Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 19 November 2008 and 17 May 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Details on test material:
- Substance type: organic
- Physical state: waxy tan solid
- Analytical purity: >99%
- Impurities (identity and concentrations): Heptane 0.007%, 2,3-dihydroxybutanedioc acid, bis oleyl amine < 1%.
- Lot/batch No.: 55-L-1434-F
- Stability under test conditions: stable
- Storage condition of test material: room temperature in the dark

Test animals

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: the males weighed 314 to 378 g; the females weighed 192 to 246 g
- Fasting period before study: Not applicable
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages withstainless steel mesh lids and softwood flake bedding (Harlan UK Ltd). During the matingphase, animals were transferred to polypropylene grid floor cages suspended over trayslined 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 floorpolypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet: The animals were allowed free access to food. A pelleted diet Rodent 2018CTeklad Global Certified Diet Harlan UK Ltd, Oxon, UK was used throughout the studyperiod. The diet was considered not to contain any contaminant at a level that mighthave 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: 7 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 19 November 2008 to 17 May 2009.
Male: up to 43 consecutive days for the control, 50, 350 and 500 mg/kg/day dose groups;;
Female: up to Day 5 post partum for control, 50, 350 and 500 mg/kg/day dose groups;
Male and female recovery groups were extended to 57 days;

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Arachis oil BP
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 Arachis oil BP. The stability and homogeneity of the
test material formulations were determined by Harlan Laboratories Ltd. Results show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately 4ºC in the dark.
Samples were taken of each test material formulation and were analysed for concentration of the test substance at Harlan Laboratories Ltd. The results indicate that the prepared formulations were within ± 9% 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): Not applicable
- Storage temperature of food: No data

VEHICLE: Arachis oil BP
- Justification for use and choice of vehicle (if other than water): Test material was poorly water soluble, so Arachis oil BP was used as vehicle to prepare test material suspension.
- Concentration in vehicle: 8.33, 58.3, 83.3 mg/ml
- Amount of vehicle (if gavage): 6 ml/kg
- Lot/batch no. (if required): Not applicable
- Purity: > 99%
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 the test substance in the test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.
The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling for homogeneity determinations was performed in triplicate.
The test material formulations were sampled and analyzed initially and then after storage at approximately +4ºC in the dark for fourteen days.
The test material formulation concentrations were sampled and analyzed within two days of preparation.
The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.
Duration of treatment / exposure:
The test material was administered to three groups each of ten male and ten female rats, for up to fifty-four consecutive days

Frequency of treatment:
Daily
Details on study schedule:
Dose Groups 1 to 4 (non-recovery)
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 fourteen 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 and urinalysis 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 Day 4 post partum for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all non-recovery females and surviving offspring were killed and examined macroscopically.
Dose groups 5 and 6 (recovery)
i) Groups of five male and five female rats were dosed according to dose group continuously up to the point of sacrifice of non-recovery males at which time treatment was discontinued.
ii) The males and females were maintained without treatment for a further fourteen days.
iii) Urinalysis was performed for all males during the final week of recovery.
iv) Blood samples were taken for haematological and blood chemical assessment on Day 56.
v) After fourteen days of recovery, males and females were killed and examined

- 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 historicallyused in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
350 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg/day. The highest dose level was reduced to 750 mg/kg/day on Day 8 due to the early termination of one male and significant actual bodyweight losses at 1000 mg/kg/day, then to 500 mg/kg/day on Day 15 due to bodyweight losses in male
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose (including control).

Control animals:
yes, concurrent no treatment
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 see .

- Rationale for animal assignment (if not random):
Random.

- Other:
Not applicable
Positive control:
Not applicable

Examinations

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 afterdosing, during the working week. Animals were observed immediately before dosing,soon after dosing, and one hour after dosing at weekends and public holidays (except forfemales during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, up to thirty minutes after dosing, and one and five hours afterdosing, during the working week. Animals were observed immediately before dosing,soon after dosing, and one hour after dosing at weekends and public holidays (except forfemales 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. Recovery animals were weighed on Day 1 (prior to dosing) and then weekly until termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes.
During the maturation period, weekly food consumption was recorded for each cage of non-recovery 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 recovery adults throughout the study period.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

FOOD EFFICIENCY: Yes.
Food efficiency (the ratio of bodyweight change/dietary intake) was calculated retrospectively for males throughout the study period and for females during maturation and the first two weeks of gestation. Due to offspring growth and milk production, food efficiency could not be accurately calculated during the final week of gestation and during lactation.
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes.
- Time schedule for examinations: A possible treatment-related effect was detected on Day 6, therefore gravimetric measurements were initiated from Day 7 throughout to study termination.

