N-isopropylmethacrylamide

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The in-life phase of the study was conducted between 06 June 2012 (first day of treatment) and 29 July 2012 (final necropsy).

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd
- Age at study initiation: Approximately twelve weeks old
- Weight at study initiation: At the start of the treatment the males weighed 301 to 358g, the females weighed 193 to 236g.
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. 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.
- Diet (e.g. ad libitum): The animals were allowed free access to food (a pelleted diet).
- Water (e.g. ad libitum): The animals were allowed free access to water (mains drinking water was supplied from polycarbonate bottles attached to the cage).
- Acclimation period: The animals were
acclimatised for seven days during which time their health status was assessed.A total of eighty animals (forty males and forty females) were accepted into the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature was set to achieve target values of 21 ± 2°C.
- Humidity (%): The humidity was set to acieve target values of 55 ± 15% .
- Air changes (per hr): The rate of air exchange was at least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a suspension in Arachis oil BP.
The stability and homogeneity of the test item formulations were previously determined in a 7 day range finding study.
Results showed the formulations to be stable for at least twenty three days. Formulations were therefore prepared fortnightly and stored at 4ºC in the dark.

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 regular intervals.

Details on mating procedure:

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 oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test item formulation were taken and analysed for concentration of N-Isopropylmethacrylamide (NIPMAA).

The concentration of N-Isopropylmethylacrylamide in the test item formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.

The results indicate that the prepared formulations were within ±6% of the nominal concentration.

Duration of treatment / exposure:

The test item was administered to rats for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females).

Frequency of treatment:

The test item was administered daily.

Details on study schedule:

Chronological Sequence of Study:
i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv) 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.
v) On completion of the pre-pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43.
ix) Blood samples were taken from five randomly selected females from each dose group for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.

Remarks:

Doses / Concentrations:
100 mg ai/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
190 mg ai/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
350 mg ai/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Ten males and ten females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were chosen based on the results of a 7-day range finding study.

Positive control:

None

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

FUNCTIONAL OBSERVATIONS:
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. 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.

BEHAVIOURAL ASSESSMENTS:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.

FUNCTIONAL PERFORMANCE TESTS:
Motor Activity: Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period
and also during the final 20% of the period (considered to be the asymptotic period, 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). Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Blink reflex , Tail pinch, Startle reflex, Finger approach.

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 mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

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 on Days 1 and 4 post partum.

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 for females during gestation and lactation.

WATER CONSUMPTION:
Water intake was measured daily throughout the study (with the exception of the pairing phase).

PREGNANCY AND PARTURITION:
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition

LABORATORY INVESTIGATIONS:
Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 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.

HAEMATOLOGY:
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices
- mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed
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
Calcium (Ca++)
Glucose
Inorganic phosphorus (P)
Total protein (Tot.Prot.)
Aspartate aminotransferase (ASAT)
Albumin
Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation)
Alkaline phosphatase (AP)
Sodium (Na+)
Creatinine (Creat)
Potassium (K+)
Total cholesterol (Chol)
Chloride (Cl-)
Total bilirubin (Bili)
Bile acids (Bile)

Oestrous cyclicity (parental animals):

During mating a vaginal smear was prepared for each female and the stage of oestrus 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).

Sperm parameters (parental animals):

During histopathology, the male testes and epididymides were examined.

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 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

PHYSICAL DEVELOPMENT:
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Postmortem examinations (parental animals):

PATHOLOGY:
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 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, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix), Pituitary (post fixation).

HISTOPATHOLOGY:
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Pituitary
Prostate
Brain (including cerebrum, cerebellum and pons)
Oesophagus
Caecum
Rectum
Coagulating gland
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides*
Skin (hind limb)
Eyes**
Spinal cord (cervical, mid-thoracic and lumbar)
Gross lesions
Heart
Spleen
Ileum (including peyer’s patches)
Stomach
Jejunum
Thyroid/parathyroid
Kidneys
Trachea
Liver
Testes
Lungs (with brochi)***
Thymus
Lymph nodes (cervical and mesenteric)
Urinary bladder
Mammary gland
Uterus/Cevix
Muscle (skeletal)
Vagina
* preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately forty-eight hours later
** eyes fixed in Davidson’s fluid
*** lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

All tissues were despatched to the histology processing Test Site for processing. The tissues from five selected control and 1000 mg/kg bw/day A.I. dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues
shown in bold from the remaining control and 350 mg/kg bw/day A.I. animals were also processed. In addition, sections of testes and epididymides from all control and 350 mg/kg bw/day A.I. males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Since there were indications of treatment-related sciatic nerve changes, examination was subsequently extended to include similarly prepared sections of the sciatic nerve from five animals per sex from the low and intermediate groups.

