Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 12 December 2016 Experimental Completion Date: 19 June 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)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(1,1-dimethylethyl)cyclohexyl methacrylate
EC Number:
256-277-5
EC Name:
4-(1,1-dimethylethyl)cyclohexyl methacrylate
Cas Number:
46729-07-1
Molecular formula:
C14H24O2
IUPAC Name:
4-(1,1-dimethylethyl)cyclohexyl methacrylate
Test material form:
liquid
Details on test material:
Batch 1307501003
Specific details on test material used for the study:
Identification: 4-(1,1-dimethylethyl)cyclohexyl methacrylate
Chemical Name: 4-tert-butylcyclohexyl methacrylate
Lot/Batch Number: DAR-16030
Description: Clear colorless liquid
Purity: 89.2%
Expiry Date: 01 August 2018
Storage: Approximately 4 ºC in the dark

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 appropriate 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 278 to 347g and were approximately eleven weeks old. The females weighed 195 to 241g 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 Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Annex 6. 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 integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively; 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 disposable 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 show the formulations to be stable for at least twenty-one days when kept at approximately 4 ºC. Formulations were therefore prepared weekly for the first two weeks and then fortnightly thereafter, and stored at approximately 4 ºC in the dark.

Samples of test item formulations were taken on four occasions and analyzed for concentration of 4-(1,1-dimethylethyl)cyclohexyl methacrylate (CAS 46729-07-1) at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within 8% of the nominal concentration.
Duration of treatment / exposure:
approximately six weeks (males) and up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females)
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). During the pre-pairing period, vaginal smears were performed for females. 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 phase, 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, 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 are 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.:
450 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 males and females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were chosen in collaboration with the Sponsor and based on the results of previous toxicity work including a fourteen day dose range-finding toxicity study in the rat (Envigo Study Number YS88CH).

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 the pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4 and 7 post partum. Body weights were also recorded at terminal kill.

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.

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.


Hematology
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++)

Thyroid Hormone Analysis
Blood samples taken to produce serum were allowed to clot, centrifuged and the serum from each blood sample stored frozen at lower than -60ºC. Blood samples taken to produce plasma were collected into K2EDTA, centrifuged, and the plasma from each blood sample stored frozen at lower than -60ºC. Samples were taken as follows:
Serum and plasma samples were taken from all adult males and females at termination.
All serum samples were dispatched to the Test Site (Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS) where the serum from adult males was analyzed for Thyroxine (T4) under the supervision of the Principal Investigator (H Bose).
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:
Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1, 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)

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.


Thyroid Hormone Analysis
Blood samples taken to produce serum were allowed to clot, centrifuged and the serum from each blood sample stored frozen at lower than -60ºC. Blood samples taken to produce plasma were collected into K2EDTA, centrifuged, and the plasma from each blood sample stored frozen at lower than -60ºC. Samples were taken as follows:
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 fewer 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.
All serum samples were dispatched to the Test Site (Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS) where the serum from Day 13 offspring was analyzed for Thyroxine (T4) under the supervision of the Principal Investigator (H Bose).
Postmortem examinations (parental animals):
Necropsy
Adult males were sacrificed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 44 or 45. Adult females were sacrificed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 14 post partum. Any females which failed to achieve pregnancy 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, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

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
Prostate
Brain
Seminal Vesicles (with Coagulating Gland)
Epididymides
Spleen
Heart
Testes
Kidneys
Thymus
Liver
Thyroid (weighed post-fixation with Parathyroid)
Ovaries
Uterus (weighed with Cervix and Oviducts)
Pituitary (weighed post-fixation)


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
Caecum
Rectum
Colon
Salivary glands (submaxillary)
Cowpers glands
Sciatic nerve
Duodenum
Seminal vesicles (with coagulating gland)
Epididymides ♦
Esophagus
Skin
Eyes *
Spinal cord (cervical, mid thoracic and lumbar)
Glans penis
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 (Propath (UK) Ltd, Willow Court, Netherwood Road, Hereford, HR2 6JU) for processing (Principal Investigator: M Dow). The tissues from five selected control and 450 mg/kg bw/day dose group animals and any animals dying during the study 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 450 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 450 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Since there were indications of treatment-related kidney changes, examination was subsequently extended to include similarly prepared sections of the kidneys from five male animals in the low and intermediate groups.