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: Haematological and blood chemical investigations were performed on five males and five females selected from each non-recovery test and control group on Day 14 (day prior to pairing) and on Day 42. In addition haematological and blood chemical investigations were performed on all recovery group animals after the fourteen day treatment free period at termination (Day 56). Blood samples were obtained from the lateral tail vein at Day 14 and by cardiac puncture at termination.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: five males and five females selected from each non-recovery test and control group
- Parameters were examined:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices
- mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed

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 non-recovery test and control group on Day 14 (day prior to pairing) and on Day 42. In addition haematological and blood chemical investigations were performed on all recovery group animals after the fourteen day treatment free period at termination (Day 56). Blood samples were obtained from the lateral tail vein at Day 14 and by cardiac puncture at termination.
- Animals fasted: No
- How many animals: five males and five females selected from each non-recovery test and control group
- Parameters were examined:
Urea
Calcium (Ca++)
Glucose
Inorganic phosphorus (P)
Total protein (Tot.Prot.)
Aspartate aminotransferase (ASAT)
Albumin
Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation)
Alkaline phosphate (AP)
Sodium (Na+)
Creatinine (Creat)
Potassium (K+)
Total cholesterol (Chol)
Chloride (Cl¯)
Total bilirubin (Bili)

URINALYSIS: Yes.
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters were examined:
Volume
Ketones
Specific Gravity
Bilirubin
Ph
Urobilinogen
Protein
Reducing Substances
Glucose
Blood

NEUROBEHAVIOURAL EXAMINATION: Yes.
- Time schedule for examinations:
- (Behavioural assessment) Prior to the start of treatment 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
Lachrymatation

- (Functional Performance Tests)
- Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were usedto assess motor activity. Animals were randomly allocated to the activity monitors. Thetests were performed at approximately the same time each day, under similar laboratoryconditions. The evaluation period was thirty minutes for each animal. The percentage oftime each animal was active and mobile was recorded for the overall thirty minute periodand 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 wasallowed to grip the proximal metal bar of the meter with its forepaws. The animal waspulled by the base of the tail until its grip was broken. The animal was drawn along thetrough of the meter by the tail until its hind paws gripped the distal metal bar. The animalwas pulled by the base of the tail until its grip was broken. A record of the force requiredto break the grip for each animal was made. Three consecutive trials were performed foreach 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
finger approach



Sacrifice and pathology
Adult non-recovery males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult non-recovery females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females that failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.
For all females the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 1% ammonium polysulphide solution.
Recovery animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination fourteen days after the termination of treatment (Day 57).
Adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

GROSS PATHOLOGY: Yes.
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 T
hyroid
Liver

HISTOPATHOLOGY: Yes.
Samples of the following tissues were preserved from five males and five females from each dose group, in buffered 10% formalin except where indicated. The tissues shown in bold were also removed from the remaining animals:
Ovaries
Pituitary
Prostate
Coagulation gland
Seminal vesicles
Epididymides♦
Testes♦
Uterus/Cervix
Mammary gland (all females)
Adrenals
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Oesophagus
Caecum
Rectum
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Skin (hind limb)
Eyes*
Spinal cord (cervical, mid-thoracic and lumbar)
Gross lesions
Spleen
Heart
Stomach
Ileum Thyroid/parathyroid
Jejunum
Trachea
Kidneys
Liver
Thymus
Lungs (with bronchi) #
Urinary bladder
Lymph nodes (cervical and mesenteric)
Vagina
Muscle (skeletal)
All tissues were despatched to Propath UK Ltd, Willow Court, Netherwood Road,
Rotherwas, Hereford, UK (Principal Investigator: E Richards). The tissues from five selected control and high 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 first 9 tissues (ovaries to mammalian gland) from the remaining control and high dose were also processed, and sections of testes and epididymides from all control and high dose males were stained with Periodic-Acid Shiff (PAS) and examined. Since there were indications of treatment-related bone marrow and thymus changes, examination was subsequently extended to include similarly prepared sections of the bone marrow and thymus from five animals per sex from the low and intermediate groups and all recovery group animals.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.

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:
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 42.

