVIOUR
(Behavioural assessment)
There were no treatment-related changes in the behavioural parameters measured.
All remaining inter and intra group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and are considered to be of no toxicological importance.
(Functional Performance Tests)
There were no treatment-related changes in the functional performance parameters measured.
(Sensory Reactivity)
There were no treatment-related changes in sensory reactivity.
Statistical analysis of the data revealed no significant inter-group differences.
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Basis for effect level:
other: other:
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day
Basis for effect level:
other: other:
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day
Basis for effect level:
other: other:
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The following assessment of litter response is generally based on those litters reared to termination on Day 5 post partum, although data available for females showing total litter loss has also been taken into consideration, where considered appropriate.

VIABILITY (OFFSPRING)
Group mean corpora lutea and implantation counts, litter size, litter weights; implantation losses and survival indices and sex ratio are given in Tables 23 to 25 attached. There was no adverse effect on litter size for treatment animals when compared to controls and there was no obvious difference in sex ratio for litters from treated females in comparison to controls.
At 500 mg/kg/day a reduction in group mean corpora lutea with a reduction in mean implantation site number and mean litter size at birth was evident in comparison to controls. This may not be considered a treatment related effect, primarily due to the reduced group size.
At 150 mg/kg/day there was a slightly lower corpora lutea count with a concomitant reduction in mean implant number and litter size at both. These differences were not statistically significant when compared with controls and therefore are likely to reflect normal biological variation.
At 50 mg/kg/day the difference in mean corpora lutea count compared with controls was not biologically or statistically significant. There was a slight increase in pre-implantation loss with a reduction in implant numbers. This was artificially increased as a consequence of two values (females 31 and 35) which had a significantly higher value when compared to other females from this group.

CLINICAL SIGNS (OFFSPRING)
Group values for surface righting reflex and the incidence of clinical signs are given in Tables 26 and 27 attached.
Of the nine pregnancies at 150 mg/kg/day one litter showed dead offspring and the remaining offspring and dam were of poor physical health on Day 2 post partum, resulting in the termination of the dam and remaining litter. The effect on offspring were considered a consequence of maternal effect. One litter had a pup with a swollen tail on Day 4 post partum which progressed to an open wound on Day 5 whilst another litter was observed with one small pup from Pre Day 1 to Day 5 post partum. In addition, three litters had one pup missing, one on Day 2 and two on Day 3 post partum. Prior to one of the pups missing on Day 3 it was seen to be weak and cold on Day 2. These effects seen are considered incidental and unrelated to treatment.
The litter responses of the 50 mg/kg/day treatment animals were considered to be of no toxicological significance.
Of the nine pregnancies at 50 mg/kg/day one litter showed one dead and one missing offspring together with two cold pups one of which was weak on Day 2 post partum. By Day 3 post partum the cold and weak pup was missing and by Day 4 post partum the previously observed cold pup was also small. This pup was subsequently missing on Day 5 post partum whilst a further pup was observed with a swollen left hindlimb. From Pre Day 1 to Day 5 post partum another litter showed two small offspring together with two dead pups on Day 2 post partum. An additional litter was observed with one small offspring between Day 4 and Day 5 post partum only. Furthermore, another litter showed one cold pup on Day 2 post partum only.
Of the eight litters produced in the controls one litter showed five dead offspring Pre Day 1, whilst another showed one small offspring between Pre Day 1 and Day 5 post partum. Another litter showed one pup with a swollen right hind paw between Days 4 and 5 post partum. In addition two animals from a further litter were observed as cold on Day 4 post partum one was also small. Furthermore, one litter showed one missing offspring on Day 2 post partum and two cold offspring on Day 5 post partum.
A statistically significant reduction in reflexological response (p<0.05) was evident in the 500 mg/kg/day offspring, in comparison to controls. The effect on offspring were considered a consequence of maternal effect.
No such effect was detected for the 150 and 50 mg/kg/day offspring.


BODY WEIGHT (OFFSPRING)
Group values for offspring bodyweight and bodyweight change, surface righting reflex and the incidence of clinical signs are given in 23 attached.
There was no significant intergroup difference in mean litter weights or offspring bodyweights for treated animals in comparison to controls.
Of the four pregnancies at 500 mg/kg/day, one litter lost 7/9 pups on Day 2 post partum after the whole litter was weak, cold with no milk in the stomach. Two litters showed one dead pup Pre Day 1, two also showed instances of cold pups, one also showed weak pups. A reduction in offspring bodyweight and bodyweight change was also seen in this treatment group, in comparison to controls. However, due to the number of surviving litters no conclusive relationship to treatment could be established. The dose group was terminated early due to toxicity to adults at this dose level.

