Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 January 2017-20 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

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
Chemical structure
Reference substance name:
2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate
EC Number:
224-116-8
EC Name:
2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate
Cas Number:
4203-89-8
Molecular formula:
C14H23NO3
IUPAC Name:
2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate
Test material form:
liquid: viscous
Specific details on test material used for the study:
Identification : 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate (CAS No.: 4203-89-8)
Supplier : Akzo Nobel Functional Chemicals bv
Product Name : Nourycryl MA 128
Chemical Name : 2-(2,2-pentamethylene-1,3-oxazolidyl-3)ethyl methacrylate
EC Name : 2-(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate
CAS Number : 4203-89-8
Batch Number : 1609501020
Purity : 94.5%
Description : Extremely pale yellow, slightly viscous liquid
Expiry Date : 05 October 2017
Receipt Date : 13 October 2016
Storage : Approximately 4 °C in the dark, used/formulated at ambient temperature in the light

No correction for purity was made.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal Information A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for nineteen days during which time their health status was assessed. Following the day of arrival, vaginal smears were performed for all females throughout the acclimatization period and females considered not showing appropriat e estrous cycling activity were excluded from treatment groups at least five days before the start of treatment. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 274 to 348g, and were approximately eleven weeks old. The females weighed 190 to 221g, and were approximately fourteen weeks old.
Animal Care and Husbandry Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing 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. The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Gl obal Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Annex 5. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or inte grity of the study. The animals were housed in a single air-conditioned room within the Envigo Research Limited, Sh ardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air c hanges per hour and the low intensity fluorescent lighting was controlled to give twelve hours cont inuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively; there were no deviations from these targets. The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
The test item was administered daily by gavage using a stainless steel cannula attached to a disposa ble plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP. The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Details on mating procedure:
On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results showed the formulations to be stable for at least 8 days at 4ºC in the dark. Formulations were therefore prepared weekly and stored at approximately 4 ºC in the dark. Samples of test item formulations were taken on three occasions and analyzed for concentration of 2(1-oxa-4-azaspiro[4.5]dec-4-yl)ethyl methacrylate (CAS No.: 4203-89-8) at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 5% of the nominal concentration.
Duration of treatment / exposure:
approximately 6 weeks (males) and up to eight weeks (females) (including a two week pre-paring phase, pairing, gestation and early lactation for females), at dose levels of 50, 150 and 375 mg/kg bw/day.
Frequency of treatment:
Daily
Details on study schedule:
Chronological Sequence of Study
i. Males and females were housed for a suitable acclimatization period which allowed at least two weeks of pre-treatment vaginal smears to be performed for females enabling the exclusion of females not showing appropriate estrous cycling.
ii. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
iii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
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 the pairing five selected males per dose group were evaluated for functional/sensory responses to various stimuli during week 6.
vii. Pregnant females were allowed to give birth and maintain their offspring until Day 13 post partum. Litter size, offspring weight and sex ratio, ano-genital distance and visible nipple counts (male offspring) and clinical signs were also recorded during this period.
viii. At Day 12 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
ix. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 43. The male dose groups were sacrificed and examined macroscopically on Day 44 or 45.
x. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 13 post partum. All females were sacrificed on Day 14 post partum and examined macroscopically. A vaginal smear was also performed for all females in the morning of the day of necropsy. Any female which did not produce a pregnancy was also sacrificed and examined macroscopically around the same time as littering females.
xi. Where possible, blood samples (to produce serum) were taken from two randomly allocated offspring on Day 4 post partum for assessment of thyroid hormones. On Day 13 post partum, where possible, blood sampling (to produce serum) was performed on two randomly allocated offspring (one male and one female) per litter for assessment of thyroid hormones. Where possible, a further two randomly allocated offspring (one male and one female) per litter were sampled (to produce plasma). On day 13 post partum all surviving offspring were sacrificed and examined externally; an internal examination was performed if abnormalities were detected externally. In addition, blood samples were taken from all adult males and females at termination. Blood samples from all adult males and Day 13 offspring were analyzed for Thyroxine (T4).
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
375 mg/kg bw/day (nominal)
Control animals:
yes, concurrent vehicle
Details on study design:
12 males and females per dose

Examinations

Parental animals: Observations and examinations:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable). All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4 and 7 post partum. Body weights were also recorded at terminal sacrifice.
Normal range data for body weight changes in pregnant and lactating females are shown in Annex 7.

Food Consumption
During the pre-pairing 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 post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded for the periods covering post partum Days 1-4, 4-7, 7-14.
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.
Normal range data for pregnant and lactating females are presented in Annex 7.