- 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 procedurewas enhanced; as necessary, by staining the uteri with a 1% ammonium polysulphide solution. In addition, the corpora lutea of all ovaries from pregnantfemales 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), mammarygland, 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 Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford, UK. The tissues from five selected control, control and high 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 in bold from the remaining control and high dose were also processed, and sections of testes and epididymides from all control and high dose males were stained with Periodic-Acid Shiff (PAS) and examined.
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. All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
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:
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.
In addition, histopathological findings were analyzed 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.
Plus (+) signs indicate positive differences from the control group and minus (-) signs indicate negative differences. Asterisks refer to overall differences between group variation which is non-directional.
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 paired ÷ Number of animals mated) x 100
Pregnancy Index (%) = (Number of animals mated ÷ Number of pregnant females) 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 pregnant females ÷ Number of females delivering live offspring) 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 Corpora Lutea) ÷ Number of implantation sites] x 100
% post – implantation loss =[(Number of implantation sites - Number of implantation sites) ÷ Total number of offspring born] x 100
ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring born ÷Number of offspring alive on Day 1) x 100
Viability Index 1 (%) = (Number of offspring alive on Day 1 ÷ Number of offspring alive on Day 4) x 100
iii) Sex Ratio (% males)
Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula:
(Number of male offspring ÷ Total number of offspring) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

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

Details on results (P0)

Results showed significant changes as comparing treated group to the concurrent control group were present in the attached Tables.
Adult Responses:
Mortality. One male treated at the high dose level was killed in extremis on Day 12. There were no further unscheduled deaths during the study.
Clinical Signs. Animals of either sex treated at the high dose level, all males and four females treated with 350 mg/kg/day and three males and one female treated with 50 mg/kg/day showed episodes of increased salivation immediately or one hour post dosing throughout the treatment period. Noisy respiration was evident for one male treated at the high dose level on Day 8. A further male from this treatment group also showed tiptoe gait on Day 15 and hunched posture between Days 15 and 21. Instances of generalised staining around the mouth, snout and eyes were detected for animals of either sex treated at the high dose level and in five males treated with 350 mg/kg/day throughout the study. The male that was killed in extremis developed hunched posture and was dehydrated between Days 9 and 12. This male also showed increased salivation between Days 10and 11, pilo-erection between Days 11 and 12 and on Day 12 had staining around the mouth/snout and was emaciated.

Bodyweights (Table 10 and 11). Males treated at the high dose level showed statistically significant reductions in bodyweight gain during the first two weeks of treatment. Significant actual bodyweight losses were evident in these males during the first two weeks of treatment. During this period animals were dosed with 1000 mg/kg/day which was reduced to 750 mg/kg/day on Day 8. Females from this treatment group also showed a statistically significant reduction in bodyweight gain during the first week of treatment. Males treated with 350 mg/kg/day showed a statistically significant reduction in bodyweight gain during Week 2 and a reduced cumulative bodyweight gain throughout the treatment period. No such effects were detected in females treated with 350 mg/kg/day or animals of either sex treated with 50 mg/kg/day.

Food Consumption and Food Efficiency. Males treated at the high dose level showed a reduction in food consumption and food efficiency during the first two weeks of treatment. During this period animals were dosed with 1000 mg/kg/day which was reduced to 750 mg/kg/day on Day 8. Females treated at the high dose level showed a reduction in food consumption and food efficiency during the first week of treatment. No such effects were detected in animals of either sex treated with 350 or 50 mg/kg/day.

Water Consumptions. No significant intergroup differences were detected.

Ophthalmoscopic Examination
Not examined.

Haematology. No treatment-related effects were detected in the haematological parameters measured.

Blood Chemistry (Table 20 and 21). Males treated at the high dose level showed a statistically significant increase in albumin/globulin ratio and a statistically significant reduction in total protein at both Day 14 and Day 42 evaluations. The effect on total protein also extended to recovery high dose males following fourteen days without treatment. Females treated at the high dose showed a similar effect on the total protein at the Day 14 observation; this effect was not seen at Day 4 post partum or in recovery females. No such effects were detected in animals of either sex treated with 350 or 50 mg/kg/day or recovery females following fourteen days without treatment.

Urinalysis. No toxicologically significant effects were detected in the parameters measured.