ORGAN WEIGHTS (OFFSPRING)
Not applicable.

GROSS PATHOLOGY (OFFSPRING)
A summary incidence of necropsy findings for offspring are given in Table 28 attached.
Two females treated with 500 mg/kg/day produced a litter with one death at birth. One pup showed autolytic changes and one pup had no milk in the stomach. On Day 2 post partum three pups in one 150 mg/kg/day and two pups in one 50 mg/kg/day litter were found dead. These pups displayed autolytic changes. In addition, on Day 2 post partum one up from a 50 mg/kg/day was found dead, however, no abnormalities were detected. Furthermore, one control female produced a litter with five deaths at birth. Two animals showed autolytic changes, two had no milk in their stomach and the remaining pup was seen to have intestines protruding into the thoracic cavity.

HISTOPATHOLOGY (OFFSPRING)
Not examined.

OTHER FINDINGS (OFFSPRING)
Not applicable.

Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1

TABULAR SUMMARY REPORT OF EFFECTS ON REPRODUCTION/DEVELOPMENT

Observations

Dose Level (mg/kg/day)

0 (Control)

50

150

500

Mated pairs

n

10

10

10

9

Females showing evidence of copulation

n

10

10

10

9

Conception Days 1-4

n

10

10

10

9

Pregnant females

n

10

9

9

7

Gestation = 22 Days

n

0

0

3

0

Gestation = 22 ½ Days

n

3

5

3

4

Gestation = 23 Days

n

2

0

2

0

Gestation = 23 ½ Days

n

3

3

1

0

Gestation = 24 Days

n

0

1

0

0

Dams with live young born

n

8

9

9

4*

Dams with live young at Day 4post partum

n

8

9

8

1

Corpora lutea/dam

14.1

13.1

12.3

11.3

Implants/dam

13.6

11.1

11.4

10.8

Live offspring/dam Day 1post partum

11.5

10.1

10.5

9.3

Live offspring/dam at Day 4post partum

11.4

9.6

10.1

2.25

Sex ratio: % males Day 1post partum

53.6

56.1

49.2

46.9

Sex ratio: %males at Day 4post partum

53.2

57.3

48.7

44.4

Litter weight (g) at Day 1post partum

65.5

57.1

59.5

46.3

Litter weight (g) at Day 4post partum

91.0

75.9

81.9

41.2

Male offspring weight (g) at Day 1post partum

5.9

6.1

6.0

5.2

Female offspring weight (g) at Day 1post partum

5.6

5.8

5.6

4.8

Male offspring weight (g) at Day 4post partum

8.4

8.6

8.5

4.8

Female offspring weight (g) at Day 4post partum

7.9

8.4

8.1

4.4

ABNORMAL OFFSPRING/DAM

 

0

n

8

9

8

4

LOSS OF OFFSPRING/DAM

 

Pre-implantation (corpora lutea minus implantations)

 

0

n

6

4

3

5

1

n

2

1

5

2

2

n

1

2

0

0

3

n

0

0

1

0

6

n

0

1

0

0

7

n

0

1

0

0

8

n

1

0

0

0

Pre-natal (implantations minus live births)

 

0

n

3

4

6

2

1

n

2

2

2

1

2

n

2

2

0

0

3

n

0

1

0

1

5

n

0

0

1

0

6

n

1

0

0

0

Post natal (live births minus offspring alive on Day 4post partum)

 

0

n

7

7

5

1

1

n

1

0

3

0

2

n

0

1

0

0

3

n

0

1

0

0

5

n

0

0

0

0

8

n

0

0

0

1

9

n

0

0

0

1

11

n

0

0

0

1

15

n

0

0

1

0

 