Microscopic examination was conducted by the Study Pathologist.

Postmortem examinations (offspring):

PATHOLGY:
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.

All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

The following parameters were subjected to statistical analysis:
Quantitative functional performance data
Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Water Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights

Reproductive indices:

Mating Performance and Fertility:

The following parameters were calculated from the individual data during the mating period of the parental generation.
i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii) Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals mated ÷ Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females ÷ Number of animals mated) x 100

Gestation and Parturition Data :
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.
i) Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii) Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of females delivering live offspring ÷ Number of pregnant females) x 100

Offspring viability indices:

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 5 of age).

i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
% pre – implantation loss = [(Number of corpora lutea - Number of implantation sites) ÷ Number of corpora lutea] x 100
% post – implantation loss =[(Number of implantation sites - Total number of offspring born) ÷ Number of implantation sites] x 100

ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1 ÷ Number of offspring born) x 100
Viability Index (%) = (Number of offspring alive on Day 4 ÷ Number of offspring alive on Day 1 ) x 100

iii) Sex Ratio (% males)
Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula:
(Number of male offspring ÷ Total number of offspring) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see details on results

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Sciatic nerve: Minimal axonal swelling with mineralisation was evident in animals of either sex from all treatment groups.

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

No treatment-related effects detected in the reproductive parameters observed.

ADULT RESPONSES:
MORTALITY:
There were no unscheduled deaths.

CLINICAL OBSERVATIONS:
Animals of either sex treated with 350 mg/kg bw/day A.I. showed episodes of increased salivation between Days 14 and 38. One male treated with 350 mg/kg bw/day A.I. showed pilo-erection and hunched posture on Day 29.
No such effects were detected in animals of either sex treated with 100 or 190 mg/kg bw/day A.I.
One control male had generalised fur loss and scab formation on Days 42 and 43. Observations of this nature are commonly observed in group housed animals and in view of this observation only being present in a control animal is considered to be incidental.

BEHAVIOURAL ASSESSMENTS:
Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls.
All inter and intra group differences in behavioural scores were considered to be a result of normal variation for rats of the strain and age used, and the differences were of no toxicological importance.

FUNCTIONAL PERFORMANCE TESTS:
Males treated with 350 mg/kg bw/day A.I. showed a statistically significant increase in fore limb grip strength. All individual values were within the normal range for rats of the strain and age used and was confined to one out of the three tests therefore the intergroup difference was considered not to be of toxicological importance. Animals of either sex treated with 350 mg/kg bw/day A.I. showed a statistically significant increase in hind limb grip strength. The statistically significant differences were confined to one out of the three tests and in the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered to be of no toxicological significance. Males treated with 100 mg/kg bw/day A.I. showed a statistically significant increase in overall activity and overall mobile activity. In the absence of a true dose related response the intergroup differences were considered not to be of toxicological importance.

SENSORY REACTIVITY ASSESSMENTS:
There were no treatment-related changes in sensory reactivity.

BODY WEIGHT:
Males treated with 350 and 190 mg/kg bw/day A.I. showed a statistically significant reduction in body weight gain during Week 1 (P<0.001, P<0.05 respectively). Males treated with 350 mg/kg bw/day A.I. also showed a statistically significant reduction in body weight gain during Week 4 (P<0.05). Overall body weight gain was also reduced in males treated with 350 mg/kg bw/day A.I. No such effects were detected in females treated with 350 or 190 mg/kg bw/day A.I. or in animals of either sex treated with 100 mg/kg bw/day A.I.

FOOD CONSUMPTION:
No adverse effects on food consumption were detected in treated animals when compared to controls. Males treated with 350 and 190mg/kg bw/day A.I. did however show a reduction in food efficiency during the first week of treatment.

Females treated with 350 and 190 mg/kg bw/day A.I. showed a statistically significant increase in food consumption during the final week of gestation. An increase in food consumption in isolation is not generally considered to be an adverse effect of treatment therefore the intergroup difference was considered not to be of toxicological importance.

WATER CONSUMPTION:
No toxicologically significant effect on water consumption was detected.

Females treated with 350 mg/kg bw/day A.I. showed an increase in overall water consumption throughout the treatment period. Males treated with 190 and 100 mg/kg bw/day A.I. also showed an increase in overall water consumption however males treated with 350 mg/kg bw/day A.I. showed a reduction in overall water consumption compared to control animals. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in the absence of a true dose related response in males the intergroup differences were considered not to be of toxicological importance.