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

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

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

Pathology.
Necropsy. No toxicologically significant effects were detected.
Organ Weights (Table 32 and 33). Males treated at the high dose level showed a statistically significant reduction in absolute thymus weight. No such effects were detected in females at the high dose level, animals of either sex treated with 350 or 50 mg/kg/day or recovery animals following fourteen days without treatment.
Histopathology - non-neoplastic. The following treatment-related microscopic changes were detected:
BONE MARROW: A greater incidence of higher grades of adipose infiltration of the marrow, indicative of marrow hypoplasia, was seen for males only treated at the high dose level compared with controls. A convincing effect was not seen at any other dose level. There was no evidence of regression of the condition among recovery high dose animals following an additional fourteen days without treatment.
THYMUS: Treatment-related lymphoid atrophy was seen in relation to treatment for four females treated at the high dose level compared with control animals. There was possibly an effect of treatment at the 350 mg/kg/day dose level with three females demonstrating atrophy, but no convincing effect at the 50 mg/kg/day dose level. Two male rats at the high dose level were also affected although one of these animals died prematurely. Atrophy of the thymus was not seen among control or treated recovery group animals and treatment-related atrophy was thus considered to have fully regressed after completion of the recovery period. Thymic atrophy is occasionally seen among post-partum and lactating female rats but the group distribution of incidence in this investigation does suggest an effect of treatment.

HISTOPATHOLOGY: NEOPLASTIC (if applicable) Not applicable.
HISTORICAL CONTROL DATA (if applicable) Not applicable.

OTHER FINDINGS:
Reproductive Performance:
Mating. One female treated with 350 mg/kg/day did not show positive evidence of mating. There were no treatment-related effects on mating or conception rates for animals of either sex treated at the high dose level or at 50 mg/kg/day.
Fertility. There were no treatment-related effects on conception rates.
Gestation Length. There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

Effect levels (P0)

Key result
Dose descriptor:
NOEC
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

Offspring Litter Size and Viability. Of the litters born, litter size at birth and subsequently on Day 1 and 4 post partum were comparable to controls.
Offspring Growth and Development. Offspring bodyweight gain and litter weights at birth and subsequently on Day 1 and 4 post partum were comparable to controls.
Litter observations. No clinically observable signs of toxicity were detected for offspring from all treatment groups.

Effect levels (F1)

Key result
Dose descriptor:
NOEC
Generation:
F1
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Resuls are showed in the attached Tables.

Applicant's summary and conclusion

Conclusions:
The oral administration of the test substance to rats by gavage, at dose levels of 50, 350 and
1000 mg/kg/day (reduced to 750 mg/kg/day on Day 8 and then 500 mg/kg/day onDay 15), no treatment-related effects were detected in the reproductive parameters measured. In view of the longer administration period the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 500 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).

Methods. The test material was administered by gavage to three groups each of ten male and ten female Wistar Han™:HsdRccHan™:WIST strain rats, for up to fifty-four consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 50, 350 and 1000 mg/kg/day. Due to the early termination of one male and significant actual bodyweight losses at 1000 mg/kg/day, the highest dose level was reduced to 750 mg/kg/day on Day 8. Males continued to show actual bodyweight losses and the high dose level was reduced again to 500 mg/kg/day on Day 15. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil). Two recovery groups, each of five males and five females, were treated with the high dose (1000/750/500 mg/kg/day) or the vehicle alone for forty-two consecutive days and then maintained without treatment for a further fourteen days. 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 non-recovery males and females from each dose group. In addition, haematology and blood chemistry were evaluated after the fourteen day treatment free period on all recovery animals. Urinalysis was evaluated on five selected non-recovery males at the end of the treatment period and for all recovery group males at the end of the treatment free period.

Pairing of animals within each non-recovery 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 nonrecovery dose group after the completion of the mating phase, and for five selected parental females from each non-recovery dose group on Day 4post partum.

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

Recovery animals were treated according to the dose group continuously up to the point of sacrifice of the non-recovery males at which time the treatment was discontinued. After fourteen days without treatment, the recovery males and females were subjected to a gross necropsy examination of selected tissues was performed.

Results

Adult Responses:

Mortality.One male treated at the high dose level was killedin extremison Day 12. There were no further unscheduled deaths during the study.

Clinical Signs.Animals of either sex treated at the high dose level, all males and four females treated with 350 mg/kg/day and three males and one female treated with 50 mg/kg/day showed episodes of increased salivation immediately or one hour post dosing throughout the treatment period. Noisy respiration was evident for one male treated at the high dose level on Day 8. A further male from this treatment group also showed tiptoe gait on Day 15 and hunched posture between Days 15 and 21. Instances of generalised staining around the mouth, snout and eyes were detected for animals of either sex treated at the high dose level and in five males treated with 350 mg/kg/day throughout the study.

The male that was killedin extremisdeveloped hunched posture and was dehydrated between Days 9 and 12. This male also showed increased salivation between Days 10 and 11, pilo-erection between Days 11 and 12 and on Day 12 had staining around the mouth/snout and was emaciated.