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

Specialist Evaluations
Functional Observations
Prior to the start of treatment and at approximately weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. These observations were performed on mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

Behavioral 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 color
Twitches
Respiration
Convulsions
Palpebral closure
Bizarre/Abnormal/Stereotypic behavior
Urination
Salivation
Defecation
Pilo-erection
Transfer arousal
Exophthalmia
Tail elevation
Lachrymation
This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

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 on each occasion (at least two hours after dosing), 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, Reiter and Macphail, 1979).
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. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).
The following parameters were observed:
Grasp response Touch escape
Vocalization Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach

Reproductive Performance

Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus 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. Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 43 for males and Day 13 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)

Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Inorganic phosphorus (P)
Glucose Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.) Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation) Creatinine (Creat)
Sodium (Na+) Total cholesterol (Chol)
Potassium (K+) Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Calcium (Ca++)



Oestrous cyclicity (parental animals):
Estrous Cycle Assessment
Vaginal smears were taken daily for females throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.
Sperm parameters (parental animals):
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.
Litter observations:
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post partum
iii. Sex of offspring on Days 1, 4, 7 and 13 post partum
iv. Clinical condition of offspring from birth to Day 13 post partum
v. Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data)
4.4.7.4 Physical Development
All live offspring were assessed for ano-genital distance on Day 1 post partum.
Additionally, visible nipple count was performed for all male offspring on Day 13 post partum.
Postmortem examinations (parental animals):
Necropsy
Adult males were sacrificed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45. Adult females were sacrificed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum. Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were terminated by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation. Any females which failed to achieve pregnancy or produce a litter were sacrificed around the same time as littering females.
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 0.5% ammonium polysulphide solution (Salewski 1964).
All adult animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. Examination of offspring was restricted to a macroscopic external examination except where abnormalities were observed, then an additional internal examination was performed.

Organ Weights
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group. Tissues shown in bold were weighed from all remaining animals:
Adrenals Pituitary (weighed post-fixation)
Brain Prostate
Epididymides Seminal Vesicles (with Coagulating Gland)
Heart Spleen
Kidneys Testes
Liver Thymus
Ovaries Thyroid (weighed partially fixed with Parathyroid)
Uterus (weighed with Cervix and Oviducts)
Normal ranges for organ weights are given in Annex 10.
On Day 13 of age, where possible, for one male and one female offspring per litter, the thyroid/parathyroid were retained in 10% Buffered Formalin.

Histopathology
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown in bold were preserved from all remaining animals:
Adrenals Muscle (skeletal)
Aorta (thoracic) Ovaries
Bone & bone marrow (femur including stifle joint) Pancreas
Bone & bone marrow (sternum) Pituitary
Brain (including cerebrum, cerebellum and pons) Prostate
Cecum Rectum
Colon Salivary glands (submaxillary)
Cowpers glands Sciatic nerve
Duodenum Seminal vesicles (with coagulating gland)
Epididymides ♦
Esophagus Skin
Eyes * Spinal cord (cervical, mid thoracic
Glans penis and lumbar)
Gross lesions Spleen
Heart Stomach
Ileum (including peyer’s patches) Testes ♦
Jejunum Thyroid/Parathyroid
Kidneys Trachea
LABC (levator ani-bulbocavernous) muscle Thymus
Liver Urinary bladder
Lungs (with bronchi)# Uterus & Cervix (with oviducts)
Lymph nodes (mandibular and mesenteric) Vagina
Mammary gland
Tissues were dispatched to the Test Site (Envigo CRS Limited, Eye, Suffolk, IP23 7PX) for processing (Principal Investigator: J Schofield). The tissues from five selected control and 375 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 375 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 375 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.
Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of kidneys from five selected animals of each sex, and adrenals from five selected male animals in the low and intermediate groups.
Postmortem examinations (offspring):
Offspring required for blood sampling were terminated by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation
Where possible from each litter, serum samples from two randomly allocated offspring on Day 4 post partum (if offspring were of the same sex, samples from the same litter were pooled). If eight or less offspring were present in a litter, then no offspring from that litter were sampled on Day 4 post partum.
Serum samples from two randomly allocated offspring (one male and one female) on Day 13 post partum. Where possible from each litter, plasma samples were also taken from two randomly allocated offspring (one male and one female) on Day 13 post partum. If required the number/sex of offspring sampled was altered depending on the litter constituents.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Implantation Sites, Post-implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Developmental Parameters, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights and Thyroid Hormone (Thyroxine).
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module.
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)
Reproductive indices:
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 paired x 100
Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii. Parturition Index
The following was calculated for each group:
Parturition Index (%) = Number of females delivering live offspring / number of pregnant females x 100
Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 13 of age).
i. Implantation Losses (%)
Group mean percentile post-implantation loss was calculated for each female/litter as follows:
Post–implantation loss (%) = number of implantation sites - total number of offspring born / number of implantation sites x100
ii. Live Birth
The following indices were calculated for each litter as follows:
Live Birth Index (%) = number of offspring aline on Day 1 / Number of offspring born x 100