n=Number

= Mean

*= females surviving through to parturition

Conclusions:
The oral administration of Cellcore QX to rats for a period of up to forty-one consecutive days for the 500 mg/kg/day dose group and up to forty-two consecutive days for the 150 and 50 mg/kg/day dose groups, resulted in treatment-related effects at 500 and 150 mg/kg/day for both sexes and males only at 50 mg/kg/day. These included deaths at 500 and 150 mg/kg/day and microscopic changes in the spleen and kidneys seen for animals of either sex at 500 and 150 mg/kg/day and males only at 50 mg/kg/day. The changes identified in the kidneys of male rats are specific for male rats only and are considered not to represent “serious damage” to health. Therefore the “No Observed Adverse Effect Level” (NOAEL) for systemic toxicity for males was considered to be 50 mg/kg/day and the “No Observed Effect Level” (NOEL) for systemic toxicity for females was considered to be 50 mg/kg/day.
As the 500 mg/kg/day treatment group was terminated early due to excessive dose level no definitive reproductive effect could be established. However there was no effect on pre-natal development in comparison to controls.
No effect of treatment was detected on reproduction or offspring development, at a treatment level up to 150 mg/kg/day therefore the ‘No Observed Effect Level’ (NOEL) for reproductive and development toxicity was considered to be 150 mg/kg/day.
Executive summary:

Introduction.The study was designed to investigate the systemic toxicity and potential adverse effects of the test material on reproduction (including offspring development) and complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

This study was designed to comply with Commission Regulation (EC) No 440/2008 of 30 May 2008, test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Regulation, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods. The test material was administered by gavage to three groups each of ten male and ten femaleWistar Han™:HsdRccHan™:WIST strain rats, for up to forty-one consecutive days for the 500 mg/kg/day dose group and up to forty-two consecutive days for the 150 and 50 mg/kg/day dose groups (including a two week maturation phase, pairing, gestation and early lactation for females). A control group of ten males and ten females was dosed with vehicle alone (distilled water). 

Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating and at termination on five selected males and females from each dose group. 

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.

Extensive functional observations were performed on five selected males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4 post partum.

Surviving males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Adult Responses:

Mortality.Two males treated with 500 mg/kg/day were found dead, one on Day 20 and one on Day 29. Two females from this dose group were also found dead, one on study Day 12 and one on Day 41. 

On Day 39, two 500 mg/kg/day females were killed in extremis whilst one 150 mg/kg/day female was killed in extremis on Day 42.

There were no further unscheduled deaths.

The 500 mg/kg/day treatment group was considered to be excessive and terminated on Day 41.

Clinical Signs.Prior to being found dead one male treated with 500 mg/kg/day showed staining of the mouth plus diarrhoea and stained ano-genital region. Whilst one female, treated with 500 mg/kg/day, displayed, pilo-erection and pallor of the extremities.

Episodes of staining of the snout, hunched posture, pilo-erection, hypothermia, pallor of the extremities, dehydration, staining of the mouth and tiptoe gait were observed in two 500 mg/kg/day females killedin extremis. Reduced respiration was also evident in one of these animals.

One female treated with 150 mg/kg/day showed episodes of hunched posture, pilo-erection, dehydration, diarrhoea, fur loss, tiptoe gait, diuresis, emaciation and lethargy prior to being killedin extremis.

Dark faeces were noted in the cages of animals of either sex treated with 500 mg/kg/day from Day 16 onwards.

Behavioural Assessment.There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests.There were no treatment-related changes in the functional performance parameters measured.

Sensory Reactivity Assessments.There were no treatment-related changes in sensory reactivity.

Bodyweights.No adverse effect on bodyweight development was detected for treated animals, in comparison to controls.

Food Consumption and Food Efficiency.No adverse effect on dietary intake was detected for treated animals, in comparison to controls.

Water Consumptions.An increase in water consumption was evident in the 500 mg/kg/day males from Week 1 onwards, whilst the females treated with 500 mg/kg/day showed an increase in water consumption from Week 2 (maturation) onwards.

No such effect was detected for animals of either sex treated with 150 and 50 mg/kg/day.

Haematology.There were no toxicologically significant changes detected in the haematological parameters measured.

Blood Chemistry.There were no toxicologically significant changes detected in the blood chemical parameters measured.

Reproductive Performance:

Mating, Gestation and Fertility.There were no intergroup differences in mating performance or gestation lengths.

Pregnancy was achieved for eight out of ten control females, nine out of ten 50 and 150 mg/kg/day females. However, only seven out of ten females treated with 500 mg/kg/day achieved pregnancy.

Litter Responses.

Offspring Litter Size and Viability.There was no adverse effect on litter size for treated animals when compared to controls and there was no obvious difference in sex ratio for litters from treated females in comparison to controls.

Offspring Growth and Development.There was no significant intergroup difference in mean litter weights and offspring bodyweights for treated animals in comparison to controls.