Females treated with 190 mg/kg bw/day A.I. showed a statistically significant reduction in water consumption during Week 3 of gestation. A -6% reduction in water consumption is not generally considered to be an adverse effect of treatment therefore the intergroup difference was considered not to be of toxicological importance.

REPRODUCTIVE PERFORMANCE
MATING:
There were no treatment-related effects on mating performance.

FERTILITY:
There were no treatment-related effects on fertility.

One female treated with 190 mg/kg bw/day A.I. did not achieve pregnancy following evidence of mating. No histopathological correlates were evident in the female reproductive organs. The male partner however showed tubular atrophy in both testes and aspermia in the epididymides, which correlated with the macroscopic observations of small testes and epididymides and flaccid testes. These lesions were considered to be the cause of the non pregnancy however are considered naturally occurring background changes in rats therefore of no toxicological significance.

GESTATION LENGTH:
There were no differences in gestation lengths. The distribution for treated females was comparable to controls. The gestation lengths were between 22 and 24 Days.


LABORATORY INVESTIGATIONS:
HEAMATOLOGY:
No toxicologically significant effects were detected in the haematological parameters examined.
Males treated with 350 mg/kg bw/day A.I. showed statistically significant reductions in haemoglobin and mean corpuscular haemoglobin concentration. All of the individual values were within normal ranges for rats of the strain and age used therefore the intergroup differences were considered not to be of toxicological importance.

BLOOD CHEMISTRY:
No toxicologically significant effects were detected in the blood chemical parameters examined.

Males from all treatment groups showed a statistically significant reduction in urea. Males treated with 350 mg/kg bw/day A.I. also showed a statistically significant increase in chloride concentration (P<0.05) and a statistically significant reduction in phosphorus concentration (P<0.05). Females treated with 350 and 190 mg/kg bw/day A.I. showed a statistically significant reduction in creatinine (P<0.05). All of the of individual values
were within the normal ranges for rats of the strain and age used and in the absence of any histology correlates the intergroup differences were considered not to be of toxicological importance.

PATHOLOGY:
NECROPSY:
Adults:
No toxicologically significant macroscopic abnormalities were detected.

One female treated with 350 mg/kg bw/day A.I. and one female treated with 100 mg/kg bw/day A.I. had reddened lungs at necropsy. One male treated with 100 mg/kg bw/day A.I. had reddened cervical lymph nodes. In the absence of any histology correlates the intergroup differences were considered not to be of toxicological importance. One male treated with 100 mg/kg bw/day A.I. had small testes and epididymides and flaccid testes
at necropsy. This male was the partner of the non pregnant female and microscopic examination revealed tubular atrophy in both testes and aspermia in the epididymides. These lesions were considered to be the cause of the non pregnancy and are considered naturally occurring background changes in rats therefore of no toxicological significance. One control male had increased renal pelvic space in the right kidney. This observation
is considered to be a congenital abnormality of no toxicological importance.


ORGAN WEIGHTS:
No toxicologically significant effects were detected in the organ weights measured.

Males treated with 350 and 190 mg/kg bw/day A.I. and females treated with 350 mg/kg bw/day A.I. showed a statistically significant increase in absolute and relative liver weight. Males treated with 350 mg/kg bw/day A.I. also showed an increase in absolute and relative kidney weight. The majority of the individual values were within normal ranges for rats of the strain and age used and in the absence of any histology correlates
the intergroup differences were considered not to be of toxicological importance. Males from all treatment groups showed a statistically significant increase in absolute and relative brain weight. Males treated with 100 mg/kg bw/day A.I. also showed a statistically significant increase in absolute and relative thyroid weight. In the absence of a true dose related response or any histology correlates the intergroup differences were considered not to be of toxicological importance.

HISTOPATHOLOGY:
The following treatment-related microscopic finding was detected:

Sciatic nerve: Minimal axonal swelling with mineralisation was evident in animals of either sex from all treatment groups (see Table 1).