Offspring viability indices:
Viability Indices
Viability Index 1 (%) = number of offspring alive on Day 4 / number of offspring aline on Day 1 x 100
Viability Index 2 (%) = number of offspring aline on Day 13 / number of offspring aline on Day 4 x 100
Viability index 2 takes into consideration the offspring used for blood sampling on Day 4 post partum. iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1, 4 and 13 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:
no effects observed
Description (incidence and severity):
There were no clinical signs observed that were considered to be indicative of systemic toxicity.
For all animals at 375 mg/kg bw/day and 9/12 males at 150 mg/kg bw/day increased post-dosing salivation was observed during the study. Such salivation is often observed when administering test formulations via the oral gavage route and is generally considered to reflect palatability or slight ir ritancy of the test item formulations rather than any systemic effect of treatment.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths on the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males
At 375 mg/kg bw/day, body weight gain of males was inferior to control throughout much of the treatment period, with differences attaining statistically significance. These lower gains led to a clear reduction in overall body weight gain for the study and resulting body weight was statistically significantly lower than control by Day 43.
At 150 mg/kg bw/day, body weight gain of males was frequently lower than control throughout the treatment period, with statistical significance being attained during Week 2 (Days 8-15) and 4 (Days 22-29). Overall body weight gain was also statistically significantly lower than control but to a lesser extent than observed at 375 mg/kg bw/day.
There was considered to be no obvious effect treatment on body weight gain for males receiving 50 mg/kg bw/day. Statistically significant lower body weight gain was apparent during Week 4 but, in the absence of any statistically significant effect on overall body weight gain, this finding was considered to be incidental and unrelated to treatment.
Females
At 375 mg/kg bw/day, body weight gain of females was lower than control during the first week of treatment with differences attaining statistical significance. Body weight gain during the second week of treatment and overall body weight gain for the pre-pairing phase of the study were also lower than control but showed no statistical significance or dose relationship. However, mean body weights were statistically significantly lower than control throughout gestation, with body weight gain being significantly lower than control during the last two weeks of gestation. Body weight during the last week of gestation was adversely influenced by a lower contribution from the gravid uterus, due to lower litter size at this dosage. Evaluation of body weight during lactation was complicated by the relatively high number of females showing total litter loss post partum at this dosage, however females generally showed lower body weight gain during the second week of lactation, compared to control, with differences attaining statistical significance. These differences were more marked when females that showed total litter loss post partum were included in the calculation of mean values and mean body weights calculated in this manner were statistically significantly lower than control from Day 4 of lactation.
At 150 mg/kg bw/day, body weight gain was statistically significantly lower than control during the first week of treatment. Subsequent body weight gain during the second week of treatment was also lower than control but showed no dosage relationship or statistical significance. By the end of the two week pre-pairing phase, mean body weight and overall body weight gain were statistically significantly lower than control, although mean values showed no dose relationship. Mean body weights values continued to be statistically significantly lower than control throughout gestation, with body weight gain being lower than control during the last two weeks of gestation. However, the lower mean gain only attained statistical significance during the second week of gestation. No statistically significant differences for body weight or body weight gain from control were apparent for females during lactation.
At 50 mg/kg bw/day, there was no obvious effect on body weight or body weight gain for females during the pre-pairing, gestation or lactation phases of the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no obvious effect of treatment on food consumption of males throughout the study at 50, 150 or 375 mg/kg bw/day.
There was no obvious effect of treatment on food consumption of females during the two week pre-pairing phase of the study at 50, 150 or 375 mg/kg bw/day.
At 375 mg/kg bw/day, food consumption was statistically significantly lower than control during the second week of gestation, this finding was consistent with lower body weight gain for this period. Mean food consumption was also lower than control during the first and third week of gestation, but differences failed to attain statistical significance and, at the level observed probably reflect normal biological variation. Food consumption for females was statistically significantly lower than control throughout lactation, when females that showed total litter loss post partum were included in the calculation of mean values. However, when these females were excluded, statistically significant lower food intake was only apparent during Days 7 -14 of lactation. These differences in food intake from control probably reflect differences in demand on the females from the growing litters.
There was no obvious effect on food consumption for females during the pre-pairing, gestation or lactation phases of the study at 50 or 150 mg/kg bw/day.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
At 375 mg/kg bw/day, food conversion efficiency for males was lower than control during the two week pre-pairing phase of the study, reflecting the lower body weight gain apparent during this stage of the study. Food conversion efficiency was also lower than control during the first two weeks of the post-pairing phase of the study.
Intergroup differences in food conversion efficiency did not indicate any consistent effect of treatment during the pre-pairing and post-pairing phases of the study for males at 50 or 150 mg/kg bw/day, although values did tend to be lower than control. At 150 mg/kg bw/day, food conversion efficiency was lower than control during the second week of treatment however, the magnitude of the difference from control was not repeated for other occasions on the study.
At 375 mg/kg bw/day, food conversion efficiency for females was lower than control during the first week of treatment but was similar to control for the following week of the pre-pairing phase.
At 150 mg/kg bw/day, food conversion efficiency for females was lower than control throughout the two week pre-pairing phase, although no dosage relationship was apparent for the second week.
