N-isopropylmethacrylamide

Endpoint summary

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 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.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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 oral 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.

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.

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.

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.

Positive control:

None.

Observations and examinations performed and frequency:

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).

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)

Sacrifice and pathology:

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. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. 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 and offspring, 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.

Other examinations:

REPRODUCTION SCREENING:
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 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.

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

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.

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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Mortality:

mortality observed, treatment-related

Description (incidence):

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):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

no toxicologically significant effects detected in the haematological parameters examined.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

no toxicologically significant effects detected in the blood chemical parameters examined.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

no toxicologically significant effects

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

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

Gross pathological findings:

no effects observed

Description (incidence and severity):

no toxicologically significant macroscopic abnromalities were detected

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.

Histopathological findings: neoplastic:

not examined

Details on 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.
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.

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.

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.

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:

NOAEL

Remarks:

systemic toxicity

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

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

other: Basis missing

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.

Critical 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

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.

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 4 post 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 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

100 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.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22-Aug-2006 to 14-Aug-2008

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.

Justification for type of information:

Please refer to the CSR for the read-across rationale.

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

adopted May 12, 1981

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
- Rat strain: HanRCC: WIST(SPF)
- Source: RCC Ltd., Füllingsdorf, Switzerland
- Age at study initiation: 8 weeks (males), 11 weeks (females)
- Weight at study initiation: males: 211.1 to 236.2 g
females: 184.0 to 211.4 g
- Fasting period before study:
- Housing: Groups of 5 in Makrolon type-4 cages with wire mesh tops and standard softwood bedding ("Lignocel", Schill AG, Muttenz, Switzerland).
- Diet (e.g. ad libitum): ad libitum, pelleted standard Kliba 3433, Batch 23/06
- Water (e.g. ad libitum): ad libitum access to tap water
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hr artificial flourescent light/ 12 hr dark, music/light period.

Route of administration:

inhalation

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: The particle size (expressed as MMAD) of the generated Methacrylamide aerosols was well within the ideal range of 1 to 3 µm in each treatment group. Details on particle size characteristics see below, Table 2 in section “Any other information on materials and methods incl. tables”

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:
Inhalation exposure was performed using a system similar to that originally described by Sachsse et al. (details see: K. Sachsse, L. Ullmann, G. Voss
and R. Hess: Measurements of Inhalation Toxicity of Aerosols in Small Laboratory Animals. In: Proceedings of the Europ. Soc. for the Study of Drug
Toxicity, Vol. XV, pp. 239-251, Zürich, June 1973.)
The design of this exposure system is based upon the fluid dynamic modelling of the aerosol flow. It ensures a uniform test item distribution,
provides a constant stream of "fresh" test item to each animal, and precludes re-breathing the exhaled air. The aerosol stream reaches the animal's
nose through ports situated at three different levels around the axis of the exposure chamber. Each level has 16 animal ports and can be rotated
allowing close observation of all the animals without interruption of exposure. In the present study the exposure chambers of groups 1 (air control)
and 3 (mid-dose) comprised three levels of animal ports. For the exposure of groups 2 (low-dose) and 4 (high-dose) chambers comprising five
levels of animal ports were utilized. All animals were placed on the top two levels during each exposure, thus minimising variation in exposure
conditions between the animals of any particular group.

- Method of holding animals in test chamber:
The animals were confined separately in Makrolon* restraint tubes which were positioned radially around the nose-only, flow-past exposure chamber (details see: Cannon, W.C., E.F. Blanton and K.E. McDonald: "The Flow-Past Chamber: An Improved Nose-Only Exposure System for Rodents", Am. Ind. Hyg. Assoc. J., 44 (12): 923-928, 1983.)

- Source and rate of air:

The design of this exposure system precludes re-breathing the exhaled air, details see above (Exposure apparatus).


- Method of conditioning air:
Oxygen Concentration
The oxygen concentration of the control and test atmospheres were monitored continuously during each 6-hour exposure period for each group
(Groups 1 to 4 inclusive using an oxygen sensor (VarioGard Transmitter 4...20 mA from Dräger AG, 8305 Dietlikon / Switzerland) connected to a data logger. In addition, the oxygen concentration was noted manually from the data logger for each group at appropriate intervals after the beginning of the daily exposure (three times during each 6-hour exposure period / group / day).


- System of generating particulates/aerosols:
Test Aerosol Generation
For each dose group (Groups 2, 3 and 4), the pre-milled test item was aerosolised using a piston feed/rotating brush aerosol generator followed by a micronising jet mill (see diagrams below). Then the aerosol output from the jet mill was discharged through a 63Ni charge neutraliser. For Groups 2
(Low Dose) and 4 (High Dose) the discharged aerosol was split, one portion of it being used for the exposure of Group 4 without further dilution, the other portion of it being further diluted with filtered dry air using an air vac device. For Group 3 (Mid Dose) the test aerosol was generated in a similar manner as for Group 4, but separately from that generated for Groups 2 and 4 without using an air vac device. In order to minimize the variation in
aerosol concentration over each exposure and remain close to the target aerosol concentrations, the piston advance speed was adjusted, as deemed necessary from the daily monitoring of aerosol concentration. The above generation and/or dilution system was chosen to achieve the required test
item concentrations. Animals of group 1 (Air Control) were exposed to compressed filtered air alone at conditions similar to those used for Groups 2 (Low Dose), 3 (Mid Dose) and 4 (High Dose).


- Temperature, humidity, pressure in air chamber:
Temperature / Relative Humidity
The temperature and relative humidity of the control and test atmospheres were monitored continuously during each 6-hour exposure period for
each group (Groups 1 to 4 inclusive) using a ROTRONIC Hygrometer (Series I-200, Rotronic AG, 8040 Zürich / Switzerland) connected to a data
logger. In addition, the temperature and relative humidity were noted manually from the data logger for each group at appropriate intervals after the
beginning of the daily exposure (three times during each 6-hour exposure period / group / day).


- Air flow rate:
Exposure Airflow Rate
The airflow rate through the exposure chambers was adjusted before inhalation exposure and monitored indirectly during each exposure through
the generation and/or dilution system using calibrated pressure gauges and/or flowmeters and were checked at least at the same intervals as the
temperature, relative humidity and oxygen concentration of the control and test atmospheres. In all dose groups, a proportion of the ports on each
exposure chamber were closed such that the airflow to each open port was approximately 1.0 L/minute/animal port during each exposure.


- Air change rate:
The design of this exposure system precludes re-breathing the exhaled air, details see above (Exposure apparatus).


- Method of particle size determination:
Particle Size Distributions and Mass Median Aerodynamic Diameters
The distribution of particle size in the generated aerosols was measured by gravimetry six times in Group 2 (Low Dose) and four times in each of
groups 3 (Mid Dose) and 4 (High Dose) during the 13-week treatment period, using a Mercer 7-stage cascade impactor (Model 02-130, In-Tox.
Products Inc. Albuquerque, New Mexico, USA). Representative samples of the test atmosphere were drawn through the impactor with a flow rate of 1.0 L/min and the particles deposited according to their aerodynamic size onto stainless steel slips and the final filter stage (Type HVLP, Polyvinylidene-
difluoride membrane, pore size 0.45 m), on each stage of the impactor. To obtain the mass deposited on each stage of the impactor, the steel slips
and the final filter stage were weighed before and after sampling using an appropriate analytical balance. For each measurement in Group 2, aerosol
was collected in the impactors over three to six consecutive treatment days, in order to accumulate sufficient particulate material in the impactor for
adequate aerodynamic particle size measurement. On the corresponding results table of gravimetric particle size data only the last sampling date is
given. For the measurements in Groups 3 and 4 sufficient test item accumulated within one treatment day. The total mass (g) deposited in the impactor was then calculated by adding together the mass deposited on each of the stainless steel slips and the final filter stage. As the Effective Cut-off
Diameters (ECD) represent the lower size limit of the particles collected on each stage, the percentages less than the indicated size were tabulated as a function of the ECD. The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) of the test aerosols were calculated on the basis of the gravimetric results from the impactors applying the built-in PROBIT function of Microsoft Excel Software. The target range for the mass median aerodynamic diameter was 1 to 3 μm. The gravimetric determination of particle size distribution is only indicative of the nonevaporated phase of the test item. Any vapour component or any fraction which might evaporate or sublimate are not accounted for.



- Treatment of exhaust air:

Re-breathing of exhaust air is precluded, deteis see above (exposure apparatus).


TEST ATMOSPHERE

- Brief description of analytical method used:
Analytical Concentrations
Sampling of test atmosphere for the determination of concentrations by chemical analysis was performed at least once weekly during most of a
6-hour inhalation exposure period in each of Groups 2 to 4 inclusive. The dust fraction of the test atmospheres was collected on Millipore® durapore filters, Type HVLP, the vapour fraction was trapped in phosphoric acid (0.033 molar). On each sampling occasion, the test atmosphere was drawn
through such a filter which was followed by two wash-bottles placed in series each containing approximately 80 mL of phosphoric acid (0.033 molar), cooled in water ice. Immediately after each atmosphere sampling and weighing, the filter was put into a light protected glass vial and covered with
10 g of phosphoric acid (0.033 molar) to minimise loss of test item by sublimation from the filter. The content of each wash-bottle was transferred
into appropriate glass flasks, the wash-bottles were rinsed with phosphoric acid (0.033 molar), and the flasks made up to 100 ml with the rinsing and tightly closed.
The filters and solutions were kept light protected at approximately +5 °C in a cool box until dispatch at this temperature to the analytical laboratory of RCC Ltd, for chemical analysis. The samples were analysed by high performance liquid chromatography (HPLC) with UV detection using the test
item, METHACRYLAMIDE (as received from the Sponsor), as reference item for the analytical calibration curves. The analytical method was based on
that which was provided by the Sponsor. An analytical phase report was provided for inclusion in the report of this study.


- Samples taken from breathing zone: yes
Test atmosphere samples were collected directly from the delivery tube in the breathing zone of the animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean chemically determined aerosol concentrations of METHACRYLAMIDE and corresponding mean gravimetrically determined aerosol
concentrations are summarised below, see Table 1 in section “Any other information on materials and methods incl. tables”


Exposure System Monitoring

The atmosphere concentrations of the test item determined gravimetrically (dust fraction, trapped on filters) and chemically (including dust fraction
trapped of filters and vapour fraction trapped in solvent), particle size distribution determined gravimetrically, relative humidity, temperature
and oxygen concentration, were measured on test atmosphere samples collected directly from the delivery tube in the breathing zone of the animals, at an empty sampling port of the exposure chamber, delivering "fresh" test item to the animal's nose. The position at which these test atmosphere
samples were taken has been considered to be representative for the breathing zone of the animals. This approach was chosen in order to obtain
representative samples of what was delivered to the animals. Airflow rates were measured during the collection of samples for the determination of
aerosol concentrations using dry-test meters and pressure gauges, calibrated with a reference dry-test meter. Airflow rates for particle size samples were calibrated using a Gilibrator bubble flow generator.

Nominal Determination of Aerosol Concentrations

Based on the proportion of primary (prior to further dilution) aerosol assigned to Group 2 (Low Dose) the quantity of test item assigned to this group was calculated and then divided by the sum of primary airflow volume assigned to Group 2 plus airflow used for further aerosol dilution to
give the nominal concentration of test item in the aerosol delivered to the animals of this group. For Group 3 (Mid Dose), the nominal concentration
was measured by weighing the generator cylinder containing the test item before and after each exposure to determine the quantity of test item used. The weight of test item used was then divided by the total airflow volume to give the nominal concentration. For Group 4 (High Dose), the nominal
concentration was measured by weighing the generator cylinder containing the test item before and after each exposure to determine the quantity of test item used for generation of the primary (prior to further dilution) aerosol. The weight of test item used was then divided by the total airflow
volume used for the jet mill, i.e. used for the primary aerosol (prior to further dilution), to give the nominal concentration of test item in the aerosol
delivered to the animals of this group.

Gravimetric Determination of Aerosol Concentrations

Gravimetric determinations of aerosol concentration (dust fraction) in the generated atmospheres were performed using a Millipore® durapore filter
(Type HVLP, Polyvinylidenedifluoride membrane, pore size 0.45 µm), loaded in a 47 mm in-line stainless steel filter sampling device.
The gravimetric sampling was performed once daily for each of groups 2 (Low Dose), 3 (Mid Dose) and 4 (High Dose) sampling aerosol during most
of the 6-hour exposure period. In addition, on a number of occasions during the study for each of these groups, a further three filter samples were
taken over shorter periods shortly after the beginning, at about the middle and towards the end of the 6-hour exposure period to give an indication
of the stability of the aerosol concentration over exposure time. Additional aerosol samples were taken for monitoring purposes as considered
necessary and the corresponding aerosol concentration data were retained in the raw data without reporting.
Technical atmosphere generation trials (not performed under GLP) led to the conclusion that in the present study the gravimetrically determined
concentrations were only approximate estimates of the true concentrations of the test atmosphere, because of sublimation losses of
METHACRYLAMIDE. Therefore, one purpose of the gravimetric determination of the aerosol concentrations was to monitor roughly the aerosol
concentration during the exposure period, as the test atmosphere concentrations (dust and vapour fraction) determined by chemical analysis were
only available post exposure. The gravimetric determination of aerosol concentration is only indicative of the non-evaporated phase of the test item. Any vapour component or any fraction which might evaporate or sublimate is not accounted for in the gravimetrically determined aerosol
concentration values.

Analytical Concentrations

Sampling of test atmosphere for the determination of concentrations by chemical analysis was performed at least once weekly during most of a
6-hour inhalation exposure period in each of Groups 2 to 4 inclusive. The dust fraction of the test atmospheres was collected on Millipore® durapore filters, Type HVLP, the vapour fraction was trapped in phosphoric acid (0.033 molar). On each sampling occasion, the test atmosphere was drawn
through such a filter which was followed by two wash-bottles placed in series each containing approximately 80 mL of phosphoric acid (0.033 molar), cooled in water ice. Immediately after each atmosphere sampling and weighing, the filter was put into a light protected glass vial and covered with
10 g of phosphoric acid (0.033 molar) to minimise loss of test item by sublimation from the filter. The content of each wash-bottle was transferred
into appropriate glass flasks, the wash-bottles were rinsed with phosphoric acid (0.033 molar), and the flasks made up to 100 ml with the rinsing and tightly closed. The filters and solutions were kept light protected at approximately +5 °C in a cool box until dispatch at this temperature to the
analytical laboratory of RCC Ltd, for chemical analysis. The samples were analysed by high performance liquid chromatography (HPLC) with
UV detection using the test item, METHACRYLAMIDE, as reference item for the analytical calibration curves.