Dose descriptor:

NOEL

Remarks:

reproductive toxicity

Effect level:

350 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: No treatment related effects detected in the reproductive parameters observed.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Histopathology treatment-related findings in animals of either sex from all treatment groups (see results section for details)

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Litter Responses:
In total ten females from the control, 190 and 350 mg/kg bw/day A.I. dose groups and nine females from 100 mg/kg bw/day A.I. dose group gave birth to a live litter and successfully reared young to Day 5 age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size, Sex Ratio and Viability:
No significant differences were detected for corpora lutea, implantation counts, litter size or litter viability for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

There were no toxicologically significant effects detected in sex ratio (percentage male offspring) for litters from treated groups compared to controls. A minimal statistically significant increase (P<0.05) in sex ratio at birth was evident for litters from females treated with 350 mg/kg bw/day A.I. The group mean percent of males at this treatment group was only 56% and no adverse effects were detected in offspring survival or sex
ration at day 1 or 4 therefore the intergroup difference was considered to be incidental and of no toxicological importance.

Offspring Growth and Development:
There were no toxicologically significant effects detected.

Statistical analysis of the litter, offspring weights or surface righting reflex data did not reveal any significant intergroup differences.

No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, weak, no milk in stomach, physical injuries, found dead or missing, were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.

Pathology:
Necropsy (offspring):
No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Dose descriptor:

NOAEL

Generation:

F1

Based on:

act. ingr.

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

not specified

Histopathology:

Table 1: Incidence and Mean Severity of Main Findings in Sciatic Nerve

Finding Incidence/severity	Group 1, Control (0 mg/kg/day)		Group 2, Low (100)		Group 3, Intermediate (190)		Group 4, High (350)
	5 M	5 F	5 M	5 F	5 M	5 F	5 M	5 F
Axonal swelling	0	0	1/1.0	2/1.0	1/1.0	3/1.0	1/1.0	2/1.0

Conclusions:

The oral administration of N-Isopropylmethacrylamide (NIPMAA) to rats by gavage, at dose levels of 100, 190 and 350 mg/kg bw/day A.I., resulted in treatment-related finding in animals of either sex from all treatment groups. A ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity has therefore not been established.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 350 mg/kg bw/day A.I.

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) and is 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 22 March 1996).

This study was also designed to be compatible with the 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 ten male and ten female Wistar Han:RccHan:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 190 and 350 mg/kg bw/day A.I. (incorporating a correction factor for 91.3% purity). A control group of ten males and ten females was

dosed with vehicle alone (Arachis oil BP).

Clinical signs, behavioural assessments, body weight change, 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 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4post partum. Haematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group.

Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on or after Day 25post coitum. All animals were subjected to a gross

necropsy examination and histopathological evaluation of selected tissues was performed.

Results.

Adult Responses:

Mortality. There were no unscheduled deaths.

Clinical Observations.Animals of either sex treated with 350 mg/kg bw/day A.I. showed episodes of increased salivation between Days 14 and 38. One male treated with 350 mg/kg bw/day A.I. showed pilo-erection and hunched posture on Day 29. No such

effects were detected in animals of either sex treated with 100 or 190 mg/kg bw/day A.I.

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

Functional Performance Tests.There were no toxicologically significant changes in functional performance.

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

Body Weight.Males treated with 350 and 190 mg/kg bw/day A.I. showed a reduction in body weight gain during Week 1. Males treated with 350 mg/kg bw/day A.I. also showed a reduction in body weight gain during Week 4. Overall body weight gain was reduced in males treated with 350 mg/kg bw/day A.I. No such effects were detected in females treated with 350 and 190 mg/kg bw/day A.I. or animals of either sex treated with 100 mg/kg bw/day A.I.

Food Consumption.No adverse effect on food consumption was detected in treated animals. Males treated with 350 and 190 mg/kg bw/day A.I. did however show a reduction in food efficiency during the first week of treatment.

Water Consumption.No toxicologically significant effect on water consumption was detected.

Reproductive Performance:

Mating.There were no treatment-related effects on mating for treated animals.

Fertility.There were no treatment-related effects on fertility.

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

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability.Of the litters born, litter size at birth and subsequently on Day 1 and 4 post partum were comparable to controls. Sex ratio was also comparable to controls.

Offspring Growth and Development.Offspring body weight gain and litter weights at birth and subsequently on Day 1 and 4 post partum were comparable to controls. Surface righting was also comparable to controls.

Laboratory Investigations:

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

Blood Chemistry.There were no toxicologically significant effects detected in the blood chemical parameters examined.

Pathology:

Necropsy.No toxicologically significant macroscopic abnormalities were detected.

Organ Weights.No toxicologically significant effects were detected in the organ weights measured.

Histopathology.The following treatment-related microscopic finding was detected:

Sciatic nerve: Minimal axonal swelling with mineralisation was evident in animals of either sex from all treatment groups.