There was no obvious effect on food conversion efficiency for females during the pre-pairing phase of the study at 50 mg/kg bw/day.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of water bottles throughout the study did not indicate any effect of treatment for either sex throughout the study.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
For males at 375 mg/kg bw/day, lower mean erythrocyte count, hemoglobin, hematocrit, mean corpuscular hemoglobin, mean corpuscular volume and mean corpuscular hemoglobin concentration and higher reticulocyte count attained statistical significance when compared with control. For erythrocyte count, hemoglobin, hematocrit, mean corpuscular hemoglobin and mean corpuscular volume, all or the majority of individual values for treated animals were outside the historical control but for mean corpuscular hemoglobin concentration and higher reticulocyte count, all individual values were within the historical control range.
For females at 375 mg/kg bw/day, lower mean hemoglobin attained statistical significance when compared with control (despite the mean control value for hemoglobin being adversely influenced by one particularly low individual value), however, only one individual hemoglobin value at 375 mg/kg bw/day was outside the historical control range. Mean reticulocyte count was statistically significantly higher than control at this dosage but mean values showed no dosage relationship and all individual values were within the historical control range.
For males at 150 mg/kg bw/day, lower mean erythrocyte count, hemoglobin, hematocrit, mean corpuscular hemoglobin and mean corpuscular volume and higher reticulocyte count attained statistical significance when compared with control. However only isolated individual values for hemoglobin, hematocrit and mean corpuscular volume were outside the historical control range. For females at this dosage, mean reticulocyte count was statistically significantly higher than control but mean values showed no dosage relationship and all individual values were within the historical control range.
For both sexes at 50 mg/kg bw/day, mean reticulocyte counts were statistically significantly higher than control however, mean values for females showed no dosage relationship and all individual values for both sexes were within their respective historical control range.
For males at 375 mg/kg bw/day, mean total leucocyte count was statistically significantly lower than control, principally due to a statistically significantly lower number of lymphocytes, however, all individual values for both parameters for the treated animals were within the historical control range. For females at 375 mg/kg bw/day, the mean number of neutrophils was statistically significantly lower than control, however this had no adverse effect on mean total leucocyte count and all individual values for the treated animals were within the historical control range.
For males at all dosages, mean platelet count was statistically significantly higher than control but mean values showed no dosage relationship. All individual values for treated animals were within the historical control range, while one control value exceeded the historical control range. For females at 375 mg/kg bw/day, mean platelet count was statistically significantly lower than control. Four individual values at 375 mg/kg bw/day were outside the historical control range, however so were two individual control values and four individual values at 50 mg/kg bw/day.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
For males at all dosages, higher albumin attained statistical significance and higher albumin/globulin ratio attained statistical significance at 150 and 375 mg/kg bw/day when compared with control, but only at 375 mg/kg bw/day was there an accompanying statistically significant increase to mean total protein, and mean values for albumin/globulin ratio showed no dosage relationship. For these parameters in treated animals, only one individual value for total protein at 375 mg/kg bw/day exceeded the historical control range, while for the control group isolated values for total protein, albumin and albumin/globulin ratio were below the historical control range.
For females at 150 and 375 mg/kg bw/day, mean values for albumin and total protein were statistically significantly higher than control, but all individual values for these treated animals were within the historical control range, while two control values for total protein and one for albumin were lower than the historical control range.
For males at 150 and 375 mg/kg bw/day, mean sodium levels were statistically significantly higher than control; one individual value at 150 mg/kg bw/day and three individual values at 375 mg/kg bw/day exceeded the historical control range.
For males at 150 and 375 mg/kg bw/day, mean chloride levels were statistically significantly higher than control; all values for treated and control males animals exceeded the historical control range. For females at 375 and 150 mg/kg bw/day, mean chloride levels were also statistically significantly higher than control but all individual values for these treated females were within the historical control range, while one control value was below the historical control range.
For males at all dosages, mean calcium levels were statistically significantly higher than control, all individual values for treated and control males animals exceeded the historical control range.
For both sexes at 375 mg/kg bw/day, alanine aminotransferase activities were statistically significantly lower than control. Individual values for these treated and control males and treated females were all within the historical control range, however the majority of control females exceeded the historical control range. A decrease in this parameter is unlikely to be of any toxicological significance.
For males at 150 and 375 mg/kg bw/day, mean creatinine levels were statistically significantly higher than control, however mean values showed no dosage relationship and only one individual value at 150 mg/kg bw/day exceeded the historical control range.
For males at 150 and 375 mg/kg bw/day, mean bile acid levels were statistically significantly higher than control, however all individual values for these treated animals were within the historical control range.
For females at 150 mg/kg bw/day, mean total bilirubin was statistically significantly higher than control, however all individual values for these treated females were within the historical control range while one control value was below the historical control range. In the absence of any similar finding a 375 mg/kg bw/day, this finding was considered to be incidental and unrelated to treatment.
For females at 375 mg/kg bw/day, mean values for glucose were statistically significantly higher than control; two individual values exceeded the historical control range.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Behavioural Assessments
Assessment of the animals in a standard arena did not reveal any obvious effects of treatment for either sex at 50, 150 or 375 mg/kg bw/day.