Pathology
Microscopic examination was conducted by the Study Pathologist (W Henderson). A peer review of the findings observed was conducted by V Mowat at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS.
Postmortem examinations (offspring):
Necropsy
Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were sacrificed by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation. Examination of offspring was restricted to a macroscopic external examination except where abnormalities were observed, then an additional internal examination was performed.

On Day 13 of age, where possible, for one male and one female offspring per litter, the whole or samples of thyroid/parathyroid were retained in 10% Buffered Formalin.

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, 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:
Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
i. Pre-coital Interval

Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii. Fertility Indices

For each group the following were calculated:

Mating Index (%) = number of animals mated / number of animals paired x 100

Pregnancy Index (%) = number of pregnant females / number of animals 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
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 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 and Viability Indices

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

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, 7 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:
effects observed, treatment-related
Description (incidence and severity):
One female treated with 450 mg/kg bw/day showed signs of occasional body tremors on Days 41 and 43 to 46 relative to the start of dosing (corresponding to Days 3 and 5 to 8 of lactation); other observations for this female included tiptoe gait and hunched posture on Day 8 of dosing, with hunched posture again noted on Day 42 (corresponding to Day 4 of lactation). Another two females from this dose group showed isolated instances of occasional body tremors on Day 43 of dosing (corresponding to Day 5 of lactation). Histopathological examination of an extended list of tissues from these females did not identify any treatment-related observations.
Sporadic instances of increased post-dose salivation were evident in males (between Days 7 and 44) and in females (between Days 6 to 53 relative to the start of dosing) treated with 450 mg/kg bw/day.
There were no clinical signs for any of the remaining animals on the study.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female treated with 150 mg/kg bw/day was found dead on Day 25. There were no clinical signs for this animal before death and macroscopic findings at necropsy included fluid-filled pale lungs with dark patches. Microscopic examination of the selected tissues from this female did not reveal any notable findings.
There were no further unscheduled terminations for adult animals during the study and this death was considered unrelated to treatment with the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 450 mg/kg bw/day showed a statistically significant reduction (p<0.01) in body weight gain during the first week of treatment. Improvement was evident during Week 2, however, slightly lower body weight gains were evident during Week 3, although statistical significance was not achieved. Recovery was evident from Week 4 onwards. Overall group mean body weight gain in these males was approximately 11% lower than controls but there was no dose-relationship.
No such effects were evident in males treated with 150 or 50 mg/kg bw/day.
During the first week of treatment, group mean body weight gain in females treated with 450 mg/kg bw/day appeared to be lower than controls but without achieving statistical significance. Recovery was evident thereafter such that the mean weight gain in these females over the second week of dosing was statistically significantly higher than controls (p<0.01). On Days 0, 7 and 20 of gestation, body weight for these females were statistically significantly reduced (p<0.05-0.01) but group mean body weight gains during gestation and lactation were similar to controls.
At 150 or 50 mg/kg bw/day, the corresponding values in pregnant females remained comparable with controls throughout the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was no effect of treatment with the test item at any dose level on dietary intake for males.
Females treated with 450 mg/kg bw/day showed slightly lower food intake during the first week of treatment with subsequent improvement evident during the second week of treatment. Between Days 0 and 14 of gestation and Days 7 and 14 of lactation, the mean food consumption for these females was statistically significantly lower (p<0.05-0.01) than controls.
No such effects were evident in females treated with 150 or 50 mg/kg bw/day.
Fluctuation in food conversion efficiency for animal of either sex (not calculated for females during gestation and lactation phases of the study) were considered to be reflective of small changes in body weight gain and/or dietary intake.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of water bottles did not indicate any overt differences for animals given the test item in comparison with controls.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the hematology parameters examined.
Males treated with 450 mg/kg bw/day showed statistically significant reductions (p<0.05) in hemoglobin and hematocrit and a statistically significant increase (p<0.05) in platelet count. The effect on hematocrit and platelet count also extended to males treated with 150 mg/kg bw/day. With the exception of one platelet count value and one hematocrit value at 150 mg/kg bw/day, the remaining individual values were within historical control ranges and one or two control values were actually above or below the historical control ranges. A true dose related response was also not evident in platelet count. In the absence of any associated histology correlates the intergroup differences were considered not to be of toxicological significance. Males from all treatment groups showed a statistically significant reduction (p<0.05-0.01) in erythrocytes and a statistically significant increase (p<0.01) in reticulocytes. With the exception of one erythrocyte value at 150 mg/kg bw/day, the remaining individual values were within historical control ranges and one control value for erythrocytes was actually above the historical control range. A true dose related response was also not evident in erythrocytes and therefore, in the absence of any associated histology correlates the intergroup differences were considered not to be of toxicological significance.
Females treated with 450 mg/kg bw/day showed statistically significant reductions (p<0.01) in total leucocyte count and lymphocyte counts and a statistically significant increase (p<0.01) in reticulocytes. The effect on reticulocytes also extended to females treated with 150 mg/kg bw/day. All of the individual values were within historical control ranges and two control values for lymphocytes were actually above the historical control range. A true dose related response was also not evident for reticulocytes and therefore, in the absence of any associated histology correlates the intergroup differences were considered not to be of toxicological significance. Females treated with 50 mg/kg bw/day showed a statistically significant increase (p<0.05) in neutrophil count. All of the individual values were within historical control range and in the absence of a similar effect at 150 or 450 mg/kg bw/day, the intergroup difference was considered not to be of toxicological importance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males from all treatment groups showed a statistically significant increase (p<0.05-0.01) in creatinine levels. Although all of the individual values were within historical control range, the increase was considered to be related to the microscopic kidney changes evident in these males.
No such effects were detected in treated females.
Males treated with 450 mg/kg bw/day showed a statistically significant increase (p<0.05) in phosphorus. All of the individual values were within historical control range and in the absence of any associated changes the intergroup difference was considered not to be of toxicological significance. Males treated with 150 mg/kg bw/day showed a statistically significant reduction in albumin/globulin ratio. The majority of individual values were within historical control range and in the absence of a similar effect at 450 mg/kg bw/day, the intergroup difference was considered of no toxicological importance.
Females treated with 450 mg/kg bw/day showed statistically significant increases (p<0.01) in phosphorus and bile acids and a statistically significant reduction (p<0.01) in calcium concentration. Although the majority of individual values were outside of the historical control ranges, all of the control calcium values, the majority of control phosphorus values and one control bile acid value were also outside of the historical control ranges. Therefore, in the absence of any associated histology correlates, the intergroup differences were considered not to be of toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Behavioral Assessments
No treatment-related changes in behavioral parameters were detected at any dose level.