Duration of treatment / exposure:

6 hours daily.

Frequency of treatment:

Once daily, 5 days per week for a total of 13 consecutive weeks.

Remarks:

Doses / Concentrations:
Test concentrations: 0 mg/m³; 10 mg/m³ ; 25 mg/m³; 62.5 mg/m³
Basis:
nominal conc.

No. of animals per sex per dose:

Group 1 – Air control: 10 Males and 10 Females
Group 2 – Low Dose: 10 Males and 10 Females
Group 3 – Mid Dose: 10 Males and 10 Females
Group 4 – High Dose: 10 Males and 10 Females

Control animals:

yes

Details on study design:

- Dose selection rationale:
The above target concentrations were chosen on the basis of the results attained in a 2-Week inhalation and neurotoxicity study in the rat via
nose-only exposures.

Rationale for animal assignment:
Selection and Randomization: The animals were allocated to groups by a body weight stratification procedure on the first day of acclimatisation
to ensure that differences in group mean body weights are minimized.


Rationale for special In-Life Observations:
Functional Observational Battery (FOB) and Motor Activity were part of the in-life observations as the critical toxic effect for methacrylamide is
neurotoxicity.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

Viability / Mortality

All animals were observed for mortality/moribundity once daily during the acclimatisation period (including the reserve animals), and twice daily,
before and after exposure, during the treatment period of the study. On weekend days of the treatment period, when there was no exposure of
animals, mortality was checked at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes

Clinical Signs – Time schedule

Clinical signs were recorded once during the acclimatisation period (including the reserve animals) and twice daily during the treatment period of the study (once pre-exposure and once post exposure outside the restraint tubes on all animals, except the reserve animals). On weekend
days of the treatment period, when there was no exposure of animals, clinical examinations were performed once daily.
During exposure, only grossly abnormal signs could be observed, as the animals were in restraint tubes. Observations were detailed and carefully
recorded using explicitly defined scales as appropriate. Observations included but were not limited to changes in behaviour, somatomotor
activity, body position, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, e.g. tremors or
convulsions, reactivity to handling or sensory stimuli, altered strength, alteration of the skin, fur, nose, eyes and mucous membranes.

BODY WEIGHT: Yes

Body Weights – Time schedule

Each animal was weighed at 7-day intervals (if appropriate), during the acclimatisation and treatment periods of the study. Reserve animals were also weighed during acclimatisation. During the treatment period the body weights were recorded before the exposure on the particular day.

FOOD CONSUMPTION:

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

Food consumption was recorded weekly per cage of five animals (reserve animals exclusive), over a 7-day period (if possible and/or appropriate),
during the acclimatisation and treatment periods of the study. The food consumption was calculated per cage and per food consumption interval. It expresses the average food consumed per animal and per day for each cage over the food consumption interval.

FC = C / AD

where
FC is food consumption (food fed - food left) in grams of food per animal and day,
C is measured food consumption in grams per cage over the consumption interval, and
AD is total consumption days over all animals in the cage during the consumption interval.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

The relative food consumption was calculated per cage at each time interval according to the
following formula:

RFC = [ FC/BW(i)] x 1000

where
RFC is relative food consumption, i.e. food consumption in grams of food per kg bodyweight and day,
FC is food consumption (food fed - food left) in grams of food per animal and day, and
BW(i) is the optimal (on day closest to the middle of food consumption period) body weight in grams.


WATER CONSUMPTION: Not recorded

OPHTHALMOSCOPIC EXAMINATION: Yes

Ophthalmoscopic Examinations – Time schedule – Dose Groups:

Ophthalmoscopic examinations were performed in all animals once during the acclimatisation period (reserve animals inclusive), and in all surviving animals once during treatment week 13. Method/Instrumentation: The observations were performed after instillation of a mydriaticum,
using a Heine Bifocal Type Miroflex ophthalmoscope (Eisenhut Vet. AG, 4123 Allschwil /Switzerland).
Dates: Acclimatisation: 24-Aug-2006
Treatment Period (Week 13): 24-Nov-2006
For unilateral findings unless otherwise indicated in the tables, the contralateral eye was without abnormalities.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 13 weeks of treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: All animals

Clinical Laboratory Investigations

Blood samples for hematology and clinical biochemistry were collected from the retro-orbital plexus from all animals (10 animals/sex/group) under
light isoflurane anaesthesia. This was followed by necropsy on the same day. The animals were fasted in metabolism cages for approximately 17 hours
before blood sampling, but water was provided ad libitum. The blood samples were collected early in the working day to reduce biological variation
caused by circadian rhythms. Date of blood sampling: After 13 weeks of treatment: 28-Nov-2006
In the summary and individual tables the names of some parameters have been abbreviated.
Clinical laboratory data are expressed, with a few exceptions, in general accordance with the International System of Units (SI).

Hematology parameters determined

Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Reticulocyte count
Reticulocyte maturity index
Total leukocyte count
Differential leukocyte count
Platelet count
Coagulation:
Prothrombin time
Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 13 weeks of treatment
- Animals fasted: Yes
- How many animals: All animals

Clinical Biochemistry parameters determined:

Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Phospholipids
Aspartate aminotransferase
Alanine aminotransferase
Lactate dehydrogenase
Glutamate dehydrogenase
Alkaline phosphatase
Gamma-glutamyl-transferase
Creatine kinase
Sodium
Potassium
Chloride
Calcium
Phosphorus inorganic
Protein, total
Albumin
Globulin
Albumin/Globulin ratio


URINALYSIS: Yes
- Time schedule for collection of urine: after 13 weeks of treatment

Date of urine sampling: After 13 weeks of treatment: 28-Nov-2006
Urine was collected during an approximately 16-hour fasting period into specimen vials using a metabolism cage.

- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

Urinalysis parameters determined:

Volume
Relative density
Colour
Appearance
pH
Protein
Nitrite
Glucose
Ketone
Urobilinogen
Bilirubin
Erythrocytes
Leukocytes


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: see below
- Dose groups that were examined: see below
- Battery of functions tested: sensory activity / grip strength / motor activity / other: see below

Functional Observation Battery (FOB)

The FOB observations were performed during treatment weeks 12 (male animals) and 13 (female animals) as from approximately two hours after the
end of the 6-hour exposure period and included hind- and forelimb grip strength, landing foot splay and Preyer’s reflex. Detailed clinical
observations were evaluated in the FOB. The FOB was conducted on all animals, as the animals were not considered to otherwise reveal signs of
toxicity to an extent that would significantly have interfered with the functional test performance. Relevant parameters from a modified Irwin screen
test were performed on all rats. Any abnormal findings were recorded and graded in severity. Hind- and forelimb grip strength measurements were
performed using a push-pull strain gauge.

Functional examinations included tests for:

Sensorimotor functions: approach, touch, vision, audition, pain, vestibular
Autonomic functions: puppilary reflex, body temperature
Sensorimotor coordination: grip strength, landing foot splay

The observations included but were not limited to:

recumbency; ease of removal; chromorhinorrhea;
posture/gait; ease of handling; piloerection
gait abnormalities; vocalization; palpebral closure
paddling movements; Straub tail; eye prominence;
muscle tone; stereotypies; fecal consistency
activity; pupil size; urination
paralysis; click response; respiratory abnormalities
fasciculations; salivation; unkempt fur;
spasms; lacrimation; emaciation;
tremor; chromodacryorrhea; dehydration;
convulsions; rhinorrhea; distended abdomen.

Motor Activity

Motor activity was assessed after the conduct of the FOB in treatment week 12 (male animals) and 13 (female animals) as from approximately 3.5 to 4 hours after the end of the 6 h exposure period using an AMS Föhr Medical Instruments GmbH (FMI) activity measurement system.
Locomotor activity was measured quantitatively. Decreased or increased activity was recorded. Activity was recorded for 10-minute intervals over a period of 60 minutes. These data and the total activity over 60 minutes have been reported.


OTHER: no

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see below)
HISTOPATHOLOGY: Yes (see below)

Necropsy

After 13 Weeks of treatment: 28-Nov-2006 (40 males / 40 females)
All animals used during the treatment period of this study were transferred to the pathology unit on the scheduled day of necropsy (day 92), were
anaesthetised by an intraperitoneal injection of Eutha® 77 at a dose of at least 320 mg sodium pentobarbitone/kg body weight, weighed and
sacrificed by exsanguination. A complete examination was performed on all animals and all macroscopic abnormalities were described and reported.
Samples of the following tissues and organs were collected from all animals and fixed in neutral phosphate buffered 4 % formaldehyde solution,
unless otherwise stated (For paired organs, the left and the right one were examined). Lungs were instilled with this fixative at a hydrostatic pressure of 30 cm.

Adrenal glands; Aorta
Brain (cerebrum, cerebellum and brain stem);
Cecum; Colon; Duodenum
Epididymides (fixed in Bouin’s solution)
Esophagus; Extraorbital lacrimal glands
Eyes with optic nerves (fixed in Davidson’s solution)
Femur, including joint
Harderian glands (fixed in Davidson’s solution)
Heart
Ileum (including Peyer’s patches)
Jejunum (including Peyer’s patches)
Kidneys
Larynx (three transversal sections,at least levels II,
III and VI)
Liver
Lungs (infused with formalin; sections from 2 lobes)
Lymph nodes (mandibular, mesenteric, tracheobronchial)
Mammary gland area
Nasal cavities with ”paranasal sinuses”(Levels I to IV)
Nasopharyngeal duct and pharynx (1 longitudinal section)
Ovaries; Pancreas; Pituitary gland; Prostate; Rectum
Salivary glands – mandibular, sublingual
Sciatic nerve; Seminal vesicles; Skeletal muscle (thigh region)
Skin; Spinal cord - cervical, midthoracic, lumbar
Spleen; Sternum with bone marrow; Stomach
Testes (fixed in Bouin’s solution); Thymus
Thyroid gland with parathyroid gland; Tongue
Trachea (one transversal section below larynx)
Tracheal bifurcation, carina and mainstem bronchi
Urinary bladder (infused with formalin)
Uterus; Vagina
All gross lesions

Organ Weights

The following organ weights were recorded from all animals on the scheduled necropsy date and their ratios to terminal body weight and to brain
weight determined (The combined weight of paired organs was recorded):
Adrenal glands; Brain; Heart; Kidneys; Liver;
Lungs; Ovaries; Spleen; Testes; Thymus

Histotechnique / Histopathology

All organs and tissue samples collected at necropsy from all animals of Groups 1 (Air Control) and 4 (High Dose), and the lungs and nasal cavities
(Levels I to IV) of all animals of Groups 2 (Low Dose) and 3 (Mid Dose) were processed, embedded in paraffin, cut at a nominal thickness of
2-4 micrometers, stained with hematoxylin and eosin and histologically examined by use of a light microscope. Processiong and examination of the
nasal cavities was extended to the intermediate dose groups, because of treatment-related changes seen in these tissues in the high dose group. In
addition, all organs and tissues with gross lesions were processed and histologically examined. Attempts were made to correlate gross observations with microscopic findings.

Other examinations:

No

Statistics:

Statistics

The following statistical methods were used to analyze food consumption, body weight, clinical laboratory data, organ weights and ratios as well as macroscopic findings:

The Dunnett-test
[Dunnett 1955, details see Dunnett, C.W.: A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).] (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups with the air control group for each sex.

The Steel-test (many-one rank test)
[Miller 1981, details see Miller, R.G.: Simultaneous Statistical Inference, Springer Verlag, New York (1981).] was applied instead of the Dunnett-test
when the data could not be assumed to follow a normal distribution.

Fisher's exact-test
[Fisher 1950, details see Fisher, R.A.: Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950)]. Group means and standard
deviations were calculated for continuous data and medians for discrete data (scores), as appropriate.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY

Viability / Mortality
There were no premature deaths.

Clinical Signs

There were no clinical signs in test item treated animals during the present study. The only finding occasionally noted was hair loss in various body
regions in three females of the control group. This finding is occasionally seen in rats of this strain and age regardless of their treatment.

BODY WEIGHT AND WEIGHT GAIN

Body Weights

There were no statistically significant differences from controls in body weight and body weight gain at the low dose level (Group 2) throughout the
13-week treatment period. At the mid and high dose levels (Groups 3 and 4, respectively), male body weights and body weight gain were statistically
significantly lower than concurrent controls during most of the treatment period. Female body weights and, with one exception on Day 22, female
body weight gain were not statistically significantly affected by treatment with the test item. By the end of the 13-week treatment period, group mean
body weights differed from concurrent controls, see Table 3 below in “remarks on results including tables and figures”



FOOD CONSUMPTION

Food Consumption

Food Consumption and relative food consumption data were very limited, since they were recorded per cage of 5 animals and there were only 2
cages/group/sex. Therefore, and since spillage of food by individual animals cannot be ruled out, conclusions drawn from these data can only be
vague and tentative. Overall mean food consumption (g/animal/day) over the 13-week treatment period is demonstrated in Table 4, see below
(“remarks on results including tables and figures”)

Although a trend to reduced food consumption in treated animals might be concluded from these data, the change was considered to be minor in
degree.

FOOD EFFICIENCY

No clear pattern was evident in the relative food consumption data (relative to body weight; g / kg body weight / day), details see Table 5 in “remarks on results including tables and figures”

WATER CONSUMPTION

not examined

OPHTHALMOSCOPIC EXAMINATION

Ophthalmoscopic Examinations

There were no ophthalmic findings attributable to treatment with the test item.

HAEMATOLOGY

No adverse effects, details see clinical chemistry.

CLINICAL CHEMISTRY

Clinical Laboratory Investigations

There were no adverse effects on haematology, clinical biochemistry and urinalysis parameters.