Functional Performance Tests
Results from functional performance tests did not reveal any obvious effects of treatment for either sex at 50, 150 or 375 mg/kg bw/day.

Sensory Reactivity Assessments
There were no differences from control in the scores for sensory reactivity for either sex at 50, 150 or 375 mg/kg bw/day.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic examination of the kidneys revealed multifocal basophilic tubules in 3/5 females treated at 375 mg/kg bw/day at a mild or minimal severity.
Minimal tubular vacuolation of the kidneys was present in 2/5 males treated at 50 mg/kg bw/day and 3/5 males treated at 150 mg/kg bw/day, at a minimal severity. At 375 mg/kg bw/day, it was present in 4/5 males, with two each of minimal and mild severity. In females, minimal tubular vacuolation was present in 3/5 females treated at 150 mg/kg bw/day at minimal severity and in 3/5 females at 375 mg/kg bw/day, two mild and one minimal severity.
Vacuolation of the cortex of the adrenal glands was above expected incidental variation in 2/5 males treated at 375 mg/kg bw/day.
There were no other microscopic changes observed that were considered to indicate an effect of treatment at 50, 150 or 375 mg/kg bw/day. In particular, there were no test item-related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries. There were no consistent differences observed for the liver that could account for the increase in weight noted at necropsy. Additionally, at 375 mg/kg bw/day, there were no pathological changes apparent to account for the increase in uterine weight noted at necropsy. Uterine changes were considered to be largely consistent with pregnancy/lactation and, in those animals that had shown total litter loss, the uterus was consistent with females returning to cyclical activity.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any effect of treatment or indication of endocrine disruption at 50, 150 or 375 mg/kg bw/day.
For adult males receiving 150 or 375 mg/kg bw/day, T4 blood levels were statistically significantly higher than control. At 150 mg/kg bw/day, all individual values were within the historical control range, whilst at 375 mg/kg bw/day only 3/10 values exceeded this historical range. For the control group, although only one individual value was below the historical group range, the control group mean for this study was lower than the control group mean for all studies in the historical data. In view of the lack of any supporting effect on thyroid weights or histopathology, the observed differences from control were considered to reflect atypically low control values rather than any effect of treatment.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Assessment of estrous cycles during the pre-pairing phase of the study or at necropsy did not indicate any obvious effect of treatment at 50, 150 or 375 mg/kg bw/day.
At 375 mg/kg bw/day, a lower incidence of females were in di-estrus at the time of necropsy but was considered to reflected the early returning of females that showed total litter loss post partum to normal estrous cycling rather than any effect of treatment on the estrous cycle.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
there were no test item-related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle)
Description (incidence and severity):
Mating
A summary of adult performance is presented in Table 1. A summary incidence for mating performance is presented in Table 12. Individual data are given in Appendix 11.
Mating performance as assessed by the number of paired animals that mated was unaffected by treatment at 50, 150 or 375 mg/kg bw/day.
Fertility
A summary of adult performance is presented in Table 1. Group values for fertility, litter data and implantation losses are given in Tables 12, 13 and 15. Individual data are given in Appendices 11, 12 and 14.
There was no obvious effect on fertility, as assessed by the number of females that achieved pregnancy, at 50, 150 or 375 mg/kg bw/day.
Gestation Length
A summary of gestation lengths is presented in Table 12. Individual lengths are given in Appendix 11.
At 375 mg/kg bw/day, approximately 50% of the littering females showed a gestation length of 24 days or longer. This high incidence of extended gestation length is unusual and is considered to be related to treatment; five of the six affected females subsequently showed total litter loss post partum.
The intergroup distribution of gestation lengths observed during the study did not indicate any obvious effect of treatment at 50 or 150 mg/kg bw/day.
Litter Responses
One control female, one female at 150 mg/kg bw/day and one female at 375 mg/kg bw/day were non-pregnant. Additionally at 375 mg/kg bw/day, a total of five females showed total litter loss post partum, this high incidence of litter losses is unusual and is considered to be related to maternal treatment. The following assessment is generally based on the 11, 12, 11 and 6 females that successfully maintained a litter to Day 13 of age at 0 (Control), 50, 150 and 375 mg/kg bw/day respectively, although the data from females showing total litter loss post partum was also taken into consideration when assessing effects at the high dosage.
Litter Size and Viability
At 375 mg/kg bw/day, the mean number of implantations was lower than control for females successfully rearing offspring to Day 13 of age, although differences did not attain statistical significance. However, the difference from control for the mean implantation count became greater, and attained statistical significance, when the number of implantations from all pregnant females was considered. For females successfully rearing offspring to Day 13 of age, subsequent mean post-implantation loss was higher than control, resulting in the mean number of delivered offspring being statistically significantly lower than control. Subsequent live birth index and offspring survival to Day 4 was also inferior to control leading to a further decrease in litter size compared to control although, thereafter offspring survival from Day 4 of age was similar to control. These effects on litter size and offspring survival for litter reared to Day 13 of age have to be viewed in the context of five post partum litter losses at this dosage and these effects are considered to be related to maternal treatment. Sex ratio of the offspring was unaffected indicating that there was no selective effect on survival for either sex.
At 50 and 150 mg/kg bw/day, there was considered to be no obvious effect of maternal treatment on the numbers of implantations, post-implantation loss, litter size at birth/Day 1 and subsequent offspring survival to Day 13 of age.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Systemic Toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Reproduciton