Functional Performance Tests
There was considered to be no effect of treatment with the test item at any dose level on functional performance in animals of either sex.
Males treated with 450 mg/kg bw/day showed a statistically significant reduction (p<0.05) in hind limb grip strength in relation to controls whilst females from this treatment group showed a statistically significant increase (p<0.05) in hind limb grip strength. The intergroup differences were confined to one out of the three tests and in isolation were considered not to be of toxicological significance.
Motor activity evaluations during the last week of dosing did not identify any treatment-related differences for animal of either sex.

Sensory Reactivity Assessments
Sensory reactivity scores across all test item-treated dose groups were similar to controls.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following microscopic findings were detected:
Kidneys: An increase in hyaline droplets was present in all males treated with the test item which were examined histologically. Multifocal basophilic tubules (mild or moderate) and proteinaceous casts (mild or moderate) were evident in nine males treated with 450 mg/kg bw/day. Multifocal basophilic tubules (minimal or mild) were evident in eight males and proteinaceous casts (minimal to moderate) were evident in six males treated with 150 mg/kg bw/day. Multifocal basophilic tubules (minimal or mild) were evident in three males and proteinaceous casts (minimal) were evident in two males treated with 50 mg/kg bw/day.
No other changes which could be related to the administration of the test item were noted.
There were no test item-related microscopic findings in the reproductive tracts following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or the evaluation of the uterus or of follicles and corpora lutea in the ovaries.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
An evaluation of Thyroxine (T4) in adult males did not identify any treatment-related findings.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There was no effect of treatment with the test item at any dose level on the nature of estrous cycle with the majority of females showing regular cycles over the pre-pairing phase of the study. There were also no intergroup differences in the stage of estrus on the day of necropsy.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of treatment on mating performance with all animals mating within four days after pairing.
One female treated with 150 mg/kg bw/day and one female treated with 450 mg/kg bw/day were found to be non-pregnant following positive evidence of mating. Histopathological examinations of these females and their respective male partners did not reveal any significant microscopic changes which could account for the lack of pregnancy therefore these were considered incidental and unrelated to treatment.
Gestation lengths were generally between 22 and 23½ days and the distribution of gestation lengths for treated females was essentially similar to control.
There was no effect of treatment with the test item at any dose level on the mean number of implantations and post-implantation losses.