A number of haematology and clinical biochemistry parameters differed statistically significantly from concurrent controls in one or three test item
treated groups. However, these differences were not considered to be toxicologically relevant, because they lacked dose relationship, were restricted to one sex and/or the respective group mean values were within the historical reference range.
The only differences from concurrent controls statistically significant in all three treated groups were slightly reduced globulin levels and slightly
increased albumin to globulin ratios. However, these changes were restricted to males and also for these parameters, all group mean values were
within the historical reference range. In addition, group mean urine volumes were lower in treated animals than in concurrent controls, but did not
attain statistical significance.

URINALYSIS

No adverse effects, details see clinical chemistry.

NEUROBEHAVIOUR

Functional Observational Battery

Functional observational battery (FOB) parameters did not appear to be affected by treatment with the test item, although the finding of Straub tail was seen in one mail and one female animal of Group 4 (High Dose). The toxicological relevance of this, rather unusual, finding remained unclear. Apart
from this, the findings noted were low in incidence, lacked dose relationship and/or were considered to be within the normal range of biological
variation.

Locomotor Activity

Intergroup comparison of the motor activity of treated animals with concurrent controls over a period of 60 minutes (analysed for 10-minute intervals) revealed statistically significantly (Steel test significant at 5%) increased motor activity in mid dose males at approximately 60 minutes,
in mid dose females at 50 and 60 minutes and for the data pooled over the total 60 minute period, and in high dose females at 50 minutes. These
increases were not attributed to treatment with the test item, because they lacked dose relationship and were evident only towards the end of the 60
minute recording period. At the low dose level, the motor activity data did not differ statistically significantly from concurrent controls throughout
the 60 minute recording period.

ORGAN WEIGHTS

After 13 weeks of treatment, terminal body weights did not differ statistically significantly from concurrent controls in males of the low dose group
(-4.7%), but were significantly lower than concurrent controls in males of the mid (-11.0%) and high dose (-12.7%) groups. Female terminal body
weights were unaffected by treatment with the test item at all dose levels. These findings were consistent with the effects on live body weight and body weight gain recorded during the treatment period. Effects on organ weight were evaluated by intergroup comparison of absolute organ weights and
their ratios to terminal body weight and to brain weight with concurrent controls. The following changes were noted:
- Absolute brain weights reduced in mid dose males (-4.3%),
- Absolute kidney weights reduced in mid dose males (-12.1%),
- Absolute lung weights reduced in mid dose males (-7.7%) and high dose males (-8.5%),
- Heart to body weight ratios increased in mid dose males (+9.3%) and high dose males (+11.7%),
- Testes to body weight ratios increased in mid dose males (+16.6%) and high dose males (+19.8%).
The reductions in absolute brain weight and kidney weight in mid dose males were considered to be incidental, because they were minor in degree,
were not dose-related and there were no confirmatory histopathology findings in the brain or kidneys in high dose animals.
The reductions in absolute lung weight and increases in heart to body weight and testes to body weight ratios in mid and high dose males were
considered be a reflection of the effects on body weight in these groups, rather than representing a primary effect of the test item on these organs.
Female absolute and relative organ weights were not affected by treatment with the test item.


GROSS PATHOLOGY

Macroscopic Findings

A number of macroscopic pathology findings were seen in male animals but none in females. These findings were considered to be within the range
of natural background findings occasionally seen in rats of this strain and age. They did not distinguish test item treated animals from concurrent
controls.


HISTOPATHOLOGY: NON-NEOPLASTIC

Microscopic Findings

The following microscopic findings in the nasal cavities were considered to distinguish test item treated rats from concurrent controls:

Nasal Cavities Level III
Intracytoplasmic hyaline droplets in olfactory epithelium,
minimal in degree in 2 low dose animals and 2 mid dose animals, and
minimal to slight in degree in 4 high dose animals.

Nasal Cavities Level IV
Intracytoplasmic hyaline droplets in olfactory and/or respiratory epithelium,
minimal to slight in degree in 5 control animals,
minimal in degree in 7 low dose animals
minimal to slight in degree in 9 mid dose animals, and
minimal to slight in degree in 14 high dose animals.
Unilateral focal degeneration of olfactory mucosa,
minimal in degree in 1 mid dose animal.
Squamous metaplasia of olfactory mucosa,
slight in degree in 1 mid dose animal, and
minimal in degree in 2 high dose animals.
Respiratory metaplasia of olfactory mucosa,
minimal in degree in 1 high dose animal.
The findings of degeneration, squamous metaplasia and respiratory metaplasia of olfactory mucosa in Level IV of the nasal cavities were considered
to represent local lesions of adverse character and were attributed to treatment with the test item.
A number of other microscopic findings noted in the present study were considered to represent findings commonly seen in rats of this strain and
age. Their incidence, distribution ormorphology did not provide any evidence for a relationship to treatment with the test item.


HISTOPATHOLOGY: NEOPLASTIC (if applicable)

not examined

HISTORICAL CONTROL DATA (if applicable)

no data

OTHER FINDINGS

No

Dose descriptor:

NOAEL

Remarks:

system

Effect level:

62.5 mg/m³ air

Sex:

male/female

Basis for effect level:

other: no adverse effects

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

10 mg/m³ air

Sex:

male/female

Basis for effect level:

other: histopathology; (organ weights)

Critical effects observed:

not specified

Table 3 - Body weight by the end of the 13-week treatment period

	Group 2 (Low Dose)	Group 3 (Mid Dose)	Group 4 (High Dose)
Males	-3.8% ns	-9.3%**	-9.3%**
Females	+1.3% ns	-0.9% ns	-4.3% ns

ns: No statistically significant difference; */** Dunnet-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 4 - Overall mean food consumption (g/animal/day) over the 13-week treatment period

	Group 1 (Air Control)	Group 2 (Low Dose)	Group 3 (Mid Dose)	Group 4 (High Dose)
Males	21.8	20.7	19.7	20.0
Females	16.2	15.4	15.0	15.1

Table 5 - Relative food consumption data (relative to body weight; g / kg body weight / day)

	Group 1 (Air Control)	Group 2 (Low Dose)	Group 3 (Mid Dose)	Group 4 (High Dose)
Males	63.1	62.1	62.5	63.4
Females	68.7	64.7	64.1	66.7

Conclusions:

The NOAEL (local) in rats exposed to METHACRYLAMIDE dust for 13 weeks is set at the lowest dose of 10 mg/m³. At higher concentrations
administered, local adverse effects as degeneration, sqamous metaplasia and respiratory metaplasia were found in nasal tissues (level IV).
In addition, a statistical significant decrease of body weight gains was observed in male animals of the mid- and high dose group.

There are no observations in this study which are assigned to systemic toxicity, especially any signs of neurotoxicity, which is a known toxic effect of METHACRYLAMIDE, are lacking. Therefore, the NOAEL (systemic) is set at the highest dose administered, 62.5 mg/m³.

Executive summary:

In a subchronic inhalation toxicity study [OECD TG 413],   METHACRYLAMIDE dust (purity 99.7 %) was administered to 40 male and 40 female wistar rats by nose only inhalation at nominal concentrations of 0, 10, 25, and 62.5  mg/m³ which is equivalent to 0, 0.01, 0.025, and 0.0625 mg/l for 6 hours per day, 5 days / week for a total of  13  weeks (65 exposures).

 The chemically determined mean aerosol concentrations were 10.24 (Group 2, Low Dose), 26.85 (Group 3, Mid Dose) or 69.95 (Group 4, High Dose) mg of METHACRYLAMIDE/m³. These aerosol concentrations were equivalent to mean aerosol concentrations of  9.45, 25.67 and 68.39 mg of METHACRYLAMIDE/m³ air in Groups 2, 3 and 4, respectively, when the attained ratios between the mean chemically determined aerosol concentrations and the respective mean gravimetric concentrations were applied to the overall mean gravimetric aerosol concentrations attained from 64 or 65 exposure days.

There were no premature deaths. Clinical signs attributable to treatment with the test item were not evident and functional observational battery (FOB) parameters did not appear to be affected by treatment with the test item. The toxicological relevance of the finding of Straub tail seen in one mail and one female animal of Group 4 (High Dose) remained unclear. Motor activity was not affected by treatment with the test item. Motor activity values on some occasions statistically significantly higher in Groups 3 (Mid Dose) and/or 4 (High Dose) than in concurrent controls were not attributed to treatment with the test item, because they lacked dose relationship and were evident only towards the end of the 60 minute recording period.

In males of Group 2 (Low Dose) and females of all treated groups relevant adverse effects on body weight and body weight gain were not evident. In males of Groups 3 (Mid Dose) and 4 (High Dose) body weight and body weight gain statistically significantly lower than concurrent controls during most of the treatment period were attributed to treatment with the test item. Absolute lung weight significantly lower and heart to body weight and testes to body weight ratios significantly higher in males of Groups 3 and 4 than in concurrent controls were considered to be a reflection of the effects on body weight in these groups, rather than representing a primary effect of the test item on these organs.

The findings of degeneration, squamous metaplasia and/or respiratory metaplasia of olfactory mucosa in Level IV of the nasal cavities affecting one female animal in Group 3 (Mid Dose) and two females in Group 4 (High Dose) were considered to represent local lesions of adverse character and were attributed to treatment with the test item. The minor increases in incidence and/or severity of intracytoplasmic hyaline droplets in nasal cavity levels III and IV were also attributed to treatment with the test item, but were not considered to be of adverse character, as this finding was also present in nasal cavity Level IV in Group 1 (Air Control).

The no-observed-adverse-effect-level for local effects [NOAEL(local)] was set at the low dose level administered, 10 mg/m³, because at the mid and high dose levels the findings of degeneration, squamous metaplasia and/or respiratory metaplasia of olfactory mucosa were evident in nasal cavity level IV and male body weight and body weight gain were statistically significantly lower than concurrent controls during most of the treatment period. However, the effects on body weight at the mid and high dose levels were considered to be only moderate in degree, as they were restricted to one gender, relevant body weight loss was not evident during the study, and by the end of the 13 week treatment period the group mean body weights of mid and high dose male animals were only 9.3% lower than that of concurrent control animals.

There were no changes observed in organs, tissues or other parameters investigated which were attributed to systemic toxicity. Especially, any evidence for neurotoxicity, which is the known critical effect of methacrylamide when administered at higher doses in oral and dermal studies, was lacking.

 

Therefore, the no-observed-adverse-effect-level for systemic effects [NOAEL(systemic)] was set at the high dose level administered, 62.5 mg/m³.

This subchronic inhalation toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic inhalation study OECD 413 in the rat. 

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

22-Aug-2006 to 14-Aug-2008

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.

Justification for type of information:

Please refer to the CSR for the read-across rationale.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

adopted May 12, 1981

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
- Rat strain: HanRCC: WIST(SPF)
- Source: RCC Ltd., Füllingsdorf, Switzerland
- Age at study initiation: 8 weeks (males), 11 weeks (females)
- Weight at study initiation: males: 211.1 to 236.2 g
females: 184.0 to 211.4 g
- Fasting period before study:
- Housing: Groups of 5 in Makrolon type-4 cages with wire mesh tops and standard softwood bedding ("Lignocel", Schill AG, Muttenz, Switzerland).
- Diet (e.g. ad libitum): ad libitum, pelleted standard Kliba 3433, Batch 23/06
- Water (e.g. ad libitum): ad libitum access to tap water
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hr artificial flourescent light/ 12 hr dark, music/light period.

Route of administration:

inhalation

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: The particle size (expressed as MMAD) of the generated Methacrylamide aerosols was well within the ideal range of 1 to 3 µm in each treatment group. Details on particle size characteristics see below, Table 2 in section “Any other information on materials and methods incl. tables”

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:
Inhalation exposure was performed using a system similar to that originally described by Sachsse et al. (details see: K. Sachsse, L. Ullmann, G. Voss
and R. Hess: Measurements of Inhalation Toxicity of Aerosols in Small Laboratory Animals. In: Proceedings of the Europ. Soc. for the Study of Drug
Toxicity, Vol. XV, pp. 239-251, Zürich, June 1973.)
The design of this exposure system is based upon the fluid dynamic modelling of the aerosol flow. It ensures a uniform test item distribution,
provides a constant stream of "fresh" test item to each animal, and precludes re-breathing the exhaled air. The aerosol stream reaches the animal's
nose through ports situated at three different levels around the axis of the exposure chamber. Each level has 16 animal ports and can be rotated
allowing close observation of all the animals without interruption of exposure. In the present study the exposure chambers of groups 1 (air control)
and 3 (mid-dose) comprised three levels of animal ports. For the exposure of groups 2 (low-dose) and 4 (high-dose) chambers comprising five
levels of animal ports were utilized. All animals were placed on the top two levels during each exposure, thus minimising variation in exposure
conditions between the animals of any particular group.

- Method of holding animals in test chamber:
The animals were confined separately in Makrolon* restraint tubes which were positioned radially around the nose-only, flow-past exposure chamber (details see: Cannon, W.C., E.F. Blanton and K.E. McDonald: "The Flow-Past Chamber: An Improved Nose-Only Exposure System for Rodents", Am. Ind. Hyg. Assoc. J., 44 (12): 923-928, 1983.)

- Source and rate of air:

The design of this exposure system precludes re-breathing the exhaled air, details see above (Exposure apparatus).


- Method of conditioning air:
Oxygen Concentration
The oxygen concentration of the control and test atmospheres were monitored continuously during each 6-hour exposure period for each group
(Groups 1 to 4 inclusive using an oxygen sensor (VarioGard Transmitter 4...20 mA from Dräger AG, 8305 Dietlikon / Switzerland) connected to a data logger. In addition, the oxygen concentration was noted manually from the data logger for each group at appropriate intervals after the beginning of the daily exposure (three times during each 6-hour exposure period / group / day).


- System of generating particulates/aerosols:
Test Aerosol Generation
For each dose group (Groups 2, 3 and 4), the pre-milled test item was aerosolised using a piston feed/rotating brush aerosol generator followed by a micronising jet mill (see diagrams below). Then the aerosol output from the jet mill was discharged through a 63Ni charge neutraliser. For Groups 2
(Low Dose) and 4 (High Dose) the discharged aerosol was split, one portion of it being used for the exposure of Group 4 without further dilution, the other portion of it being further diluted with filtered dry air using an air vac device. For Group 3 (Mid Dose) the test aerosol was generated in a similar manner as for Group 4, but separately from that generated for Groups 2 and 4 without using an air vac device. In order to minimize the variation in
aerosol concentration over each exposure and remain close to the target aerosol concentrations, the piston advance speed was adjusted, as deemed necessary from the daily monitoring of aerosol concentration. The above generation and/or dilution system was chosen to achieve the required test
item concentrations. Animals of group 1 (Air Control) were exposed to compressed filtered air alone at conditions similar to those used for Groups 2 (Low Dose), 3 (Mid Dose) and 4 (High Dose).