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
At 375 mg/kg bw/day, there was a higher incidence of clinical signs apparent for the offspring during Day 1-4 of age, although the findings apparent were generally typical of the age observed. To a lesser extent, this higher incidence persisted to termination on Day 13 of age and the signs observed (e.g. small, cold, weak, no milk visible in stomach) later in the study are suggestive of poor maternal care for occasional litters.
At 50 or 150 mg/kg bw/day, neither the type, incidence nor distribution of clinical signs apparent for the offspring indicate any obvious effect of maternal treatment.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There were no obvious morphological changes apparent that could account for the higher mortality observed for post-natal offspring at 375 mg/kg bw/day.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
At 375 mg/kg bw/day, there was no obvious effect of maternal treatment on offspring body weight on Day 1 and subsequent body weight gain to Day 4. However, this observation has to be treated with caution due to the comparatively high offspring mortality apparent at this dosage compared to control, that could be removing smaller offspring from this assessment. It should also be noted that extended gestation length was observed for a number of females at this dosage and would be expected to lead to heavier offspring body weight at Day 1 of age. Offspring body weight gain during Days 4-7 and 7-13 were clearly lower than control and attained statistical significance during Days 7-13. These lower gains resulted in statistically significant lower overall body weight gain on Day 13 of age. Litter weights were statistically significantly lower than control throughout, reflecting initially lower litter size and later, also, the lower offspring body weight gain at this dosage.
At 50 or 150 mg/kg bw/day, there was no obvious effect of maternal treatment on offspring body weight, offspring body weight gain or litter weight.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid Hormone Analysis
Evaluation of Thyroxine (T4) in maleoffspring at Day 13 of age did not identify any effect of treatment or indication of endocrine disruption at 50, 150 or 375 mg/kg bw/day.
For female offspring at 150 mg/kg bw/day, T4 blood levels were statistically significantly higher than control, but all individual values for these treated offspring were within the historical control range. In the absence of any similar statistically significant increase for offspring at 375 mg/kg bw/day, this finding was considered incidental and unrelated to maternal treatment.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any obvious effect of maternal treatment at 50, 150 or 375 mg/kg bw/day. There were no obvious morphological changes apparent that could account for the higher mortality observed for post-natal offspring at 375 mg/kg bw/day.
Evaluation of ano-genital distance for offspring on Day 1 post partum and visible nipple count for male offspring on Day 13 post partum did not reveal any effect of maternal treatment at 50, 150 or 375 mg/kg bw/day. At 375 mg/kg bw/day, ano-genital distance for female offspring (including total litter losses) did attain statistical significance when compared to control, but as no statistically significant difference was apparent when values were adjusted for body weight, this finding was considered to be incidental and of no toxicological significance
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

At 375 mg/kg bw/day, the mean number of implantations was lower than control for females successfully rearing offspring to Day 13 of age, although differences did not attain statistical significance. However, the difference from control for the mean implantation count became greater, and attained statistical significance, when the number of implantations from all pregnant females was considered. For females successfully rearing offspring to Day 13 of age, subsequent mean post-implantation loss was higher than control, resulting in the mean number of delivered offspring being statistically significantly lower than control. Subsequent live birth index and offspring survival to Day 4 was also inferior to control leading to a further decrease in litter size compared to control although, thereafter offspring survival from Day 4 of age was similar to control. These effects on litter size and offspring survival for litter reared to Day 13 of age have to be viewed in the context of five post partum litter losses at this dosage and these effects are considered to be related to maternal treatment. Sex ratio of the offspring was unaffected indicating that there was no selective effect on survival for either sex.
At 375 mg/kg bw/day, there was no obvious effect of maternal treatment on offspring body weight on Day 1 and subsequent body weight gain to Day 4. However, this observation has to be treated with caution due to the comparatively high offspring mortality apparent at this dosage compared to control, that could be removing smaller offspring from this assessment. It should also be noted that extended gestation length was observed for a number of females at this dosage and would be expected to lead to heavier offspring body weight at Day 1 of age. Offspring body weight gain during Days 4-7 and 7-13 were clearly lower than control and attained statistical significance during Days 7-13. These lower gains resulted in statistically significant lower overall body weight gain on Day 13 of age. Litter weights were statistically significantly lower than control throughout, reflecting initially lower litter size and later, also, the lower offspring body weight gain at this dosage.
At 50 or 150 mg/kg bw/day, there was no obvious effect of maternal treatment on offspring body weight, offspring body weight gain or litter weight.
At 375 mg/kg bw/day, there was a higher incidence of clinical signs apparent for the offspring during Day 1-4 of age, although the findings apparent were generally typical of the age observed. To a lesser extent, this higher incidence persisted to termination on Day 13 of age and the signs observed (e.g. small, cold, weak, no milk visible in stomach) later in the study are suggestive of poor maternal care for occasional litters.
At 50 or 150 mg/kg bw/day, neither the type, incidence nor distribution of clinical signs apparent for the offspring indicate any obvious effect of maternal treatment.
Evaluation of ano-genital distance for offspring on Day 1 post partum and visible nipple count for male offspring on Day 13 post partum did not reveal any effect of maternal treatment at 50, 150 or 375 mg/kg bw/day. At 375 mg/kg bw/day, ano-genital distance for female offspring (including total litter losses) did attain statistical significance when compared to control, but as no statistically significant difference was apparent when values were adjusted for body weight, this finding was considered to be incidental and of no toxicological significance.