A reduction in surface righting was evident in the 500 mg/kg/day offspring in comparison to controls. This was considered to be attributed to maternal nurturing.

No such effect was detected at 150 and 50 mg/kg/day.

Offspring Observations. Of the four litters produced at 500 mg/kg/day, one litter had one dead offspring. No abnormalities were detected in the remaining animals. Another litter had one dead offspring and four of the remaining offspring were cold and weak. All the animals in a further litter were cold. The whole of the remaining litter were cold with no milk in the stomach, they were also weak and two pups were found dead and five were missing.

Of the nine pregnancies at 150 mg/kg/day one litter had a number of dead offspring and the remaining offspring and dam were of poor physical health resulting in their termination.

The incidental findings detected in the control, 50 and remaining 150 mg/kg/day dose group were considered to be incidental.

Pathology.

Necropsy.

Offspring:Two females treated with 500 mg/kg/day produced a litter with one death at birth. One pup displayed autolytic changes and one pup had no milk in the stomach. On Day 2 post partum three pups in one 150 mg/kg/day and two pups in one 50 mg/kg/day litter were found dead. These pups displayed autolytic changes.

The incidental signs detected in litters from the remaining dose groups were of no toxicological importance.

Adults:One 500 mg/kg/day male showed small seminal vesicles, whilst another, showed a mass attached to the mandibular lymph nodes. In addition three females from this treatment group had dark contents of the gastrointestinal tract, two of these also showed a distended stomach. A dark content of the stomach was seen in two of the females with enlarged adrenals also noted in one of these. One female showed a dark mass on the left horn of the uterus.

A yellow fluid filled mass on the right epididymis was evident in one 50 mg/kg/day male. Two females treated with 50 mg/kg/day showed epithelial sloughing of the glandular region of the stomach, whilst two additional females showed epithelial sloughing of both regions of the stomach. In addition, reddened lungs were seen in one 50 mg/kg/day female.

Epithelial sloughing of the glandular region of the stomach was seen in two control females one of which also showed both ovaries encased in a clear fluid filled sac. One additional control female displayed dark viscous fluid filled uterus.

One interim death male treated with 500 mg/kg/day showed a distended stomach with autolysis of the gastrointestinal tract and small seminal vesicles. One male showed distended stomach with fluid filled dark viscous contents and a small caecum. In addition, one interim death female treated with 500 mg/kg/day was found partially cannibalised, with enlarged adrenals and an enlarged stomach, with dark contents. A distended stomach, with autolysis of the gastrointestinal tract was seen in another interim death female. Dark coloured contents of the stomach and enlarged adrenals were evident in the remaining interim death females treated with 500 mg/kg/day. One also showed dark contents of the gastrointestinal tract.

One interim death female treated with 150 mg/kg/day showed dark coloured contents of the stomach with the remaining gastrointestinal tract empty.

Uterine Examination.From evaluation of the corpora lutea and implantation data, two females treated with 500 mg/kg/day, one female treated with 150 mg/kg/day and one female treated with 50 mg/kg/day showed no corpora lutea or implantation sites, therefore were never pregnant. Two control females that did not produce litters displayed corpora lutea and implantation sites, suggesting post-implantation losses.

Organ Weights.No abnormalities were detected for treatment animals, in comparison to controls.

Histopathology.The following treatment-related changes were observed:

The following treatment-related changes were observed:

SPLEEN:Generally higher grades of severity of extramedullary haemopoiesis were seen in relation to treatment for males rats, and possibly also for females treated with 500 mg/kg/day which died or were killed prematurely. Animals of either sex treated with 150 mg/kg/day and surviving until scheduled termination of the study showed a similar effect compared with controls, although females demonstrated a stronger effect than males at this dose level. There was no evidence of a convincing effect of treatment on extramedullary haemopoiesis among animals of either sex treated with 50 mg/kg/day. 

KIDNEY:Globular accumulations of eosinophilic material were observed in the tubular epithelium of 3/5 males treated with 500 mg/kg/day all of which died before scheduled termination; one control was similarly affected. 3/5 surviving males treated with 150 mg/kg/day also demonstrated globular accumulations of eosinophilic material in the tubular epithelium, but female treatment animals were not affected. 2/5 males treated with 50 mg/kg/day were similarly affected, one with moderate severity.

Hypertrophy of the collecting duct epithelium was seen in 3/5 high dose females treated with 500 mg/kg/day, but all were found dead prior to scheduled termination of the study. The condition was not observed among females treated with 150 mg/kg/day. 