- Temperature, humidity, pressure in air chamber:
Temperature / Relative Humidity
The temperature and relative humidity of the control and test atmospheres were monitored continuously during each 6-hour exposure period for
each group (Groups 1 to 4 inclusive) using a ROTRONIC Hygrometer (Series I-200, Rotronic AG, 8040 Zürich / Switzerland) connected to a data
logger. In addition, the temperature and relative humidity were noted manually from the data logger for each group at appropriate intervals after the
beginning of the daily exposure (three times during each 6-hour exposure period / group / day).


- Air flow rate:
Exposure Airflow Rate
The airflow rate through the exposure chambers was adjusted before inhalation exposure and monitored indirectly during each exposure through
the generation and/or dilution system using calibrated pressure gauges and/or flowmeters and were checked at least at the same intervals as the
temperature, relative humidity and oxygen concentration of the control and test atmospheres. In all dose groups, a proportion of the ports on each
exposure chamber were closed such that the airflow to each open port was approximately 1.0 L/minute/animal port during each exposure.


- Air change rate:
The design of this exposure system precludes re-breathing the exhaled air, details see above (Exposure apparatus).


- Method of particle size determination:
Particle Size Distributions and Mass Median Aerodynamic Diameters
The distribution of particle size in the generated aerosols was measured by gravimetry six times in Group 2 (Low Dose) and four times in each of
groups 3 (Mid Dose) and 4 (High Dose) during the 13-week treatment period, using a Mercer 7-stage cascade impactor (Model 02-130, In-Tox.
Products Inc. Albuquerque, New Mexico, USA). Representative samples of the test atmosphere were drawn through the impactor with a flow rate of 1.0 L/min and the particles deposited according to their aerodynamic size onto stainless steel slips and the final filter stage (Type HVLP, Polyvinylidene-
difluoride membrane, pore size 0.45 m), on each stage of the impactor. To obtain the mass deposited on each stage of the impactor, the steel slips
and the final filter stage were weighed before and after sampling using an appropriate analytical balance. For each measurement in Group 2, aerosol
was collected in the impactors over three to six consecutive treatment days, in order to accumulate sufficient particulate material in the impactor for
adequate aerodynamic particle size measurement. On the corresponding results table of gravimetric particle size data only the last sampling date is
given. For the measurements in Groups 3 and 4 sufficient test item accumulated within one treatment day. The total mass (g) deposited in the impactor was then calculated by adding together the mass deposited on each of the stainless steel slips and the final filter stage. As the Effective Cut-off
Diameters (ECD) represent the lower size limit of the particles collected on each stage, the percentages less than the indicated size were tabulated as a function of the ECD. The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) of the test aerosols were calculated on the basis of the gravimetric results from the impactors applying the built-in PROBIT function of Microsoft Excel Software. The target range for the mass median aerodynamic diameter was 1 to 3 μm. The gravimetric determination of particle size distribution is only indicative of the nonevaporated phase of the test item. Any vapour component or any fraction which might evaporate or sublimate are not accounted for.



- Treatment of exhaust air:

Re-breathing of exhaust air is precluded, deteis see above (exposure apparatus).


TEST ATMOSPHERE

- Brief description of analytical method used:
Analytical Concentrations
Sampling of test atmosphere for the determination of concentrations by chemical analysis was performed at least once weekly during most of a
6-hour inhalation exposure period in each of Groups 2 to 4 inclusive. The dust fraction of the test atmospheres was collected on Millipore® durapore filters, Type HVLP, the vapour fraction was trapped in phosphoric acid (0.033 molar). On each sampling occasion, the test atmosphere was drawn
through such a filter which was followed by two wash-bottles placed in series each containing approximately 80 mL of phosphoric acid (0.033 molar), cooled in water ice. Immediately after each atmosphere sampling and weighing, the filter was put into a light protected glass vial and covered with
10 g of phosphoric acid (0.033 molar) to minimise loss of test item by sublimation from the filter. The content of each wash-bottle was transferred
into appropriate glass flasks, the wash-bottles were rinsed with phosphoric acid (0.033 molar), and the flasks made up to 100 ml with the rinsing and tightly closed.
The filters and solutions were kept light protected at approximately +5 °C in a cool box until dispatch at this temperature to the analytical laboratory of RCC Ltd, for chemical analysis. The samples were analysed by high performance liquid chromatography (HPLC) with UV detection using the test
item, METHACRYLAMIDE (as received from the Sponsor), as reference item for the analytical calibration curves. The analytical method was based on
that which was provided by the Sponsor. An analytical phase report was provided for inclusion in the report of this study.


- Samples taken from breathing zone: yes
Test atmosphere samples were collected directly from the delivery tube in the breathing zone of the animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean chemically determined aerosol concentrations of METHACRYLAMIDE and corresponding mean gravimetrically determined aerosol
concentrations are summarised below, see Table 1 in section “Any other information on materials and methods incl. tables”


Exposure System Monitoring

The atmosphere concentrations of the test item determined gravimetrically (dust fraction, trapped on filters) and chemically (including dust fraction
trapped of filters and vapour fraction trapped in solvent), particle size distribution determined gravimetrically, relative humidity, temperature
and oxygen concentration, were measured on test atmosphere samples collected directly from the delivery tube in the breathing zone of the animals, at an empty sampling port of the exposure chamber, delivering "fresh" test item to the animal's nose. The position at which these test atmosphere
samples were taken has been considered to be representative for the breathing zone of the animals. This approach was chosen in order to obtain
representative samples of what was delivered to the animals. Airflow rates were measured during the collection of samples for the determination of
aerosol concentrations using dry-test meters and pressure gauges, calibrated with a reference dry-test meter. Airflow rates for particle size samples were calibrated using a Gilibrator bubble flow generator.

Nominal Determination of Aerosol Concentrations

Based on the proportion of primary (prior to further dilution) aerosol assigned to Group 2 (Low Dose) the quantity of test item assigned to this group was calculated and then divided by the sum of primary airflow volume assigned to Group 2 plus airflow used for further aerosol dilution to
give the nominal concentration of test item in the aerosol delivered to the animals of this group. For Group 3 (Mid Dose), the nominal concentration
was measured by weighing the generator cylinder containing the test item before and after each exposure to determine the quantity of test item used. The weight of test item used was then divided by the total airflow volume to give the nominal concentration. For Group 4 (High Dose), the nominal
concentration was measured by weighing the generator cylinder containing the test item before and after each exposure to determine the quantity of test item used for generation of the primary (prior to further dilution) aerosol. The weight of test item used was then divided by the total airflow
volume used for the jet mill, i.e. used for the primary aerosol (prior to further dilution), to give the nominal concentration of test item in the aerosol
delivered to the animals of this group.

Gravimetric Determination of Aerosol Concentrations

Gravimetric determinations of aerosol concentration (dust fraction) in the generated atmospheres were performed using a Millipore® durapore filter
(Type HVLP, Polyvinylidenedifluoride membrane, pore size 0.45 µm), loaded in a 47 mm in-line stainless steel filter sampling device.
The gravimetric sampling was performed once daily for each of groups 2 (Low Dose), 3 (Mid Dose) and 4 (High Dose) sampling aerosol during most
of the 6-hour exposure period. In addition, on a number of occasions during the study for each of these groups, a further three filter samples were
taken over shorter periods shortly after the beginning, at about the middle and towards the end of the 6-hour exposure period to give an indication
of the stability of the aerosol concentration over exposure time. Additional aerosol samples were taken for monitoring purposes as considered
necessary and the corresponding aerosol concentration data were retained in the raw data without reporting.
Technical atmosphere generation trials (not performed under GLP) led to the conclusion that in the present study the gravimetrically determined
concentrations were only approximate estimates of the true concentrations of the test atmosphere, because of sublimation losses of
METHACRYLAMIDE. Therefore, one purpose of the gravimetric determination of the aerosol concentrations was to monitor roughly the aerosol
concentration during the exposure period, as the test atmosphere concentrations (dust and vapour fraction) determined by chemical analysis were
only available post exposure. The gravimetric determination of aerosol concentration is only indicative of the non-evaporated phase of the test item. Any vapour component or any fraction which might evaporate or sublimate is not accounted for in the gravimetrically determined aerosol
concentration values.

Analytical Concentrations

Sampling of test atmosphere for the determination of concentrations by chemical analysis was performed at least once weekly during most of a
6-hour inhalation exposure period in each of Groups 2 to 4 inclusive. The dust fraction of the test atmospheres was collected on Millipore® durapore filters, Type HVLP, the vapour fraction was trapped in phosphoric acid (0.033 molar). On each sampling occasion, the test atmosphere was drawn
through such a filter which was followed by two wash-bottles placed in series each containing approximately 80 mL of phosphoric acid (0.033 molar), cooled in water ice. Immediately after each atmosphere sampling and weighing, the filter was put into a light protected glass vial and covered with
10 g of phosphoric acid (0.033 molar) to minimise loss of test item by sublimation from the filter. The content of each wash-bottle was transferred
into appropriate glass flasks, the wash-bottles were rinsed with phosphoric acid (0.033 molar), and the flasks made up to 100 ml with the rinsing and tightly closed. The filters and solutions were kept light protected at approximately +5 °C in a cool box until dispatch at this temperature to the
analytical laboratory of RCC Ltd, for chemical analysis. The samples were analysed by high performance liquid chromatography (HPLC) with
UV detection using the test item, METHACRYLAMIDE, as reference item for the analytical calibration curves.

Duration of treatment / exposure:

6 hours daily.

Frequency of treatment:

Once daily, 5 days per week for a total of 13 consecutive weeks.

Remarks:

Doses / Concentrations:
Test concentrations: 0 mg/m³; 10 mg/m³ ; 25 mg/m³; 62.5 mg/m³
Basis:
nominal conc.

No. of animals per sex per dose:

Group 1 – Air control: 10 Males and 10 Females
Group 2 – Low Dose: 10 Males and 10 Females
Group 3 – Mid Dose: 10 Males and 10 Females
Group 4 – High Dose: 10 Males and 10 Females

Control animals:

yes

Details on study design:

- Dose selection rationale:
The above target concentrations were chosen on the basis of the results attained in a 2-Week inhalation and neurotoxicity study in the rat via
nose-only exposures.

Rationale for animal assignment:
Selection and Randomization: The animals were allocated to groups by a body weight stratification procedure on the first day of acclimatisation
to ensure that differences in group mean body weights are minimized.


Rationale for special In-Life Observations:
Functional Observational Battery (FOB) and Motor Activity were part of the in-life observations as the critical toxic effect for methacrylamide is
neurotoxicity.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

Viability / Mortality

All animals were observed for mortality/moribundity once daily during the acclimatisation period (including the reserve animals), and twice daily,
before and after exposure, during the treatment period of the study. On weekend days of the treatment period, when there was no exposure of
animals, mortality was checked at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes

Clinical Signs – Time schedule

Clinical signs were recorded once during the acclimatisation period (including the reserve animals) and twice daily during the treatment period of the study (once pre-exposure and once post exposure outside the restraint tubes on all animals, except the reserve animals). On weekend
days of the treatment period, when there was no exposure of animals, clinical examinations were performed once daily.
During exposure, only grossly abnormal signs could be observed, as the animals were in restraint tubes. Observations were detailed and carefully
recorded using explicitly defined scales as appropriate. Observations included but were not limited to changes in behaviour, somatomotor
activity, body position, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, e.g. tremors or
convulsions, reactivity to handling or sensory stimuli, altered strength, alteration of the skin, fur, nose, eyes and mucous membranes.

BODY WEIGHT: Yes

Body Weights – Time schedule

Each animal was weighed at 7-day intervals (if appropriate), during the acclimatisation and treatment periods of the study. Reserve animals were also weighed during acclimatisation. During the treatment period the body weights were recorded before the exposure on the particular day.

FOOD CONSUMPTION:

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

Food consumption was recorded weekly per cage of five animals (reserve animals exclusive), over a 7-day period (if possible and/or appropriate),
during the acclimatisation and treatment periods of the study. The food consumption was calculated per cage and per food consumption interval. It expresses the average food consumed per animal and per day for each cage over the food consumption interval.

FC = C / AD

where
FC is food consumption (food fed - food left) in grams of food per animal and day,
C is measured food consumption in grams per cage over the consumption interval, and
AD is total consumption days over all animals in the cage during the consumption interval.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

The relative food consumption was calculated per cage at each time interval according to the
following formula:

RFC = [ FC/BW(i)] x 1000

where
RFC is relative food consumption, i.e. food consumption in grams of food per kg bodyweight and day,
FC is food consumption (food fed - food left) in grams of food per animal and day, and
BW(i) is the optimal (on day closest to the middle of food consumption period) body weight in grams.


WATER CONSUMPTION: Not recorded

OPHTHALMOSCOPIC EXAMINATION: Yes

Ophthalmoscopic Examinations – Time schedule – Dose Groups:

Ophthalmoscopic examinations were performed in all animals once during the acclimatisation period (reserve animals inclusive), and in all surviving animals once during treatment week 13. Method/Instrumentation: The observations were performed after instillation of a mydriaticum,
using a Heine Bifocal Type Miroflex ophthalmoscope (Eisenhut Vet. AG, 4123 Allschwil /Switzerland).
Dates: Acclimatisation: 24-Aug-2006
Treatment Period (Week 13): 24-Nov-2006
For unilateral findings unless otherwise indicated in the tables, the contralateral eye was without abnormalities.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 13 weeks of treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: All animals

Clinical Laboratory Investigations

Blood samples for hematology and clinical biochemistry were collected from the retro-orbital plexus from all animals (10 animals/sex/group) under
light isoflurane anaesthesia. This was followed by necropsy on the same day. The animals were fasted in metabolism cages for approximately 17 hours
before blood sampling, but water was provided ad libitum. The blood samples were collected early in the working day to reduce biological variation
caused by circadian rhythms. Date of blood sampling: After 13 weeks of treatment: 28-Nov-2006
In the summary and individual tables the names of some parameters have been abbreviated.
Clinical laboratory data are expressed, with a few exceptions, in general accordance with the International System of Units (SI).