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

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

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

Bodyweights.Males treated at the high dose level showed statistically significant reductions in bodyweight gain during the first two weeks of treatment. Significant actual bodyweight losses were evident in these males during the first two weeks of treatment. During this period animals were dosed with 1000 mg/kg/day which was reduced to 750 mg/kg/day on Day 8. Females from this treatment group also showed a statistically significant reduction in bodyweight gain during the first week of treatment. Males treated with 350 mg/kg/day showed a statistically significant reduction in bodyweight gain during Week 2 and a reduced cumulative bodyweight gain throughout the treatment period. No such effects were detected in females treated with 350 mg/kg/day or animals of either sex treated with 50 mg/kg/day.

Food Consumption and Food Efficiency.Males treated at the high dose level showed a reduction in food consumption and food efficiency during the first two weeks of treatment. During this period animals were dosed with 1000 mg/kg/day which was reduced to 750 mg/kg/day on Day 8. Females treated at the high dose level showed a reduction in food consumption and food efficiency during the first week of treatment. No such effects were detected in animals of either sex treated with 350 or 50 mg/kg/day.

Water Consumptions.No significant intergroup differences were detected.

Haematology.No treatment-related effects were detected in the haematological parameters measured.

Blood Chemistry.Males treated at the high dose level showed a statistically significant increase in albumin/globulin ratio and a statistically significant reduction in total protein at both Day 14 and Day 42 evaluations. The effect on total protein also extended to recovery high dose males following fourteen days without treatment. Females treated at the high dose showed a similar effect on the total protein at the Day 14 observation; this effect was not seen at Day 4post partumor in recovery females. No such effects were detected in animals of either sex treated with 350 or 50 mg/kg/day or recovery females following fourteen days without treatment.

Urinalysis.No toxicologically significant effects were detected in the parameters measured.

Reproductive Performance:

Mating.One female treated with 350 mg/kg/day did not show positive evidence of mating. There were no treatment-related effects on mating or conception rates for animals of either sex treated at the high dose level or at 50 mg/kg/day.

Fertility.There were no treatment-related effects on conception rates.

Gestation Length.There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

Litter Responses.

Offspring Litter Size and Viability.Of the litters born, litter size at birth and subsequently on Day 1 and 4post partumwere comparable to controls.

Offspring Growth and Development.Offspring bodyweight gain and litter weights at birth and subsequently on Day 1 and 4post partumwere comparable to controls.

Litter observations.No clinically observable signs of toxicity were detected for offspring from all treatment groups.

Pathology.

Necropsy.No toxicologically significant effects were detected.

Organ Weights.Males treated at the high dose level showed a statistically significant reduction in absolute thymus weight. No such effects were detected in females at the high dose level, animals of either sex treated with 350 or 50 mg/kg/day or recovery animals following fourteen days without treatment.

Histopathology.The following treatment-related microscopic changes were detected:BONE MARROW: A greater incidence of higher grades of adipose infiltration of the marrow, indicative of marrow hypoplasia, was seen for males only treated at the high dose level compared with controls. A convincing effect was not seen at any other dose level. There was no evidence of regression of the condition among recovery high dose animals following an additional fourteen days without treatment.

THYMUS: Treatment-related lymphoid atrophy was seen in relation to treatment for four females treated at the high dose level compared with control animals. There was possibly an effect of treatment at the 350 mg/kg/day dose level with three females demonstrating atrophy, but no convincing effect at the 50 mg/kg/day dose level. Two male rats at the high dose level were also affected although one of these animals died prematurely.

Atrophy of the thymus was not seen among control or treated recovery group animals and treatment-related atrophy was thus considered to have fully regressed after completion of the recovery period.

Thymic atrophy is occasionally seen among post-partum and lactating female rats but

the group distribution of incidence in this investigation does suggest an effect of

treatment.

Conclusion.The oral administration of the test substance to rats by gavage, at dose levels of 50, 350 and 1000 mg/kg/day (reduced to 750 mg/kg/day on Day 8 and then 500 mg/kg/day on Day 15), resulted in treatment-related effects in animals of either sex treated at the high dose level and at 350 mg/kg/day. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 50 mg/kg/day. The effects detected at 350 mg/kg/day were confined to a slight reduction in bodyweight development for males, an increase in albumin/globulin ratio for males and lymphoid atrophy in the thymus for three females. In the absence of any associated changes

these effects were considered not to represent an adverse health effect. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore considered to be 350 mg/kg/day.

No treatment-related effects were detected in the reproductive parameters measured. In view of the longer administration period the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 500 mg/kg/day.