Details on results (P0)

In total all females from the control and eleven females from the 50, 150 and 450 mg/kg bw/day dose groups gave birth to a live litter and successfully reared young to Day 13 of age. The following assessment of litter response is based on all litters reared to termination on Day 13 of lactation/age.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: systemic toxicity
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: systemic toxicity
Remarks:
Although the kidney findings of tubular basophilia and proteinaceous casts in male kidneys could be considered an adverse effect, these findings were considered to be associated with alpha 2u-globulin and formation of hyaline droplets, an effect recognized as being both species and sex specific and not relevant for humans. In terms of risk assessment, these findings observed on this study would suggest that a ‘No Observed Adverse Effect Level’ (NOAEL) can be established at 150 mg/kg bw/day for males because the findings do not reflect true systemic toxicity.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: reproductive toxicity

Target system / organ toxicity (P0)

Critical effects observed:
not specified
Lowest effective dose / conc.:
50 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs detected in pups from all dose groups including controls included small size, cold, no milk in stomach, physical injury, found dead, terminated for welfare reasons and/or missing and were considered to be low incidence findings observed in offspring in studies of this type and as such unlikely to be related to the toxicity of the test item.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
litter size at birth and subsequently on Days 1, 4, 7 and 13 post partum from all treated dose groups was comparable with controls indicating the lack of any effect on offspring viability.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 450 mg/kg bw/day, group mean body weight gains in both male and female offspring were statistically significantly lower (p<0.05-0.01) than controls between Days 4 and 13 post partum. This resulted in statistically significantly lower (p<0.05) body weights on Day 13 post partum and statistically significantly lower cumulative weight gains (p<0.05-0.01) and litter weights between Days 4 and 13 post partum (statistical significance (p<0.01) for litter weights only achieved on Day 13).
No such effects were evident in litters from females treated with 150 or 50 mg/kg bw/day.
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:
not examined
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 adverse effect of maternal treatment on offspring development at 50, 150 or 450 mg/kg bw/day.
Histopathological findings:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
There were no treatment-related intergroup differences in sex ratio (percentage male offspring) for litters from test item-treated groups when compared with controls.
When compared with controls, evaluation of ano-genital distance on Day 1 post partum (male and female offspring) and visible nipple count on Day 13 post partum (male offspring) did not reveal any treatment-related intergroup differences.

An evaluation of Thyroxine (T4) in male/female offspring (Day 13 of age) did not identify any treatment-related findings.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
yes
Lowest effective dose / conc.:
450 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
not specified
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
The oral administration of 4-(1,1-dimethylethyl)cyclohexyl methacrylate (CAS 46729-07-1) to rats for a period of up to eight weeks (including two weeks pre-pairing, gestation and early lactation for females) at dose levels 50, 150 and 450 mg/kg bw/day resulted in treatment related effects in animals of either sex treated with 450 mg/kg bw/day and in males treated with 150 and 50 mg/kg bw/day. These included reduced initial body weight gains in either sex at 450 mg/kg bw/day, reduced food consumption in females at 450 mg/kg bw/day, organ weight changes in males at 450 mg/kg bw/day, macroscopic changes in males at 450 mg/kg bw/day and microscopic changes in males at 450, 150 and 50 mg/kg bw/day. A ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore not established for males but was considered to be 150 mg/kg bw/day for females. The ‘No Observed Adverse Effect Level’ (NOAEL) for females was also considered to be 150 mg/kg bw/day.
Although the kidney findings of tubular basophilia and proteinaceous casts in male kidneys could be considered an adverse effect, these findings were considered to be associated with alpha 2u-globulin and formation of hyaline droplets, an effect recognized as being both species and sex specific and not relevant for humans. In terms of risk assessment, these findings observed on this study would suggest that a ‘No Observed Adverse Effect Level’ (NOAEL) can be established at 150 mg/kg bw/day for males because the findings do not reflect true systemic toxicity.
The ‘No Observed Effect Level’ (NOEL) and the ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive and developmental toxicity was considered to be 150 mg/kg bw/day based on reduced offspring body weight gain and litter weights from Day 4 post partum at 450 mg/kg bw/day.
Executive summary:

Introduction

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 28 July 2015).

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-pairing phase, pairing, gestation and early lactation for females), at dose levels of 50, 150 and 450 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP).

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 parental 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 13 offspring 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 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.

Results

Adult Responses

Mortality

One female treated with 150 mg/kg bw/day was found dead on Day 25. There were no clinical signs for this animal before death and macroscopic findings at necropsy included fluid-filled pale lungs with dark patches. Microscopic examination of the selected tissues from this female did not reveal any notable findings. There were no further unscheduled deaths for adult animals during the study and this death was considered unrelated to treatment with the test item.

Clinical Observations

One female treated with 450 mg/kg bw/day showed signs of occasional body tremors on Days 41 and 43 to 46; other observations for this female included tiptoe gait and hunched posture on Day 8 of dosing, with hunched posture again noted on Day 42. Another two females from this dose group showed isolated instances of occasional body tremors on Day 43. Sporadic instances of increased post-dose salivation were evident in animals of either sex treated with 450 mg/kg bw/day from Week 1, which persisted throughout the treatment period. There were no clinical signs for any of the remaining animals on the study.

Behavioral Assessment

No treatment-related changes in behavioral parameters were detected at any dose level.

Functional Performance Tests

There was considered to be no effect of treatment with the test item at any dose level on functional performance in animals of either sex.

Sensory Reactivity Assessments

Sensory reactivity scores across all test item-treated dose groups were similar to controls.


Body Weight

Males treated with 450 mg/kg bw/day showed a reduction in body weight gain during the first and third week of treatment. Improvement was evident during Week 2 and from Week 4 onwards. During the first week of treatment, females treated with 450 mg/kg bw/day showed lower body weight gains, however, recovery was evident thereafter. On Days 0, 7 and 20 of gestation, body weight for these females were lower than controls but group mean body weight gain during the lactation period were similar to controls. At 150 or 50 mg/kg bw/day, the corresponding values in pregnant females remained comparable with controls throughout the study.

Food Consumption

There was no effect of treatment with the test item at any dose level on dietary intake for males. Females treated with 450 mg/kg bw/day showed slightly lower food intake during the first week of the dosing period with subsequent improvement noted. Over Days 0 to 14 of gestation and Days 7 to 14 of lactation, the mean food consumption for these females was again lower than controls. Treatment with the test item at 150 or 50 mg/kg bw/day did not result in any effect on food intake for the pregnant females.

Fluctuation in food conversion efficiency for animals of either sex (not calculated for females during gestation and lactation phases of the study) were considered to be reflective of small changes in body weight gain and/or dietary intake.

Water Consumption

Visual inspection of water bottles did not indicate any overt differences for animals given the test item in comparison with controls.

Reproductive Performance

Mating

There was no effect of treatment on mating performance with all animals mating within four days after pairing.

Fertility

There was no effect of treatment with the test item on fertility. With the exception of one female each from the 50 or 450 mg/kg bw/day dose groups, all females were pregnant with the surviving females rearing offspring to Day 13post partum.

Gestation Lengths

Gestation lengths were between 22 and 23½ days and the distribution of gestation lengths for females receiving the test item was generally comparable with controls.


Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There did not appear to be any detrimental effect of treatment with the test item at any dose level on implantation sites or post-implantation loss. Of the litters born, litter size at birth and subsequently on Days 1, 4, 7 and 13post partumfrom all dose groups were comparable with controls. There were no intergroup differences in sex ratio (percentage male offspring) for litters from test item-treated dose groups when compared with controls.