Hematology parameters determined

Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Reticulocyte count
Reticulocyte maturity index
Total leukocyte count
Differential leukocyte count
Platelet count
Coagulation:
Prothrombin time
Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 13 weeks of treatment
- Animals fasted: Yes
- How many animals: All animals

Clinical Biochemistry parameters determined:

Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Phospholipids
Aspartate aminotransferase
Alanine aminotransferase
Lactate dehydrogenase
Glutamate dehydrogenase
Alkaline phosphatase
Gamma-glutamyl-transferase
Creatine kinase
Sodium
Potassium
Chloride
Calcium
Phosphorus inorganic
Protein, total
Albumin
Globulin
Albumin/Globulin ratio


URINALYSIS: Yes
- Time schedule for collection of urine: after 13 weeks of treatment

Date of urine sampling: After 13 weeks of treatment: 28-Nov-2006
Urine was collected during an approximately 16-hour fasting period into specimen vials using a metabolism cage.

- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

Urinalysis parameters determined:

Volume
Relative density
Colour
Appearance
pH
Protein
Nitrite
Glucose
Ketone
Urobilinogen
Bilirubin
Erythrocytes
Leukocytes


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: see below
- Dose groups that were examined: see below
- Battery of functions tested: sensory activity / grip strength / motor activity / other: see below

Functional Observation Battery (FOB)

The FOB observations were performed during treatment weeks 12 (male animals) and 13 (female animals) as from approximately two hours after the
end of the 6-hour exposure period and included hind- and forelimb grip strength, landing foot splay and Preyer’s reflex. Detailed clinical
observations were evaluated in the FOB. The FOB was conducted on all animals, as the animals were not considered to otherwise reveal signs of
toxicity to an extent that would significantly have interfered with the functional test performance. Relevant parameters from a modified Irwin screen
test were performed on all rats. Any abnormal findings were recorded and graded in severity. Hind- and forelimb grip strength measurements were
performed using a push-pull strain gauge.

Functional examinations included tests for:

Sensorimotor functions: approach, touch, vision, audition, pain, vestibular
Autonomic functions: puppilary reflex, body temperature
Sensorimotor coordination: grip strength, landing foot splay

The observations included but were not limited to:

recumbency; ease of removal; chromorhinorrhea;
posture/gait; ease of handling; piloerection
gait abnormalities; vocalization; palpebral closure
paddling movements; Straub tail; eye prominence;
muscle tone; stereotypies; fecal consistency
activity; pupil size; urination
paralysis; click response; respiratory abnormalities
fasciculations; salivation; unkempt fur;
spasms; lacrimation; emaciation;
tremor; chromodacryorrhea; dehydration;
convulsions; rhinorrhea; distended abdomen.

Motor Activity

Motor activity was assessed after the conduct of the FOB in treatment week 12 (male animals) and 13 (female animals) as from approximately 3.5 to 4 hours after the end of the 6 h exposure period using an AMS Föhr Medical Instruments GmbH (FMI) activity measurement system.
Locomotor activity was measured quantitatively. Decreased or increased activity was recorded. Activity was recorded for 10-minute intervals over a period of 60 minutes. These data and the total activity over 60 minutes have been reported.


OTHER: no

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see below)
HISTOPATHOLOGY: Yes (see below)

Necropsy

After 13 Weeks of treatment: 28-Nov-2006 (40 males / 40 females)
All animals used during the treatment period of this study were transferred to the pathology unit on the scheduled day of necropsy (day 92), were
anaesthetised by an intraperitoneal injection of Eutha® 77 at a dose of at least 320 mg sodium pentobarbitone/kg body weight, weighed and
sacrificed by exsanguination. A complete examination was performed on all animals and all macroscopic abnormalities were described and reported.
Samples of the following tissues and organs were collected from all animals and fixed in neutral phosphate buffered 4 % formaldehyde solution,
unless otherwise stated (For paired organs, the left and the right one were examined). Lungs were instilled with this fixative at a hydrostatic pressure of 30 cm.

Adrenal glands; Aorta
Brain (cerebrum, cerebellum and brain stem);
Cecum; Colon; Duodenum
Epididymides (fixed in Bouin’s solution)
Esophagus; Extraorbital lacrimal glands
Eyes with optic nerves (fixed in Davidson’s solution)
Femur, including joint
Harderian glands (fixed in Davidson’s solution)
Heart
Ileum (including Peyer’s patches)
Jejunum (including Peyer’s patches)
Kidneys
Larynx (three transversal sections,at least levels II,
III and VI)
Liver
Lungs (infused with formalin; sections from 2 lobes)
Lymph nodes (mandibular, mesenteric, tracheobronchial)
Mammary gland area
Nasal cavities with ”paranasal sinuses”(Levels I to IV)
Nasopharyngeal duct and pharynx (1 longitudinal section)
Ovaries; Pancreas; Pituitary gland; Prostate; Rectum
Salivary glands – mandibular, sublingual
Sciatic nerve; Seminal vesicles; Skeletal muscle (thigh region)
Skin; Spinal cord - cervical, midthoracic, lumbar
Spleen; Sternum with bone marrow; Stomach
Testes (fixed in Bouin’s solution); Thymus
Thyroid gland with parathyroid gland; Tongue
Trachea (one transversal section below larynx)
Tracheal bifurcation, carina and mainstem bronchi
Urinary bladder (infused with formalin)
Uterus; Vagina
All gross lesions

Organ Weights

The following organ weights were recorded from all animals on the scheduled necropsy date and their ratios to terminal body weight and to brain
weight determined (The combined weight of paired organs was recorded):
Adrenal glands; Brain; Heart; Kidneys; Liver;
Lungs; Ovaries; Spleen; Testes; Thymus

Histotechnique / Histopathology

All organs and tissue samples collected at necropsy from all animals of Groups 1 (Air Control) and 4 (High Dose), and the lungs and nasal cavities
(Levels I to IV) of all animals of Groups 2 (Low Dose) and 3 (Mid Dose) were processed, embedded in paraffin, cut at a nominal thickness of
2-4 micrometers, stained with hematoxylin and eosin and histologically examined by use of a light microscope. Processiong and examination of the
nasal cavities was extended to the intermediate dose groups, because of treatment-related changes seen in these tissues in the high dose group. In
addition, all organs and tissues with gross lesions were processed and histologically examined. Attempts were made to correlate gross observations with microscopic findings.

Other examinations:

No

Statistics:

Statistics

The following statistical methods were used to analyze food consumption, body weight, clinical laboratory data, organ weights and ratios as well as macroscopic findings:

The Dunnett-test
[Dunnett 1955, details see Dunnett, C.W.: A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).] (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups with the air control group for each sex.

The Steel-test (many-one rank test)
[Miller 1981, details see Miller, R.G.: Simultaneous Statistical Inference, Springer Verlag, New York (1981).] was applied instead of the Dunnett-test
when the data could not be assumed to follow a normal distribution.

Fisher's exact-test
[Fisher 1950, details see Fisher, R.A.: Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950)]. Group means and standard
deviations were calculated for continuous data and medians for discrete data (scores), as appropriate.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY

Viability / Mortality
There were no premature deaths.

Clinical Signs

There were no clinical signs in test item treated animals during the present study. The only finding occasionally noted was hair loss in various body
regions in three females of the control group. This finding is occasionally seen in rats of this strain and age regardless of their treatment.

BODY WEIGHT AND WEIGHT GAIN

Body Weights

There were no statistically significant differences from controls in body weight and body weight gain at the low dose level (Group 2) throughout the
13-week treatment period. At the mid and high dose levels (Groups 3 and 4, respectively), male body weights and body weight gain were statistically
significantly lower than concurrent controls during most of the treatment period. Female body weights and, with one exception on Day 22, female
body weight gain were not statistically significantly affected by treatment with the test item. By the end of the 13-week treatment period, group mean
body weights differed from concurrent controls, see Table 3 below in “remarks on results including tables and figures”



FOOD CONSUMPTION

Food Consumption

Food Consumption and relative food consumption data were very limited, since they were recorded per cage of 5 animals and there were only 2
cages/group/sex. Therefore, and since spillage of food by individual animals cannot be ruled out, conclusions drawn from these data can only be
vague and tentative. Overall mean food consumption (g/animal/day) over the 13-week treatment period is demonstrated in Table 4, see below
(“remarks on results including tables and figures”)

Although a trend to reduced food consumption in treated animals might be concluded from these data, the change was considered to be minor in
degree.

FOOD EFFICIENCY

No clear pattern was evident in the relative food consumption data (relative to body weight; g / kg body weight / day), details see Table 5 in “remarks on results including tables and figures”

WATER CONSUMPTION

not examined

OPHTHALMOSCOPIC EXAMINATION

Ophthalmoscopic Examinations

There were no ophthalmic findings attributable to treatment with the test item.

HAEMATOLOGY

No adverse effects, details see clinical chemistry.

CLINICAL CHEMISTRY

Clinical Laboratory Investigations

There were no adverse effects on haematology, clinical biochemistry and urinalysis parameters.

A number of haematology and clinical biochemistry parameters differed statistically significantly from concurrent controls in one or three test item
treated groups. However, these differences were not considered to be toxicologically relevant, because they lacked dose relationship, were restricted to one sex and/or the respective group mean values were within the historical reference range.
The only differences from concurrent controls statistically significant in all three treated groups were slightly reduced globulin levels and slightly
increased albumin to globulin ratios. However, these changes were restricted to males and also for these parameters, all group mean values were
within the historical reference range. In addition, group mean urine volumes were lower in treated animals than in concurrent controls, but did not
attain statistical significance.

URINALYSIS

No adverse effects, details see clinical chemistry.

NEUROBEHAVIOUR

Functional Observational Battery

Functional observational battery (FOB) parameters did not appear to be affected by treatment with the test item, although the finding of Straub tail was seen in one mail and one female animal of Group 4 (High Dose). The toxicological relevance of this, rather unusual, finding remained unclear. Apart
from this, the findings noted were low in incidence, lacked dose relationship and/or were considered to be within the normal range of biological
variation.

Locomotor Activity

Intergroup comparison of the motor activity of treated animals with concurrent controls over a period of 60 minutes (analysed for 10-minute intervals) revealed statistically significantly (Steel test significant at 5%) increased motor activity in mid dose males at approximately 60 minutes,
in mid dose females at 50 and 60 minutes and for the data pooled over the total 60 minute period, and in high dose females at 50 minutes. These
increases were not attributed to treatment with the test item, because they lacked dose relationship and were evident only towards the end of the 60
minute recording period. At the low dose level, the motor activity data did not differ statistically significantly from concurrent controls throughout
the 60 minute recording period.

ORGAN WEIGHTS

After 13 weeks of treatment, terminal body weights did not differ statistically significantly from concurrent controls in males of the low dose group
(-4.7%), but were significantly lower than concurrent controls in males of the mid (-11.0%) and high dose (-12.7%) groups. Female terminal body
weights were unaffected by treatment with the test item at all dose levels. These findings were consistent with the effects on live body weight and body weight gain recorded during the treatment period. Effects on organ weight were evaluated by intergroup comparison of absolute organ weights and
their ratios to terminal body weight and to brain weight with concurrent controls. The following changes were noted:
- Absolute brain weights reduced in mid dose males (-4.3%),
- Absolute kidney weights reduced in mid dose males (-12.1%),
- Absolute lung weights reduced in mid dose males (-7.7%) and high dose males (-8.5%),
- Heart to body weight ratios increased in mid dose males (+9.3%) and high dose males (+11.7%),
- Testes to body weight ratios increased in mid dose males (+16.6%) and high dose males (+19.8%).
The reductions in absolute brain weight and kidney weight in mid dose males were considered to be incidental, because they were minor in degree,
were not dose-related and there were no confirmatory histopathology findings in the brain or kidneys in high dose animals.
The reductions in absolute lung weight and increases in heart to body weight and testes to body weight ratios in mid and high dose males were
considered be a reflection of the effects on body weight in these groups, rather than representing a primary effect of the test item on these organs.
Female absolute and relative organ weights were not affected by treatment with the test item.


GROSS PATHOLOGY

Macroscopic Findings

A number of macroscopic pathology findings were seen in male animals but none in females. These findings were considered to be within the range
of natural background findings occasionally seen in rats of this strain and age. They did not distinguish test item treated animals from concurrent
controls.


HISTOPATHOLOGY: NON-NEOPLASTIC

Microscopic Findings

The following microscopic findings in the nasal cavities were considered to distinguish test item treated rats from concurrent controls:

Nasal Cavities Level III
Intracytoplasmic hyaline droplets in olfactory epithelium,
minimal in degree in 2 low dose animals and 2 mid dose animals, and
minimal to slight in degree in 4 high dose animals.

Nasal Cavities Level IV
Intracytoplasmic hyaline droplets in olfactory and/or respiratory epithelium,
minimal to slight in degree in 5 control animals,
minimal in degree in 7 low dose animals
minimal to slight in degree in 9 mid dose animals, and
minimal to slight in degree in 14 high dose animals.
Unilateral focal degeneration of olfactory mucosa,
minimal in degree in 1 mid dose animal.
Squamous metaplasia of olfactory mucosa,
slight in degree in 1 mid dose animal, and
minimal in degree in 2 high dose animals.
Respiratory metaplasia of olfactory mucosa,
minimal in degree in 1 high dose animal.
The findings of degeneration, squamous metaplasia and respiratory metaplasia of olfactory mucosa in Level IV of the nasal cavities were considered
to represent local lesions of adverse character and were attributed to treatment with the test item.
A number of other microscopic findings noted in the present study were considered to represent findings commonly seen in rats of this strain and
age. Their incidence, distribution ormorphology did not provide any evidence for a relationship to treatment with the test item.