Conclusion.The oral administration of N-Isopropylmethacrylamide (NIPMAA) to rats by gavage, at dose levels of 100, 190 and 350 mg/kg bw/day A.I., resulted in treatmentrelated finding in animals of either sex from all treatment groups. A ‘No Observed

Adverse Effect Level’ (NOAEL) for systemic toxicity has therefore not been established.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 350 mg/kg bw/day A.I.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The study has been conducted according to OECD Guideline 422 and GLP and is adequately reported. The study has been assigned a reliability 1.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) on NIPMAA

There were no treatment-related effects detected in the reproductive parameters observed.

Reproductive Performance:

Mating: There were no treatment-related effects on mating for treated animals.

Fertility: There were no treatment-related effects on fertility.

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

Justification for selection of Effect on fertility via oral route:
The available OECD 422 study is assigned as reliability study 1 and is the only oral reproduction/developmental study available on the substance itself.

Effects on developmental toxicity

Description of key information

The oral administration of N-Isopropylmethacrylamide (NIPMAA) to rats by gavage, at dose levels of 100, 190 and 350 mg/kg bw/day A.I., resulted in no treatment-related finding in animals of either sex from all treatment groups in the reproductive parameters observed.
The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 350 mg/kg bw/day A.I.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The in-life phase of the study was conducted between 06 June 2012 (first day of treatment) and 29 July 2012 (final necropsy).

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: 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

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd
- Age at study initiation: Approximately twelve weeks old
- Weight at study initiation: At the start of the treatment the males weighed 301 to 358g, the females weighed 193 to 236g.
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. 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.
- Diet (e.g. ad libitum): The animals were allowed free access to food (a pelleted diet).
- Water (e.g. ad libitum): The animals were allowed free access to water (mains drinking water was supplied from polycarbonate bottles attached to the cage).
- Acclimation period: The animals were
acclimatised for seven days during which time their health status was assessed.A total of eighty animals (forty males and forty females) were accepted into the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature was set to achieve target values of 21 ± 2°C.
- Humidity (%): The humidity was set to acieve target values of 55 ± 15% .
- Air changes (per hr): The rate of air exchange was at least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a suspension in Arachis oil BP.
The stability and homogeneity of the test item formulations were previously determined in a 7 day range finding study.
Results showed the formulations to be stable for at least twenty three days. Formulations were therefore prepared fortnightly and stored at 4ºC in the dark.

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 regular intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test item formulation were taken and analysed for concentration of N-Isopropylmethacrylamide (NIPMAA).

The concentration of N-Isopropylmethylacrylamide in the test item formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.

The results indicate that the prepared formulations were within ±6% of the nominal concentration.

Details on mating procedure:

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 oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Duration of treatment / exposure:

The test item was administered to rats for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females).

Frequency of treatment:

The test item was administered daily.

Duration of test:

Up to eight weeks

No. of animals per sex per dose:

Ten males and ten females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were chosen based on the results of a 7-day range finding study.

Chronological Sequence of Study:
i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv) 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.
v) On completion of the pre-pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43.
ix) Blood samples were taken from five randomly selected females from each dose group for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.

Maternal examinations:

CLINICAL OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

FUNCTIONAL OBSERVATIONS:
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. 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.

BEHAVIOURAL ASSESSMENTS:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.

FUNCTIONAL PERFORMANCE TESTS:
Motor Activity: Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period
and also during the final 20% of the period (considered to be the asymptotic period, 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). Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Blink reflex , Tail pinch, Startle reflex, Finger approach.

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 mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

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 on Days 1 and 4 post partum.

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 for females during gestation and lactation.

WATER CONSUMPTION:
Water intake was measured daily throughout the study (with the exception of the pairing phase).

PREGNANCY AND PARTURITION:
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition

LABORATORY INVESTIGATIONS:
Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 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.

HAEMATOLOGY:
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices
- mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed
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
Calcium (Ca++)
Glucose
Inorganic phosphorus (P)
Total protein (Tot.Prot.)
Aspartate aminotransferase (ASAT)
Albumin
Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation)
Alkaline phosphatase (AP)
Sodium (Na+)
Creatinine (Creat)
Potassium (K+)
Total cholesterol (Chol)
Chloride (Cl-)
Total bilirubin (Bili)
Bile acids (Bile)

Ovaries and uterine content:

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).

Fetal examinations:

Not performed.