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality
body weight and weight gain
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
150 mg/kg bw/day (nominal)
System:
other: various

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
150 mg/kg bw/day (nominal)
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, a dosage of 150 mg/kg bw/day is considered to be a No Observed Adverse Effect Level (NOAEL) for systemic toxicity and a No Observed Effect Level (NOEL) for reproduction.
Executive summary:

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development), to evaluate some endocrine disruptor relevant endpoints and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 29 July 2016).

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

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, forapproximately 6 weeks (males) and up to eight weeks (females)(including a two week pre-paring phase, pairing, gestation and early lactation for females), at dose levels of 50, 150 and 375 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP) over the same treatment period.

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 13 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 ano-genital distance and visible nipple count (male offspring only).

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 12post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13post partum, for thyroid hormone analysis; samples from all adult males and Day 13post partumoffspring were analyzed for Thyroxine (T4).

Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all treated females including controls through pre-pairing, pairing and up to confirmation of mating.

Vaginal smears were also performed in the morning on the day of termination for all treated females.

Adult males were terminated on Day 44 or 45, followed by the termination of all surviving offspring and adult females on Days 13 and 14post partum, respectively. Any female which did not show positive evidence of mating or did not produce a pregnancy was terminated around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed. Examination of offspring was limited to an external examination; an internal examination was performed if abnormalities were detected externally. 

Results…….

Adult Responses

Mortality

There were no unscheduled deaths during the study.

Clinical Observations

There were no clinical signs observed that were considered to indicate a systemic effect of treatment, findings being restricted to increased post-dosing salivation for the majority of males at 150 mg/kg bw/day and all animals at 375 mg/kg bw/day. 

Functional Observations

Behavioral Assessment

There was no effect of treatment on behavioural assessment for either sex at 50, 150 or 375 mg/kg bw/day.

Functional Performance Tests

There was no effect of treatment on functional performance tests for either sex at 50, 150 or 375 mg/kg bw/day.

Sensory Reactivity Assessments

There was no effect of treatment on sensory reactivity assessments for either sex at 50, 150 or 375 mg/kg bw/day.

Body Weight

At 375 mg/kg bw/day, male body weight gain was statistically significantly lower than control throughout most of the treatment period, leading to a statistically significant reduction in overall body weight gain and statistically significant lower mean body weight at termination. For females at this dosage, lower body weight gain was apparent for the first week of the study leading to lower overall body weight gain for the two week pre-pairing phase. Mean body weights were statistically significantly lower than control throughout gestation, with statistically significantly lower body weight gain being apparent during the last two weeks of gestation. However, body weight gain during the last week of gestation was adversely influenced by a lower contribution from the gravid uterus, due to lower litter size. Evaluation of body weight during lactation was complicated by a high number of females showingpost partumlitter loss, however females generally showed lower body weight gain during lactation.    

At 150 mg/kg bw/day, male body weight gain was frequently lower than control throughout the treatment period and, although differences only occasionally attained statistical significance, this led to a statistically significant lower overall body weight gain. For females at this dosage, body weight gain was lower than control during the two week pre-pairing phase, although no dose dependency or statistical significance was apparent during the second week, leading to statistically significant lower mean body weight and overall body weight gain. Mean body weights values were statistically significantly lower than control throughout gestation, with body weight gain being statistically significantly lower than control during the second week of gestation. No statistically significant differences for body weight or body weight gain from control were apparent for females during lactation.        

At 50 mg/kg bw/day, there was no effect treatment on body weight gain for males during the study or for females during the pre-pairing, gestation or lactation phases of the study.  

Food Consumption

There was no obvious effect of treatment on food consumption of males throughout the study at 50, 150 or 375 mg/kg bw/day.

At 375 mg/kg bw/day, food consumption of females was unaffected by treatment during the two week pre-pairing phase but lower food consumption was apparent during gestation, although differences from control only attained statistical significance during the second week of gestation. Food consumption was also lower than control throughout lactation. 

There was no effect on food consumption for females during the pre-pairing, gestation or lactation phases of the study at 50 or 150 mg/kg bw/day.

Food Conversion Efficiency 

At 375 mg/kg bw/day, food conversion efficiency for males was lower than control during the two week pre-pairing phase of the study and the first two weeks of the post-pairing phase of the study. For females at this dosage, food conversion efficiency was lower than control during the first week of treatment. 

At 150 mg/kg bw/day, food conversion efficiency for females was lower than control throughout the two week pre-pairing phase, but showed no dosage relationship. Intergroup differences in food conversion efficiency for males at 150 mg/kg bw/day and both sexes at 50 mg/kg bw/day, did not indicate any consistent effect of treatment.