HISTOPATHOLOGY: NEOPLASTIC (if applicable)

not examined

HISTORICAL CONTROL DATA (if applicable)

no data

OTHER FINDINGS

No

Dose descriptor:

NOAEL

Remarks:

system

Effect level:

62.5 mg/m³ air

Sex:

male/female

Basis for effect level:

other: no adverse effects

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

10 mg/m³ air

Sex:

male/female

Basis for effect level:

other: histopathology; (organ weights)

Critical effects observed:

not specified

Table 3 - Body weight by the end of the 13-week treatment period

	Group 2 (Low Dose)	Group 3 (Mid Dose)	Group 4 (High Dose)
Males	-3.8% ns	-9.3%**	-9.3%**
Females	+1.3% ns	-0.9% ns	-4.3% ns

ns: No statistically significant difference; */** Dunnet-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 4 - Overall mean food consumption (g/animal/day) over the 13-week treatment period

	Group 1 (Air Control)	Group 2 (Low Dose)	Group 3 (Mid Dose)	Group 4 (High Dose)
Males	21.8	20.7	19.7	20.0
Females	16.2	15.4	15.0	15.1

Table 5 - Relative food consumption data (relative to body weight; g / kg body weight / day)

	Group 1 (Air Control)	Group 2 (Low Dose)	Group 3 (Mid Dose)	Group 4 (High Dose)
Males	63.1	62.1	62.5	63.4
Females	68.7	64.7	64.1	66.7

Conclusions:

The NOAEL (local) in rats exposed to METHACRYLAMIDE dust for 13 weeks is set at the lowest dose of 10 mg/m³. At higher concentrations
administered, local adverse effects as degeneration, sqamous metaplasia and respiratory metaplasia were found in nasal tissues (level IV).
In addition, a statistical significant decrease of body weight gains was observed in male animals of the mid- and high dose group.

There are no observations in this study which are assigned to systemic toxicity, especially any signs of neurotoxicity, which is a known toxic effect of METHACRYLAMIDE, are lacking. Therefore, the NOAEL (systemic) is set at the highest dose administered, 62.5 mg/m³.

Executive summary:

In a subchronic inhalation toxicity study [OECD TG 413],   METHACRYLAMIDE dust (purity 99.7 %) was administered to 40 male and 40 female wistar rats by nose only inhalation at nominal concentrations of 0, 10, 25, and 62.5  mg/m³ which is equivalent to 0, 0.01, 0.025, and 0.0625 mg/l for 6 hours per day, 5 days / week for a total of  13  weeks (65 exposures).

 The chemically determined mean aerosol concentrations were 10.24 (Group 2, Low Dose), 26.85 (Group 3, Mid Dose) or 69.95 (Group 4, High Dose) mg of METHACRYLAMIDE/m³. These aerosol concentrations were equivalent to mean aerosol concentrations of  9.45, 25.67 and 68.39 mg of METHACRYLAMIDE/m³ air in Groups 2, 3 and 4, respectively, when the attained ratios between the mean chemically determined aerosol concentrations and the respective mean gravimetric concentrations were applied to the overall mean gravimetric aerosol concentrations attained from 64 or 65 exposure days.

There were no premature deaths. Clinical signs attributable to treatment with the test item were not evident and functional observational battery (FOB) parameters did not appear to be affected by treatment with the test item. The toxicological relevance of the finding of Straub tail seen in one mail and one female animal of Group 4 (High Dose) remained unclear. Motor activity was not affected by treatment with the test item. Motor activity values on some occasions statistically significantly higher in Groups 3 (Mid Dose) and/or 4 (High Dose) than in concurrent controls were not attributed to treatment with the test item, because they lacked dose relationship and were evident only towards the end of the 60 minute recording period.

In males of Group 2 (Low Dose) and females of all treated groups relevant adverse effects on body weight and body weight gain were not evident. In males of Groups 3 (Mid Dose) and 4 (High Dose) body weight and body weight gain statistically significantly lower than concurrent controls during most of the treatment period were attributed to treatment with the test item. Absolute lung weight significantly lower and heart to body weight and testes to body weight ratios significantly higher in males of Groups 3 and 4 than in concurrent controls were considered to be a reflection of the effects on body weight in these groups, rather than representing a primary effect of the test item on these organs.

The findings of degeneration, squamous metaplasia and/or respiratory metaplasia of olfactory mucosa in Level IV of the nasal cavities affecting one female animal in Group 3 (Mid Dose) and two females in Group 4 (High Dose) were considered to represent local lesions of adverse character and were attributed to treatment with the test item. The minor increases in incidence and/or severity of intracytoplasmic hyaline droplets in nasal cavity levels III and IV were also attributed to treatment with the test item, but were not considered to be of adverse character, as this finding was also present in nasal cavity Level IV in Group 1 (Air Control).

The no-observed-adverse-effect-level for local effects [NOAEL(local)] was set at the low dose level administered, 10 mg/m³, because at the mid and high dose levels the findings of degeneration, squamous metaplasia and/or respiratory metaplasia of olfactory mucosa were evident in nasal cavity level IV and male body weight and body weight gain were statistically significantly lower than concurrent controls during most of the treatment period. However, the effects on body weight at the mid and high dose levels were considered to be only moderate in degree, as they were restricted to one gender, relevant body weight loss was not evident during the study, and by the end of the 13 week treatment period the group mean body weights of mid and high dose male animals were only 9.3% lower than that of concurrent control animals.

There were no changes observed in organs, tissues or other parameters investigated which were attributed to systemic toxicity. Especially, any evidence for neurotoxicity, which is the known critical effect of methacrylamide when administered at higher doses in oral and dermal studies, was lacking.

 

Therefore, the no-observed-adverse-effect-level for systemic effects [NOAEL(systemic)] was set at the high dose level administered, 62.5 mg/m³.

This subchronic inhalation toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic inhalation study OECD 413 in the rat. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

62.5 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22-Aug-2006 to 14-Aug-2008

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.

Justification for type of information:

Please refer to the CSR for the read-across rationale.

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

adopted May 12, 1981

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
- Rat strain: HanRCC: WIST(SPF)
- Source: RCC Ltd., Füllingsdorf, Switzerland
- Age at study initiation: 8 weeks (males), 11 weeks (females)
- Weight at study initiation: males: 211.1 to 236.2 g
females: 184.0 to 211.4 g
- Fasting period before study:
- Housing: Groups of 5 in Makrolon type-4 cages with wire mesh tops and standard softwood bedding ("Lignocel", Schill AG, Muttenz, Switzerland).
- Diet (e.g. ad libitum): ad libitum, pelleted standard Kliba 3433, Batch 23/06
- Water (e.g. ad libitum): ad libitum access to tap water
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hr artificial flourescent light/ 12 hr dark, music/light period.

Route of administration:

inhalation

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: The particle size (expressed as MMAD) of the generated Methacrylamide aerosols was well within the ideal range of 1 to 3 µm in each treatment group. Details on particle size characteristics see below, Table 2 in section “Any other information on materials and methods incl. tables”

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:
Inhalation exposure was performed using a system similar to that originally described by Sachsse et al. (details see: K. Sachsse, L. Ullmann, G. Voss
and R. Hess: Measurements of Inhalation Toxicity of Aerosols in Small Laboratory Animals. In: Proceedings of the Europ. Soc. for the Study of Drug
Toxicity, Vol. XV, pp. 239-251, Zürich, June 1973.)
The design of this exposure system is based upon the fluid dynamic modelling of the aerosol flow. It ensures a uniform test item distribution,
provides a constant stream of "fresh" test item to each animal, and precludes re-breathing the exhaled air. The aerosol stream reaches the animal's
nose through ports situated at three different levels around the axis of the exposure chamber. Each level has 16 animal ports and can be rotated
allowing close observation of all the animals without interruption of exposure. In the present study the exposure chambers of groups 1 (air control)
and 3 (mid-dose) comprised three levels of animal ports. For the exposure of groups 2 (low-dose) and 4 (high-dose) chambers comprising five
levels of animal ports were utilized. All animals were placed on the top two levels during each exposure, thus minimising variation in exposure
conditions between the animals of any particular group.

- Method of holding animals in test chamber:
The animals were confined separately in Makrolon* restraint tubes which were positioned radially around the nose-only, flow-past exposure chamber (details see: Cannon, W.C., E.F. Blanton and K.E. McDonald: "The Flow-Past Chamber: An Improved Nose-Only Exposure System for Rodents", Am. Ind. Hyg. Assoc. J., 44 (12): 923-928, 1983.)

- Source and rate of air:

The design of this exposure system precludes re-breathing the exhaled air, details see above (Exposure apparatus).


- Method of conditioning air:
Oxygen Concentration
The oxygen concentration of the control and test atmospheres were monitored continuously during each 6-hour exposure period for each group
(Groups 1 to 4 inclusive using an oxygen sensor (VarioGard Transmitter 4...20 mA from Dräger AG, 8305 Dietlikon / Switzerland) connected to a data logger. In addition, the oxygen concentration was noted manually from the data logger for each group at appropriate intervals after the beginning of the daily exposure (three times during each 6-hour exposure period / group / day).


- System of generating particulates/aerosols:
Test Aerosol Generation
For each dose group (Groups 2, 3 and 4), the pre-milled test item was aerosolised using a piston feed/rotating brush aerosol generator followed by a micronising jet mill (see diagrams below). Then the aerosol output from the jet mill was discharged through a 63Ni charge neutraliser. For Groups 2
(Low Dose) and 4 (High Dose) the discharged aerosol was split, one portion of it being used for the exposure of Group 4 without further dilution, the other portion of it being further diluted with filtered dry air using an air vac device. For Group 3 (Mid Dose) the test aerosol was generated in a similar manner as for Group 4, but separately from that generated for Groups 2 and 4 without using an air vac device. In order to minimize the variation in
aerosol concentration over each exposure and remain close to the target aerosol concentrations, the piston advance speed was adjusted, as deemed necessary from the daily monitoring of aerosol concentration. The above generation and/or dilution system was chosen to achieve the required test
item concentrations. Animals of group 1 (Air Control) were exposed to compressed filtered air alone at conditions similar to those used for Groups 2 (Low Dose), 3 (Mid Dose) and 4 (High Dose).


- Temperature, humidity, pressure in air chamber:
Temperature / Relative Humidity
The temperature and relative humidity of the control and test atmospheres were monitored continuously during each 6-hour exposure period for
each group (Groups 1 to 4 inclusive) using a ROTRONIC Hygrometer (Series I-200, Rotronic AG, 8040 Zürich / Switzerland) connected to a data
logger. In addition, the temperature and relative humidity were noted manually from the data logger for each group at appropriate intervals after the
beginning of the daily exposure (three times during each 6-hour exposure period / group / day).


- Air flow rate:
Exposure Airflow Rate
The airflow rate through the exposure chambers was adjusted before inhalation exposure and monitored indirectly during each exposure through
the generation and/or dilution system using calibrated pressure gauges and/or flowmeters and were checked at least at the same intervals as the
temperature, relative humidity and oxygen concentration of the control and test atmospheres. In all dose groups, a proportion of the ports on each
exposure chamber were closed such that the airflow to each open port was approximately 1.0 L/minute/animal port during each exposure.


- Air change rate:
The design of this exposure system precludes re-breathing the exhaled air, details see above (Exposure apparatus).


- Method of particle size determination:
Particle Size Distributions and Mass Median Aerodynamic Diameters
The distribution of particle size in the generated aerosols was measured by gravimetry six times in Group 2 (Low Dose) and four times in each of
groups 3 (Mid Dose) and 4 (High Dose) during the 13-week treatment period, using a Mercer 7-stage cascade impactor (Model 02-130, In-Tox.
Products Inc. Albuquerque, New Mexico, USA). Representative samples of the test atmosphere were drawn through the impactor with a flow rate of 1.0 L/min and the particles deposited according to their aerodynamic size onto stainless steel slips and the final filter stage (Type HVLP, Polyvinylidene-
difluoride membrane, pore size 0.45 m), on each stage of the impactor. To obtain the mass deposited on each stage of the impactor, the steel slips
and the final filter stage were weighed before and after sampling using an appropriate analytical balance. For each measurement in Group 2, aerosol
was collected in the impactors over three to six consecutive treatment days, in order to accumulate sufficient particulate material in the impactor for
adequate aerodynamic particle size measurement. On the corresponding results table of gravimetric particle size data only the last sampling date is
given. For the measurements in Groups 3 and 4 sufficient test item accumulated within one treatment day. The total mass (g) deposited in the impactor was then calculated by adding together the mass deposited on each of the stainless steel slips and the final filter stage. As the Effective Cut-off
Diameters (ECD) represent the lower size limit of the particles collected on each stage, the percentages less than the indicated size were tabulated as a function of the ECD. The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) of the test aerosols were calculated on the basis of the gravimetric results from the impactors applying the built-in PROBIT function of Microsoft Excel Software. The target range for the mass median aerodynamic diameter was 1 to 3 μm. The gravimetric determination of particle size distribution is only indicative of the nonevaporated phase of the test item. Any vapour component or any fraction which might evaporate or sublimate are not accounted for.



- Treatment of exhaust air:

Re-breathing of exhaust air is precluded, deteis see above (exposure apparatus).


TEST ATMOSPHERE

- Brief description of analytical method used:
Analytical Concentrations
Sampling of test atmosphere for the determination of concentrations by chemical analysis was performed at least once weekly during most of a
6-hour inhalation exposure period in each of Groups 2 to 4 inclusive. The dust fraction of the test atmospheres was collected on Millipore® durapore filters, Type HVLP, the vapour fraction was trapped in phosphoric acid (0.033 molar). On each sampling occasion, the test atmosphere was drawn
through such a filter which was followed by two wash-bottles placed in series each containing approximately 80 mL of phosphoric acid (0.033 molar), cooled in water ice. Immediately after each atmosphere sampling and weighing, the filter was put into a light protected glass vial and covered with
10 g of phosphoric acid (0.033 molar) to minimise loss of test item by sublimation from the filter. The content of each wash-bottle was transferred
into appropriate glass flasks, the wash-bottles were rinsed with phosphoric acid (0.033 molar), and the flasks made up to 100 ml with the rinsing and tightly closed.
The filters and solutions were kept light protected at approximately +5 °C in a cool box until dispatch at this temperature to the analytical laboratory of RCC Ltd, for chemical analysis. The samples were analysed by high performance liquid chromatography (HPLC) with UV detection using the test
item, METHACRYLAMIDE (as received from the Sponsor), as reference item for the analytical calibration curves. The analytical method was based on
that which was provided by the Sponsor. An analytical phase report was provided for inclusion in the report of this study.