Statistics:

The following parameters were subjected to statistical analysis:
Quantitative functional performance data
Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Water Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights

Indices:

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 5 of age).

i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
% pre – implantation loss = [(Number of corpora lutea - Number of implantation sites) ÷ Number of corpora lutea] x 100
% post – implantation loss =[(Number of implantation sites - Total number of offspring born) ÷ Number of implantation sites] x 100

ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1 ÷ Number of offspring born) x 100
Viability Index (%) = (Number of offspring alive on Day 4 ÷ Number of offspring alive on Day 1 ) x 100

Historical control data:

Normal range data for the different parameters examined were used in comparison against the test data.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
ADULT RESPONSES:
MORTALITY:
There were no unscheduled deaths.

CLINICAL OBSERVATIONS:
Animals of either sex treated with 350 mg/kg bw/day A.I. showed episodes of increased salivation between Days 14 and 38. One male treated with 350 mg/kg bw/day A.I. showed pilo-erection and hunched posture on Day 29.
No such effects were detected in animals of either sex treated with 100 or 190 mg/kg bw/day A.I.
One control male had generalised fur loss and scab formation on Days 42 and 43. Observations of this nature are commonly observed in group housed animals and in view of this observation only being present in a control animal is considered to be incidental.

BEHAVIOURAL ASSESSMENTS:
Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls.
All inter and intra group differences in behavioural scores were considered to be a result of normal variation for rats of the strain and age used, and the differences were of no toxicological importance.

FUNCTIONAL PERFORMANCE TESTS:
Males treated with 350 mg/kg bw/day A.I. showed a statistically significant increase in fore limb grip strength. All individual values were within the normal range for rats of the strain and age used and was confined to one out of the three tests therefore the intergroup difference was considered not to be of toxicological importance. Animals of either sex treated with 350 mg/kg bw/day A.I. showed a statistically significant increase in hind limb grip strength. The statistically significant differences were confined to one out of the three tests and in the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered to be of no toxicological significance. Males treated with 100 mg/kg bw/day A.I. showed a statistically significant increase in overall activity and overall mobile activity. In the absence of a true dose related response the intergroup differences were considered not to be of toxicological importance.

SENSORY REACTIVITY ASSESSMENTS:
There were no treatment-related changes in sensory reactivity.

BODY WEIGHT:
Males treated with 350 and 190 mg/kg bw/day A.I. showed a statistically significant reduction in body weight gain during Week 1 (P<0.001, P<0.05 respectively). Males treated with 350 mg/kg bw/day A.I. also showed a statistically significant reduction in body weight gain during Week 4 (P<0.05). Overall body weight gain was also reduced in males treated with 350 mg/kg bw/day A.I. No such effects were detected in females treated with 350 or 190 mg/kg bw/day A.I. or in animals of either sex treated with 100 mg/kg bw/day A.I.

FOOD CONSUMPTION:
No adverse effects on food consumption were detected in treated animals when compared to controls. Males treated with 350 and 190mg/kg bw/day A.I. did however show a reduction in food efficiency during the first week of treatment.

Females treated with 350 and 190 mg/kg bw/day A.I. showed a statistically significant increase in food consumption during the final week of gestation. An increase in food consumption in isolation is not generally considered to be an adverse effect of treatment therefore the intergroup difference was considered not to be of toxicological importance.

WATER CONSUMPTION:
No toxicologically significant effect on water consumption was detected.

Females treated with 350 mg/kg bw/day A.I. showed an increase in overall water consumption throughout the treatment period. Males treated with 190 and 100 mg/kg bw/day A.I. also showed an increase in overall water consumption however males treated with 350 mg/kg bw/day A.I. showed a reduction in overall water consumption compared to control animals. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in the absence of a true dose related response in males the intergroup differences were considered not to be of toxicological importance.

Females treated with 190 mg/kg bw/day A.I. showed a statistically significant reduction in water consumption during Week 3 of gestation. A -6% reduction in water consumption is not generally considered to be an adverse effect of treatment therefore the intergroup difference was considered not to be of toxicological importance.

REPRODUCTIVE PERFORMANCE
MATING:
There were no treatment-related effects on mating performance.

FERTILITY:
There were no treatment-related effects on fertility.

One female treated with 190 mg/kg bw/day A.I. did not achieve pregnancy following evidence of mating. No histopathological correlates were evident in the female reproductive organs. The male partner however showed tubular atrophy in both testes and aspermia in the epididymides, which correlated with the macroscopic observations of small testes and epididymides and flaccid testes. These lesions were considered to be the cause of the non pregnancy however are considered naturally occurring background changes in rats therefore of no toxicological significance.