Offspring Growth and Development

At 450 mg/kg bw/day, group mean body weight gains in both male and female offspring were lower than controls between Days 4 and 13post partum, which resulted in lower body weights on Day 13post partumand lower cumulative weight gains and litter weights between Days 4 and 13post partum. The corresponding values for the 150 or 50 mg/kg bw/day dose groups were comparable with controls.

There was no detrimental effect of treatment with the test item indicated by ano-genital distance on Day 1post partumor visible nipple count in male offspring on Day 13post partumat 50, 150 or 450 mg/kg bw/day.

Clinical signs detected in pups from all dose groups including controls included small size, cold, no milk in stomach, physical injury, found dead, terminated for welfare reasons and/or missing and were considered to be low incidence findings observed in offspring in studies of this type and as such unlikely to be related to the toxicity of the test item.

Laboratory Investigations

Hematology

There were no toxicologically significant effects detected in the hematological parameters examined. 

Blood Chemistry

Males from all treatment groups showed an increase in creatinine levels. No such effects were detected in treated females.

Pathology

Necropsy

At necropsy, most males treated with the test item and one control male showed mottled kidneys. Increased pelvic space in both kidneys was also evident in one male treated with 50 mg/kg bw/day and in one male treated with 450 mg/kg bw/day. 

Other minor changes were noted in individual animals, however these were considered not to be related to the administration of the test item.

Organ Weights

Males treated with 450 mg/kg bw/day showed an increase in kidney weight both absolute and relative to terminal body weight. This change was considered likely to be related to the microscopic findings noted in male kidneys at this dose level.

No such effects were detected in males treated with 150 or 50 mg/kg bw/day. 

No treatment-related effects were detected in treated females in the organ weights measured.

Histopathology

The following microscopic findings were detected:

Kidneys: An increase in hyaline droplets was present in all males treated with the test item which were examined histologically. Multifocal basophilic tubules (mild or moderate) and proteinaceous casts (mild or moderate) were evident in nine males treated with 450 mg/kg bw/day. Multifocal basophilic tubules (minimal or mild) were evident in eight males and proteinaceous casts (minimal to moderate) were evident in six males treated with 150 mg/kg bw/day. Multifocal basophilic tubules (minimal or mild) were evident in three males and proteinaceous casts (minimal) were evident in two males treated with 50 mg/kg bw/day.

No other changes which could be related to the administration of the test item were noted.

Thyroid Hormone Analysis

An evaluation of Thyroxine (T4) in adult males and male/female offspring (Day 13 of age) did not identify any treatment-related findings.

Conclusion

The oral administration of 4-(1,1-dimethylethyl)cyclohexyl methacrylate (CAS 46729-07-1) to rats for a period of up to eight weeks (including two weeks pre-pairing, gestation and early lactation for females) at dose levels 50, 150 and 450 mg/kg bw/day resulted in treatment related effects in animals of either sex treated with 450 mg/kg bw/day and in males treated with 150 and 50 mg/kg bw/day. These included reduced initial body weight gains in either sex at 450 mg/kg bw/day, reduced food consumption in females at 450 mg/kg bw/day, organ weight changes in males at 450 mg/kg bw/day, macroscopic changes in males at 450 mg/kg bw/day and microscopic changes in males at 450, 150 and 50 mg/kg bw/day. A ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore not established for males but was considered to be 150 mg/kg bw/day for females. The ‘No Observed Adverse Effect Level’ (NOAEL) for females was also considered to be 150 mg/kg bw/day.

Although the kidney findings of tubular basophilia and proteinaceous casts in male kidneys could be considered an adverse effect, these findings were considered to be associated with alpha 2u-globulin and formation of hyaline droplets, an effect recognized as being both species and sex specific and not relevant for humans. In terms of risk assessment, these findings observed on this study would suggest that a ‘No Observed Adverse Effect Level’ (NOAEL) can be established at 150 mg/kg bw/day for males because the findings do not reflect true systemic toxicity.

The ‘No Observed Effect Level’ (NOEL) and the ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive and developmental toxicity was considered to be 150 mg/kg bw/day based on reduced offspring body weight gain and litter weights from Day 4post partumat 450 mg/kg bw/day.