- Samples taken from breathing zone: yes
Test atmosphere samples were collected directly from the delivery tube in the breathing zone of the animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean chemically determined aerosol concentrations of METHACRYLAMIDE and corresponding mean gravimetrically determined aerosol
concentrations are summarised below, see Table 1 in section “Any other information on materials and methods incl. tables”


Exposure System Monitoring

The atmosphere concentrations of the test item determined gravimetrically (dust fraction, trapped on filters) and chemically (including dust fraction
trapped of filters and vapour fraction trapped in solvent), particle size distribution determined gravimetrically, relative humidity, temperature
and oxygen concentration, were measured on test atmosphere samples collected directly from the delivery tube in the breathing zone of the animals, at an empty sampling port of the exposure chamber, delivering "fresh" test item to the animal's nose. The position at which these test atmosphere
samples were taken has been considered to be representative for the breathing zone of the animals. This approach was chosen in order to obtain
representative samples of what was delivered to the animals. Airflow rates were measured during the collection of samples for the determination of
aerosol concentrations using dry-test meters and pressure gauges, calibrated with a reference dry-test meter. Airflow rates for particle size samples were calibrated using a Gilibrator bubble flow generator.

Nominal Determination of Aerosol Concentrations

Based on the proportion of primary (prior to further dilution) aerosol assigned to Group 2 (Low Dose) the quantity of test item assigned to this group was calculated and then divided by the sum of primary airflow volume assigned to Group 2 plus airflow used for further aerosol dilution to
give the nominal concentration of test item in the aerosol delivered to the animals of this group. For Group 3 (Mid Dose), the nominal concentration
was measured by weighing the generator cylinder containing the test item before and after each exposure to determine the quantity of test item used. The weight of test item used was then divided by the total airflow volume to give the nominal concentration. For Group 4 (High Dose), the nominal
concentration was measured by weighing the generator cylinder containing the test item before and after each exposure to determine the quantity of test item used for generation of the primary (prior to further dilution) aerosol. The weight of test item used was then divided by the total airflow
volume used for the jet mill, i.e. used for the primary aerosol (prior to further dilution), to give the nominal concentration of test item in the aerosol
delivered to the animals of this group.

Gravimetric Determination of Aerosol Concentrations

Gravimetric determinations of aerosol concentration (dust fraction) in the generated atmospheres were performed using a Millipore® durapore filter
(Type HVLP, Polyvinylidenedifluoride membrane, pore size 0.45 µm), loaded in a 47 mm in-line stainless steel filter sampling device.
The gravimetric sampling was performed once daily for each of groups 2 (Low Dose), 3 (Mid Dose) and 4 (High Dose) sampling aerosol during most
of the 6-hour exposure period. In addition, on a number of occasions during the study for each of these groups, a further three filter samples were
taken over shorter periods shortly after the beginning, at about the middle and towards the end of the 6-hour exposure period to give an indication
of the stability of the aerosol concentration over exposure time. Additional aerosol samples were taken for monitoring purposes as considered
necessary and the corresponding aerosol concentration data were retained in the raw data without reporting.
Technical atmosphere generation trials (not performed under GLP) led to the conclusion that in the present study the gravimetrically determined
concentrations were only approximate estimates of the true concentrations of the test atmosphere, because of sublimation losses of
METHACRYLAMIDE. Therefore, one purpose of the gravimetric determination of the aerosol concentrations was to monitor roughly the aerosol
concentration during the exposure period, as the test atmosphere concentrations (dust and vapour fraction) determined by chemical analysis were
only available post exposure. The gravimetric determination of aerosol concentration is only indicative of the non-evaporated phase of the test item. Any vapour component or any fraction which might evaporate or sublimate is not accounted for in the gravimetrically determined aerosol
concentration values.

Analytical Concentrations

Sampling of test atmosphere for the determination of concentrations by chemical analysis was performed at least once weekly during most of a
6-hour inhalation exposure period in each of Groups 2 to 4 inclusive. The dust fraction of the test atmospheres was collected on Millipore® durapore filters, Type HVLP, the vapour fraction was trapped in phosphoric acid (0.033 molar). On each sampling occasion, the test atmosphere was drawn
through such a filter which was followed by two wash-bottles placed in series each containing approximately 80 mL of phosphoric acid (0.033 molar), cooled in water ice. Immediately after each atmosphere sampling and weighing, the filter was put into a light protected glass vial and covered with
10 g of phosphoric acid (0.033 molar) to minimise loss of test item by sublimation from the filter. The content of each wash-bottle was transferred
into appropriate glass flasks, the wash-bottles were rinsed with phosphoric acid (0.033 molar), and the flasks made up to 100 ml with the rinsing and tightly closed. The filters and solutions were kept light protected at approximately +5 °C in a cool box until dispatch at this temperature to the
analytical laboratory of RCC Ltd, for chemical analysis. The samples were analysed by high performance liquid chromatography (HPLC) with
UV detection using the test item, METHACRYLAMIDE, as reference item for the analytical calibration curves.

Duration of treatment / exposure:

6 hours daily.

Frequency of treatment:

Once daily, 5 days per week for a total of 13 consecutive weeks.

Remarks:

Doses / Concentrations:
Test concentrations: 0 mg/m³; 10 mg/m³ ; 25 mg/m³; 62.5 mg/m³
Basis:
nominal conc.

No. of animals per sex per dose:

Group 1 – Air control: 10 Males and 10 Females
Group 2 – Low Dose: 10 Males and 10 Females
Group 3 – Mid Dose: 10 Males and 10 Females
Group 4 – High Dose: 10 Males and 10 Females

Control animals:

yes

Details on study design:

- Dose selection rationale:
The above target concentrations were chosen on the basis of the results attained in a 2-Week inhalation and neurotoxicity study in the rat via
nose-only exposures.

Rationale for animal assignment:
Selection and Randomization: The animals were allocated to groups by a body weight stratification procedure on the first day of acclimatisation
to ensure that differences in group mean body weights are minimized.


Rationale for special In-Life Observations:
Functional Observational Battery (FOB) and Motor Activity were part of the in-life observations as the critical toxic effect for methacrylamide is
neurotoxicity.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

Viability / Mortality

All animals were observed for mortality/moribundity once daily during the acclimatisation period (including the reserve animals), and twice daily,
before and after exposure, during the treatment period of the study. On weekend days of the treatment period, when there was no exposure of
animals, mortality was checked at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes

Clinical Signs – Time schedule

Clinical signs were recorded once during the acclimatisation period (including the reserve animals) and twice daily during the treatment period of the study (once pre-exposure and once post exposure outside the restraint tubes on all animals, except the reserve animals). On weekend
days of the treatment period, when there was no exposure of animals, clinical examinations were performed once daily.
During exposure, only grossly abnormal signs could be observed, as the animals were in restraint tubes. Observations were detailed and carefully
recorded using explicitly defined scales as appropriate. Observations included but were not limited to changes in behaviour, somatomotor
activity, body position, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, e.g. tremors or
convulsions, reactivity to handling or sensory stimuli, altered strength, alteration of the skin, fur, nose, eyes and mucous membranes.

BODY WEIGHT: Yes

Body Weights – Time schedule

Each animal was weighed at 7-day intervals (if appropriate), during the acclimatisation and treatment periods of the study. Reserve animals were also weighed during acclimatisation. During the treatment period the body weights were recorded before the exposure on the particular day.

FOOD CONSUMPTION:

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

Food consumption was recorded weekly per cage of five animals (reserve animals exclusive), over a 7-day period (if possible and/or appropriate),
during the acclimatisation and treatment periods of the study. The food consumption was calculated per cage and per food consumption interval. It expresses the average food consumed per animal and per day for each cage over the food consumption interval.

FC = C / AD

where
FC is food consumption (food fed - food left) in grams of food per animal and day,
C is measured food consumption in grams per cage over the consumption interval, and
AD is total consumption days over all animals in the cage during the consumption interval.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

The relative food consumption was calculated per cage at each time interval according to the
following formula:

RFC = [ FC/BW(i)] x 1000

where
RFC is relative food consumption, i.e. food consumption in grams of food per kg bodyweight and day,
FC is food consumption (food fed - food left) in grams of food per animal and day, and
BW(i) is the optimal (on day closest to the middle of food consumption period) body weight in grams.


WATER CONSUMPTION: Not recorded

OPHTHALMOSCOPIC EXAMINATION: Yes

Ophthalmoscopic Examinations – Time schedule – Dose Groups:

Ophthalmoscopic examinations were performed in all animals once during the acclimatisation period (reserve animals inclusive), and in all surviving animals once during treatment week 13. Method/Instrumentation: The observations were performed after instillation of a mydriaticum,
using a Heine Bifocal Type Miroflex ophthalmoscope (Eisenhut Vet. AG, 4123 Allschwil /Switzerland).
Dates: Acclimatisation: 24-Aug-2006
Treatment Period (Week 13): 24-Nov-2006
For unilateral findings unless otherwise indicated in the tables, the contralateral eye was without abnormalities.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 13 weeks of treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: All animals

Clinical Laboratory Investigations

Blood samples for hematology and clinical biochemistry were collected from the retro-orbital plexus from all animals (10 animals/sex/group) under
light isoflurane anaesthesia. This was followed by necropsy on the same day. The animals were fasted in metabolism cages for approximately 17 hours
before blood sampling, but water was provided ad libitum. The blood samples were collected early in the working day to reduce biological variation
caused by circadian rhythms. Date of blood sampling: After 13 weeks of treatment: 28-Nov-2006
In the summary and individual tables the names of some parameters have been abbreviated.
Clinical laboratory data are expressed, with a few exceptions, in general accordance with the International System of Units (SI).

Hematology parameters determined

Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Reticulocyte count
Reticulocyte maturity index
Total leukocyte count
Differential leukocyte count
Platelet count
Coagulation:
Prothrombin time
Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 13 weeks of treatment
- Animals fasted: Yes
- How many animals: All animals

Clinical Biochemistry parameters determined:

Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Phospholipids
Aspartate aminotransferase
Alanine aminotransferase
Lactate dehydrogenase
Glutamate dehydrogenase
Alkaline phosphatase
Gamma-glutamyl-transferase
Creatine kinase
Sodium
Potassium
Chloride
Calcium
Phosphorus inorganic
Protein, total
Albumin
Globulin
Albumin/Globulin ratio


URINALYSIS: Yes
- Time schedule for collection of urine: after 13 weeks of treatment

Date of urine sampling: After 13 weeks of treatment: 28-Nov-2006
Urine was collected during an approximately 16-hour fasting period into specimen vials using a metabolism cage.

- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

Urinalysis parameters determined:

Volume
Relative density
Colour
Appearance
pH
Protein
Nitrite
Glucose
Ketone
Urobilinogen
Bilirubin
Erythrocytes
Leukocytes


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: see below
- Dose groups that were examined: see below
- Battery of functions tested: sensory activity / grip strength / motor activity / other: see below

Functional Observation Battery (FOB)

The FOB observations were performed during treatment weeks 12 (male animals) and 13 (female animals) as from approximately two hours after the
end of the 6-hour exposure period and included hind- and forelimb grip strength, landing foot splay and Preyer’s reflex. Detailed clinical
observations were evaluated in the FOB. The FOB was conducted on all animals, as the animals were not considered to otherwise reveal signs of
toxicity to an extent that would significantly have interfered with the functional test performance. Relevant parameters from a modified Irwin screen
test were performed on all rats. Any abnormal findings were recorded and graded in severity. Hind- and forelimb grip strength measurements were
performed using a push-pull strain gauge.

Functional examinations included tests for:

Sensorimotor functions: approach, touch, vision, audition, pain, vestibular
Autonomic functions: puppilary reflex, body temperature
Sensorimotor coordination: grip strength, landing foot splay

The observations included but were not limited to:

recumbency; ease of removal; chromorhinorrhea;
posture/gait; ease of handling; piloerection
gait abnormalities; vocalization; palpebral closure
paddling movements; Straub tail; eye prominence;
muscle tone; stereotypies; fecal consistency
activity; pupil size; urination
paralysis; click response; respiratory abnormalities
fasciculations; salivation; unkempt fur;
spasms; lacrimation; emaciation;
tremor; chromodacryorrhea; dehydration;
convulsions; rhinorrhea; distended abdomen.

Motor Activity

Motor activity was assessed after the conduct of the FOB in treatment week 12 (male animals) and 13 (female animals) as from approximately 3.5 to 4 hours after the end of the 6 h exposure period using an AMS Föhr Medical Instruments GmbH (FMI) activity measurement system.
Locomotor activity was measured quantitatively. Decreased or increased activity was recorded. Activity was recorded for 10-minute intervals over a period of 60 minutes. These data and the total activity over 60 minutes have been reported.


OTHER: no

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see below)
HISTOPATHOLOGY: Yes (see below)

Necropsy

After 13 Weeks of treatment: 28-Nov-2006 (40 males / 40 females)
All animals used during the treatment period of this study were transferred to the pathology unit on the scheduled day of necropsy (day 92), were
anaesthetised by an intraperitoneal injection of Eutha® 77 at a dose of at least 320 mg sodium pentobarbitone/kg body weight, weighed and
sacrificed by exsanguination. A complete examination was performed on all animals and all macroscopic abnormalities were described and reported.
Samples of the following tissues and organs were collected from all animals and fixed in neutral phosphate buffered 4 % formaldehyde solution,
unless otherwise stated (For paired organs, the left and the right one were examined). Lungs were instilled with this fixative at a hydrostatic pressure of 30 cm.