GESTATION LENGTH:
There were no differences in gestation lengths. The distribution for treated females was comparable to controls. The gestation lengths were between 22 and 24 Days.


LABORATORY INVESTIGATIONS:
HEAMATOLOGY:
No toxicologically significant effects were detected in the haematological parameters examined.
Males treated with 350 mg/kg bw/day A.I. showed statistically significant reductions in haemoglobin and mean corpuscular haemoglobin concentration. All of the individual values were within normal ranges for rats of the strain and age used therefore the intergroup differences were considered not to be of toxicological importance.

BLOOD CHEMISTRY:
No toxicologically significant effects were detected in the blood chemical parameters examined.

Males from all treatment groups showed a statistically significant reduction in urea. Males treated with 350 mg/kg bw/day A.I. also showed a statistically significant increase in chloride concentration (P<0.05) and a statistically significant reduction in phosphorus concentration (P<0.05). Females treated with 350 and 190 mg/kg bw/day A.I. showed a statistically significant reduction in creatinine (P<0.05). All of the of individual values
were within the normal ranges for rats of the strain and age used and in the absence of any histology correlates the intergroup differences were considered not to be of toxicological importance.

PATHOLOGY:
NECROPSY:
Adults:
No toxicologically significant macroscopic abnormalities were detected.

One female treated with 350 mg/kg bw/day A.I. and one female treated with 100 mg/kg bw/day A.I. had reddened lungs at necropsy. One male treated with 100 mg/kg bw/day A.I. had reddened cervical lymph nodes. In the absence of any histology correlates the intergroup differences were considered not to be of toxicological importance. One male treated with 100 mg/kg bw/day A.I. had small testes and epididymides and flaccid testes
at necropsy. This male was the partner of the non pregnant female and microscopic examination revealed tubular atrophy in both testes and aspermia in the epididymides. These lesions were considered to be the cause of the non pregnancy and are considered naturally occurring background changes in rats therefore of no toxicological significance. One control male had increased renal pelvic space in the right kidney. This observation
is considered to be a congenital abnormality of no toxicological importance.


ORGAN WEIGHTS:
No toxicologically significant effects were detected in the organ weights measured.

Males treated with 350 and 190 mg/kg bw/day A.I. and females treated with 350 mg/kg bw/day A.I. showed a statistically significant increase in absolute and relative liver weight. Males treated with 350 mg/kg bw/day A.I. also showed an increase in absolute and relative kidney weight. The majority of the individual values were within normal ranges for rats of the strain and age used and in the absence of any histology correlates
the intergroup differences were considered not to be of toxicological importance. Males from all treatment groups showed a statistically significant increase in absolute and relative brain weight. Males treated with 100 mg/kg bw/day A.I. also showed a statistically significant increase in absolute and relative thyroid weight. In the absence of a true dose related response or any histology correlates the intergroup differences were considered not to be of toxicological importance.

HISTOPATHOLOGY:
The following treatment-related microscopic finding was detected:

Sciatic nerve: Minimal axonal swelling with mineralisation was evident in animals of either sex from all treatment groups (see Table 1).

Dose descriptor:

NOEL

Effect level:

350 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Basis for effect level:

other: other:

Dose descriptor:

NOAEL

Basis for effect level:

other: other:

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:not examined

Dose descriptor:

NOAEL

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

not specified

Developmental effects observed:

not specified

Litter Responses:

In total ten females from the control, 190 and 350 mg/kg bw/day A.I. dose groups and nine females from 100 mg/kg bw/day A.I. dose group gave birth to a live litter and successfully reared young to Day 5 age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size, Sex Ratio and Viability:

No significant differences were detected for corpora lutea, implantation counts, litter size or litter viability for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

There were no toxicologically significant effects detected in sex ratio (percentage male offspring) for litters from treated groups compared to controls. A minimal statistically significant increase (P<0.05) in sex ratio at birth was evident for litters from females treated with 350 mg/kg bw/day A.I. The group mean percent of males at this treatment group was only 56% and no adverse effects were detected in offspring survival or sex

ration at day 1 or 4 therefore the intergroup difference was considered to be incidental and of no toxicological importance.

Offspring Growth and Development:

There were no toxicologically significant effects detected.

Statistical analysis of the litter, offspring weights or surface righting reflex data did not reveal any significant intergroup differences.

No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, weak, no milk in stomach, physical injuries, found dead or missing, were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.