 

Water Consumption

There was no effect of treatment on water consumption throughout the study for both sexes at 50, 150 or 375 mg/kg bw/day. 

Reproductive Performance

Estrous Cycle

There was no effect of treatment on estrous cycles for females at 50, 150 or 375 mg/kg bw/day. 

Mating

There was no effect of treatment on mating performance for females at 50, 150 or 375 mg/kg bw/day. 

Fertility

There was no effect of treatment on the number of females that achieved pregnancy at 50, 150 or 375 mg/kg bw/day.

Gestation Lengths

At 375 mg/kg bw/day, there was a high incidence of extended gestation lengths for pregnant females, which probably adversely affected post-natal offspring survival. 

There was no effect of treatment on gestation length at 50 or 150 mg/kg bw/day.

Litter Responses

Litter Size and Viability

At 375 mg/kg bw/day, a lower mean number of implantations, compared to control and a higher mean post-implantation loss lead to a statistically significantly lower mean number of delivered offspring. Subsequent live birth index and offspring survival to Day 4 at this dosage was also inferior to control leading to a further decrease in comparative litter size, although subsequent offspring survival from Day 4 of age was similar to control. The observed effects on offspring survival have to be viewed in the context of fivepost partumlitter losses at this dosage and were considered to be related to maternal treatment. Sex ratio of the offspring was unaffected indicating that there was no selective effect on survival for either sex.

At 50 and 150 mg/kg bw/day, there was considered to be no effect of maternal treatment on the numbers of implantations, post-implantation loss, litter size at birth/Day 1 and subsequent offspring survival to Day 13 of age.

 

Offspring Growth and Development

At 375 mg/kg bw/day, there was no clear effect of maternal treatment on offspring body weight on Day 1 although body weight was lower than may have been expected considering the extended gestation length at this dosage. Offspring body weight gain was lower from Day 4 of age leading to statistically significant lower overall body weight gain on Day 13 of age. Litter weights were statistically significantly lower than control throughout, although initially this only reflected lower litter size. There was a higher incidence of clinical signs apparent for the offspring during Days 1-4 of age which persisted, to a lesser extent, to termination at Day 13 of age. The clinical signs observed later in the study were suggestive of poor maternal care for occasional litters. 

At 50 or 150 mg/kg bw/day, there was no effect of maternal treatment on offspring body weight, offspring body weight gain or litter weight. Clinical signs at these dosages did not indicate any effect of maternal treatment. 

Evaluation of ano-genital distance for offspring on Day 1post partumand visible nipple count for male offspring on Day 13post partumdid not reveal any effect of maternal treatment at 50, 150 or 375 mg/kg bw/day. 

In-Life sampling and Analysis

Hematology

For males at 375 mg/kg bw/day, lower mean erythrocyte count, hemoglobin, hematocrit, mean corpuscular hemoglobin, corpuscular hemoglobin concentration and mean corpuscular volume and higher reticulocyte count attained statistical significance when compared with control.

Blood Chemistry

There were no statistically significant differences from control for blood chemistry parameters that indicated any clear association with treatment.

Terminal Investigations:

Necropsy

Offspring

Offspring necropsy findings did not indicate any effect of treatment at 50, 150 or 375 mg/kg bw/day, in particular, there were no morphological changes apparent that could account for the higher post-natal mortality observed for offspring at 375 mg/kg bw/day.     

Adults

Macroscopic necropsy findings did not indicate any obvious effect of treatment for either sex at 50, 150 or 375 mg/kg bw/day.

Organ Weights

Absolute and body weight relative kidney weights were statistically significantly higher than control for females at 50 mg/kg bw/day and both sexes at 150 and 375 mg/kg bw/day.

Absolute and body weight relative liver weights were statistically significantly higher than control for females at 50 mg/kg bw/day and both sexes at 150 and 375 mg/kg bw/day. 

For females at 375 mg/kg bw/day, absolute and body weight relative uterus weights were statistically significantly higher than control. There was no obvious correlation between uterus weight and the incidence of females showing total litter loss at 375 mg/kg bw/day.

Histopathology

Microscopic examination of the kidneys revealed multifocal basophilic tubules in 3/5 females treated at 375 mg/kg bw/day. 

Minimal tubular vacuolation of the kidneys was present in 2/5 males treated at 50 mg/kg bw/day, 3/5 male and 3/5 females treated at 150 mg/kg bw/day and 4/5 males and 3/5 females at 375 mg/kg bw/day.

Vacuolation of the cortex of the adrenal glands was above expected incidental variation in 2/5 males treated at 375 mg/kg bw/day.

Thyroid Hormone Analysis

Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any effect of treatment or indication of endocrine disruption at 50, 150 or 375 mg/kg bw/day. 

Conclusion

Based on the results of this study, a dosage of 150 mg/kg bw/day is considered to be a No Observed Adverse Effect Level (NOAEL) for systemic toxicity and a No Observed Effect Level (NOEL) for reproduction.