There were no treatment-related changes in the reproductive organs examined.

Conclusion.The oral administration of Cellcore QX to rats for a period of up to forty-one consecutive days for the 500 mg/kg/day dose group and up to forty-two consecutive days for the 150 and 50 mg/kg/day dose groups,resulted in treatment-related effects at 500 and 150 mg/kg/day for both sexes and males only at 50 mg/kg/day. These included deaths at 500 and 150 mg/kg/day and microscopic changes in the spleen and kidneys seen for animals of either sex at 500 and 150 mg/kg/day and males only at 50 mg/kg/day. The changes identified in the kidneys of male rats are specific for male rats only and are considered not to represent “serious damage” to health. Therefore the “No Observed Adverse Effect Level” (NOAEL) for systemic toxicity for males was considered to be 50 mg/kg/day and the “No Observed Effect Level” (NOEL) for systemic toxicity for females was considered to be 50 mg/kg/day.

As the 500 mg/kg/day treatment group was terminated early due to excessive toxicity no definitive reproductive effect could be established. However there was no effect on pre-natal development in comparison to controls.

No effect of treatment was detected on reproduction or offspring development, at a treatment level up to 150 mg/kg/day therefore the ‘No Observed Effect Level’ (NOEL) for reproductive and development toxicity was considered to be 150 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The key study was conducted in accordance with the standardised guideline OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).It complies with GLP and was awarded a reliability score of 1 in accordance with the principles laid out by Klimisch (1997).
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

The systemic toxicity and potential adverse effects of the test material on reproduction (including offspring development) was investigated in accordance with the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

 

The test material was administered by gavage to three groups each of ten male and ten female Wistar strain rats, for up to forty-one consecutive days at 500 mg/kg/day and up to forty-two consecutive days at 150 and 50 mg/kg/day (including a two week maturation phase, pairing, gestation and early lactation for females). A control group of ten males and ten females was dosed with vehicle alone (distilled water). 

Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating and at termination on five selected males and females from each dose group. 

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.

Extensive functional observations were performed on five selected males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4post partum.

Surviving males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Adult Responses

Mortality

Two males treated with 500 mg/kg/day were found dead, one on Day 20 and one on Day 29. Two females from this dose group were also found dead, one on study Day 12 and one on Day 41. 

On Day 39, two 500 mg/kg/day females were killed in extremis whilst one 150 mg/kg/day female was killed in extremis on Day 42.

There were no further unscheduled deaths.

The 500 mg/kg/day treatment group was considered to be excessive and terminated on Day 41.

 

Litter Responses

Offspring Litter Size and Viability

There was no adverse effect on litter size for treated animals when compared to controls and there was no obvious difference in sex ratio for litters from treated females in comparison to controls.

 

Offspring Growth and Development

There was no significant intergroup difference in mean litter weights and offspring bodyweights for treated animals in comparison to controls.

A reduction in surface righting was evident in the 500 mg/kg/day offspring in comparison to controls. This was considered to be attributed to maternal nurturing.

No such effect was detected at 150 and 50 mg/kg/day.

 

Offspring Observations

Of the four litters produced at 500 mg/kg/day, one litter had one dead offspring. No abnormalities were detected in the remaining animals. Another litter had one dead offspring and four of the remaining offspring were cold and weak. All the animals in a further litter were cold. The whole of the remaining litter were cold with no milk in the stomach, they were also weak and two pups were found dead and five were missing.

Of the nine pregnancies at 150 mg/kg/day one litter had a number of dead offspring and the remaining offspring and dam were of poor physical health resulting in their termination.

 

Pathology

Two females treated with 500 mg/kg/day produced a litter with one death at birth. One pup displayed autolytic changes and one pup had no milk in the stomach. On Day 2 post partum three pups in one 150 mg/kg/day and two pups in one 50 mg/kg/day litter were found dead. These pups displayed autolytic changes.

 

As the 500 mg/kg/day treatment group was terminated early due to excessive toxicity no definitive reproductive effect could be established. However there was no effect on pre-natal development in comparison to controls.

No effect of treatment was detected on offspring development, at a treatment level up to 150 mg/kg/day therefore the ‘No Observed Effect Level’ (NOEL) for developmental toxicity was considered to be 150 mg/kg/day.


Justification for selection of Effect on developmental toxicity: via oral route:
Only one study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation 1272/2008, NCA requires a classification for reproductive and developmental toxicity (H360FD).

Additional information