Pathology:

Necropsy (offspring):

No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Conclusions:

The oral administration of N-Isopropylmethacrylamide (NIPMAA) to rats by gavage, at dose levels of 100, 190 and 350 mg/kg bw/day A.I., resulted in treatment-related finding in animals of either sex from all treatment groups. A ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity has therefore not been established.
The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 350 mg/kg bw/day A.I.

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) and is 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 22 March 1996).

This study was also designed to be compatible with the 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 ten male and ten female Wistar Han:RccHan:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 190 and 350 mg/kg bw/day A.I. (incorporating a correction factor for 91.3% purity). A control group of ten males and ten females was

dosed with vehicle alone (Arachis oil BP).

Clinical signs, behavioural assessments, body weight change, 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 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4post partum. Haematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group.

Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on or after Day 25post coitum. All animals were subjected to a gross

necropsy examination and histopathological evaluation of selected tissues was performed.

Results.

Adult Responses:

Mortality. There were no unscheduled deaths.

Clinical Observations.Animals of either sex treated with 350 mg/kg bw/day A.I. showed episodes of increased salivation between Days 14 and 38. One male treated with 350 mg/kg bw/day A.I. showed pilo-erection and hunched posture on Day 29. No such

effects were detected in animals of either sex treated with 100 or 190 mg/kg bw/day A.I.

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

Functional Performance Tests.There were no toxicologically significant changes in functional performance.

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

Body Weight.Males treated with 350 and 190 mg/kg bw/day A.I. showed a reduction in body weight gain during Week 1. Males treated with 350 mg/kg bw/day A.I. also showed a reduction in body weight gain during Week 4. Overall body weight gain was reduced in males treated with 350 mg/kg bw/day A.I. No such effects were detected in females treated with 350 and 190 mg/kg bw/day A.I. or animals of either sex treated with 100 mg/kg bw/day A.I.

Food Consumption.No adverse effect on food consumption was detected in treated animals. Males treated with 350 and 190 mg/kg bw/day A.I. did however show a reduction in food efficiency during the first week of treatment.

Water Consumption.No toxicologically significant effect on water consumption was detected.

Reproductive Performance:

Mating.There were no treatment-related effects on mating for treated animals.

Fertility.There were no treatment-related effects on fertility.

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

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability.Of the litters born, litter size at birth and subsequently on Day 1 and 4 post partum were comparable to controls. Sex ratio was also comparable to controls.

Offspring Growth and Development.Offspring body weight gain and litter weights at birth and subsequently on Day 1 and 4 post partum were comparable to controls. Surface righting was also comparable to controls.

Laboratory Investigations:

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

Blood Chemistry.There were no toxicologically significant effects detected in the blood chemical parameters examined.

Pathology:

Necropsy.No toxicologically significant macroscopic abnormalities were detected.

Organ Weights.No toxicologically significant effects were detected in the organ weights measured.

Histopathology.The following treatment-related microscopic finding was detected:

Sciatic nerve: Minimal axonal swelling with mineralisation was evident in animals of either sex from all treatment groups.

Conclusion.The oral administration of N-Isopropylmethacrylamide (NIPMAA) to rats by gavage, at dose levels of 100, 190 and 350 mg/kg bw/day A.I., resulted in treatmentrelated finding in animals of either sex from all treatment groups. A ‘No Observed

Adverse Effect Level’ (NOAEL) for systemic toxicity has therefore not been established.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 350 mg/kg bw/day A.I.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The study has been conducted according to OECD Guideline 422 and GLP and is adequately reported. The study has been assigned a reliability 1.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) on NIPMAA

There were no treatment-related effects detected in the reproductive parameters observed.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability: Of the litters born, litter size at birth and subsequently on Day 1 and 4 post partum were comparable to controls. Sex ratio was also comparable to controls.

Offspring Growth and Development: Offspring body weight gain and litter weights at birth and subsequently on Day 1 and 4 post partum were comparable to controls. Surface righting was also comparable to controls.

Effect on developmental toxicity: via oral route:
The available OECD 422 study is assigned as reliability study 1 and is the only oral reproduction/developmental study available on the substance itself.

Justification for classification or non-classification

The oral administration of N-Isopropylmethacrylamide (NIPMAA) to rats by gavage, at dose levels of 100, 190 and 350 mg/kg bw/day A.I. resulted in no treatment-related effects detected in the reproductive parameters observed. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 350 mg/kg bw/day A.I.

Therefore, based on the results of the OECD 422 study there is no evidence that the substance has an adverse effect on sexual function and fertility, or on development at the highest dose level. The substance is therefore not classified for reproductive toxicity based on this study.