Adrenal glands; Aorta
Brain (cerebrum, cerebellum and brain stem);
Cecum; Colon; Duodenum
Epididymides (fixed in Bouin’s solution)
Esophagus; Extraorbital lacrimal glands
Eyes with optic nerves (fixed in Davidson’s solution)
Femur, including joint
Harderian glands (fixed in Davidson’s solution)
Heart
Ileum (including Peyer’s patches)
Jejunum (including Peyer’s patches)
Kidneys
Larynx (three transversal sections,at least levels II,
III and VI)
Liver
Lungs (infused with formalin; sections from 2 lobes)
Lymph nodes (mandibular, mesenteric, tracheobronchial)
Mammary gland area
Nasal cavities with ”paranasal sinuses”(Levels I to IV)
Nasopharyngeal duct and pharynx (1 longitudinal section)
Ovaries; Pancreas; Pituitary gland; Prostate; Rectum
Salivary glands – mandibular, sublingual
Sciatic nerve; Seminal vesicles; Skeletal muscle (thigh region)
Skin; Spinal cord - cervical, midthoracic, lumbar
Spleen; Sternum with bone marrow; Stomach
Testes (fixed in Bouin’s solution); Thymus
Thyroid gland with parathyroid gland; Tongue
Trachea (one transversal section below larynx)
Tracheal bifurcation, carina and mainstem bronchi
Urinary bladder (infused with formalin)
Uterus; Vagina
All gross lesions

Organ Weights

The following organ weights were recorded from all animals on the scheduled necropsy date and their ratios to terminal body weight and to brain
weight determined (The combined weight of paired organs was recorded):
Adrenal glands; Brain; Heart; Kidneys; Liver;
Lungs; Ovaries; Spleen; Testes; Thymus

Histotechnique / Histopathology

All organs and tissue samples collected at necropsy from all animals of Groups 1 (Air Control) and 4 (High Dose), and the lungs and nasal cavities
(Levels I to IV) of all animals of Groups 2 (Low Dose) and 3 (Mid Dose) were processed, embedded in paraffin, cut at a nominal thickness of
2-4 micrometers, stained with hematoxylin and eosin and histologically examined by use of a light microscope. Processiong and examination of the
nasal cavities was extended to the intermediate dose groups, because of treatment-related changes seen in these tissues in the high dose group. In
addition, all organs and tissues with gross lesions were processed and histologically examined. Attempts were made to correlate gross observations with microscopic findings.

Other examinations:

No

Statistics:

Statistics

The following statistical methods were used to analyze food consumption, body weight, clinical laboratory data, organ weights and ratios as well as macroscopic findings:

The Dunnett-test
[Dunnett 1955, details see Dunnett, C.W.: A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).] (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups with the air control group for each sex.

The Steel-test (many-one rank test)
[Miller 1981, details see Miller, R.G.: Simultaneous Statistical Inference, Springer Verlag, New York (1981).] was applied instead of the Dunnett-test
when the data could not be assumed to follow a normal distribution.

Fisher's exact-test
[Fisher 1950, details see Fisher, R.A.: Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950)]. Group means and standard
deviations were calculated for continuous data and medians for discrete data (scores), as appropriate.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY

Viability / Mortality
There were no premature deaths.

Clinical Signs

There were no clinical signs in test item treated animals during the present study. The only finding occasionally noted was hair loss in various body
regions in three females of the control group. This finding is occasionally seen in rats of this strain and age regardless of their treatment.

BODY WEIGHT AND WEIGHT GAIN

Body Weights

There were no statistically significant differences from controls in body weight and body weight gain at the low dose level (Group 2) throughout the
13-week treatment period. At the mid and high dose levels (Groups 3 and 4, respectively), male body weights and body weight gain were statistically
significantly lower than concurrent controls during most of the treatment period. Female body weights and, with one exception on Day 22, female
body weight gain were not statistically significantly affected by treatment with the test item. By the end of the 13-week treatment period, group mean
body weights differed from concurrent controls, see Table 3 below in “remarks on results including tables and figures”



FOOD CONSUMPTION

Food Consumption

Food Consumption and relative food consumption data were very limited, since they were recorded per cage of 5 animals and there were only 2
cages/group/sex. Therefore, and since spillage of food by individual animals cannot be ruled out, conclusions drawn from these data can only be
vague and tentative. Overall mean food consumption (g/animal/day) over the 13-week treatment period is demonstrated in Table 4, see below
(“remarks on results including tables and figures”)

Although a trend to reduced food consumption in treated animals might be concluded from these data, the change was considered to be minor in
degree.

FOOD EFFICIENCY

No clear pattern was evident in the relative food consumption data (relative to body weight; g / kg body weight / day), details see Table 5 in “remarks on results including tables and figures”

WATER CONSUMPTION

not examined

OPHTHALMOSCOPIC EXAMINATION

Ophthalmoscopic Examinations

There were no ophthalmic findings attributable to treatment with the test item.

HAEMATOLOGY

No adverse effects, details see clinical chemistry.

CLINICAL CHEMISTRY

Clinical Laboratory Investigations

There were no adverse effects on haematology, clinical biochemistry and urinalysis parameters.

A number of haematology and clinical biochemistry parameters differed statistically significantly from concurrent controls in one or three test item
treated groups. However, these differences were not considered to be toxicologically relevant, because they lacked dose relationship, were restricted to one sex and/or the respective group mean values were within the historical reference range.
The only differences from concurrent controls statistically significant in all three treated groups were slightly reduced globulin levels and slightly
increased albumin to globulin ratios. However, these changes were restricted to males and also for these parameters, all group mean values were
within the historical reference range. In addition, group mean urine volumes were lower in treated animals than in concurrent controls, but did not
attain statistical significance.

URINALYSIS

No adverse effects, details see clinical chemistry.

NEUROBEHAVIOUR

Functional Observational Battery

Functional observational battery (FOB) parameters did not appear to be affected by treatment with the test item, although the finding of Straub tail was seen in one mail and one female animal of Group 4 (High Dose). The toxicological relevance of this, rather unusual, finding remained unclear. Apart
from this, the findings noted were low in incidence, lacked dose relationship and/or were considered to be within the normal range of biological
variation.

Locomotor Activity

Intergroup comparison of the motor activity of treated animals with concurrent controls over a period of 60 minutes (analysed for 10-minute intervals) revealed statistically significantly (Steel test significant at 5%) increased motor activity in mid dose males at approximately 60 minutes,
in mid dose females at 50 and 60 minutes and for the data pooled over the total 60 minute period, and in high dose females at 50 minutes. These
increases were not attributed to treatment with the test item, because they lacked dose relationship and were evident only towards the end of the 60
minute recording period. At the low dose level, the motor activity data did not differ statistically significantly from concurrent controls throughout
the 60 minute recording period.

ORGAN WEIGHTS

After 13 weeks of treatment, terminal body weights did not differ statistically significantly from concurrent controls in males of the low dose group
(-4.7%), but were significantly lower than concurrent controls in males of the mid (-11.0%) and high dose (-12.7%) groups. Female terminal body
weights were unaffected by treatment with the test item at all dose levels. These findings were consistent with the effects on live body weight and body weight gain recorded during the treatment period. Effects on organ weight were evaluated by intergroup comparison of absolute organ weights and
their ratios to terminal body weight and to brain weight with concurrent controls. The following changes were noted:
- Absolute brain weights reduced in mid dose males (-4.3%),
- Absolute kidney weights reduced in mid dose males (-12.1%),
- Absolute lung weights reduced in mid dose males (-7.7%) and high dose males (-8.5%),
- Heart to body weight ratios increased in mid dose males (+9.3%) and high dose males (+11.7%),
- Testes to body weight ratios increased in mid dose males (+16.6%) and high dose males (+19.8%).
The reductions in absolute brain weight and kidney weight in mid dose males were considered to be incidental, because they were minor in degree,
were not dose-related and there were no confirmatory histopathology findings in the brain or kidneys in high dose animals.
The reductions in absolute lung weight and increases in heart to body weight and testes to body weight ratios in mid and high dose males were
considered be a reflection of the effects on body weight in these groups, rather than representing a primary effect of the test item on these organs.
Female absolute and relative organ weights were not affected by treatment with the test item.


GROSS PATHOLOGY

Macroscopic Findings

A number of macroscopic pathology findings were seen in male animals but none in females. These findings were considered to be within the range
of natural background findings occasionally seen in rats of this strain and age. They did not distinguish test item treated animals from concurrent
controls.


HISTOPATHOLOGY: NON-NEOPLASTIC

Microscopic Findings

The following microscopic findings in the nasal cavities were considered to distinguish test item treated rats from concurrent controls:

Nasal Cavities Level III
Intracytoplasmic hyaline droplets in olfactory epithelium,
minimal in degree in 2 low dose animals and 2 mid dose animals, and
minimal to slight in degree in 4 high dose animals.

Nasal Cavities Level IV
Intracytoplasmic hyaline droplets in olfactory and/or respiratory epithelium,
minimal to slight in degree in 5 control animals,
minimal in degree in 7 low dose animals
minimal to slight in degree in 9 mid dose animals, and
minimal to slight in degree in 14 high dose animals.
Unilateral focal degeneration of olfactory mucosa,
minimal in degree in 1 mid dose animal.
Squamous metaplasia of olfactory mucosa,
slight in degree in 1 mid dose animal, and
minimal in degree in 2 high dose animals.
Respiratory metaplasia of olfactory mucosa,
minimal in degree in 1 high dose animal.
The findings of degeneration, squamous metaplasia and respiratory metaplasia of olfactory mucosa in Level IV of the nasal cavities were considered
to represent local lesions of adverse character and were attributed to treatment with the test item.
A number of other microscopic findings noted in the present study were considered to represent findings commonly seen in rats of this strain and
age. Their incidence, distribution ormorphology did not provide any evidence for a relationship to treatment with the test item.


HISTOPATHOLOGY: NEOPLASTIC (if applicable)

not examined

HISTORICAL CONTROL DATA (if applicable)

no data

OTHER FINDINGS

No

Dose descriptor:

NOAEL

Remarks:

system

Effect level:

62.5 mg/m³ air

Sex:

male/female

Basis for effect level:

other: no adverse effects

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

10 mg/m³ air

Sex:

male/female

Basis for effect level:

other: histopathology; (organ weights)

Critical effects observed:

not specified

Table 3 - Body weight by the end of the 13-week treatment period

	Group 2 (Low Dose)	Group 3 (Mid Dose)	Group 4 (High Dose)
Males	-3.8% ns	-9.3%**	-9.3%**
Females	+1.3% ns	-0.9% ns	-4.3% ns

ns: No statistically significant difference; */** Dunnet-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 4 - Overall mean food consumption (g/animal/day) over the 13-week treatment period

	Group 1 (Air Control)	Group 2 (Low Dose)	Group 3 (Mid Dose)	Group 4 (High Dose)
Males	21.8	20.7	19.7	20.0
Females	16.2	15.4	15.0	15.1

Table 5 - Relative food consumption data (relative to body weight; g / kg body weight / day)

	Group 1 (Air Control)	Group 2 (Low Dose)	Group 3 (Mid Dose)	Group 4 (High Dose)
Males	63.1	62.1	62.5	63.4
Females	68.7	64.7	64.1	66.7

Conclusions:

The NOAEL (local) in rats exposed to METHACRYLAMIDE dust for 13 weeks is set at the lowest dose of 10 mg/m³. At higher concentrations
administered, local adverse effects as degeneration, sqamous metaplasia and respiratory metaplasia were found in nasal tissues (level IV).
In addition, a statistical significant decrease of body weight gains was observed in male animals of the mid- and high dose group.

There are no observations in this study which are assigned to systemic toxicity, especially any signs of neurotoxicity, which is a known toxic effect of METHACRYLAMIDE, are lacking. Therefore, the NOAEL (systemic) is set at the highest dose administered, 62.5 mg/m³.

Executive summary:

In a subchronic inhalation toxicity study [OECD TG 413],   METHACRYLAMIDE dust (purity 99.7 %) was administered to 40 male and 40 female wistar rats by nose only inhalation at nominal concentrations of 0, 10, 25, and 62.5  mg/m³ which is equivalent to 0, 0.01, 0.025, and 0.0625 mg/l for 6 hours per day, 5 days / week for a total of  13  weeks (65 exposures).

 The chemically determined mean aerosol concentrations were 10.24 (Group 2, Low Dose), 26.85 (Group 3, Mid Dose) or 69.95 (Group 4, High Dose) mg of METHACRYLAMIDE/m³. These aerosol concentrations were equivalent to mean aerosol concentrations of  9.45, 25.67 and 68.39 mg of METHACRYLAMIDE/m³ air in Groups 2, 3 and 4, respectively, when the attained ratios between the mean chemically determined aerosol concentrations and the respective mean gravimetric concentrations were applied to the overall mean gravimetric aerosol concentrations attained from 64 or 65 exposure days.

There were no premature deaths. Clinical signs attributable to treatment with the test item were not evident and functional observational battery (FOB) parameters did not appear to be affected by treatment with the test item. The toxicological relevance of the finding of Straub tail seen in one mail and one female animal of Group 4 (High Dose) remained unclear. Motor activity was not affected by treatment with the test item. Motor activity values on some occasions statistically significantly higher in Groups 3 (Mid Dose) and/or 4 (High Dose) than in concurrent controls were not attributed to treatment with the test item, because they lacked dose relationship and were evident only towards the end of the 60 minute recording period.

In males of Group 2 (Low Dose) and females of all treated groups relevant adverse effects on body weight and body weight gain were not evident. In males of Groups 3 (Mid Dose) and 4 (High Dose) body weight and body weight gain statistically significantly lower than concurrent controls during most of the treatment period were attributed to treatment with the test item. Absolute lung weight significantly lower and heart to body weight and testes to body weight ratios significantly higher in males of Groups 3 and 4 than in concurrent controls were considered to be a reflection of the effects on body weight in these groups, rather than representing a primary effect of the test item on these organs.

The findings of degeneration, squamous metaplasia and/or respiratory metaplasia of olfactory mucosa in Level IV of the nasal cavities affecting one female animal in Group 3 (Mid Dose) and two females in Group 4 (High Dose) were considered to represent local lesions of adverse character and were attributed to treatment with the test item. The minor increases in incidence and/or severity of intracytoplasmic hyaline droplets in nasal cavity levels III and IV were also attributed to treatment with the test item, but were not considered to be of adverse character, as this finding was also present in nasal cavity Level IV in Group 1 (Air Control).

The no-observed-adverse-effect-level for local effects [NOAEL(local)] was set at the low dose level administered, 10 mg/m³, because at the mid and high dose levels the findings of degeneration, squamous metaplasia and/or respiratory metaplasia of olfactory mucosa were evident in nasal cavity level IV and male body weight and body weight gain were statistically significantly lower than concurrent controls during most of the treatment period. However, the effects on body weight at the mid and high dose levels were considered to be only moderate in degree, as they were restricted to one gender, relevant body weight loss was not evident during the study, and by the end of the 13 week treatment period the group mean body weights of mid and high dose male animals were only 9.3% lower than that of concurrent control animals.

There were no changes observed in organs, tissues or other parameters investigated which were attributed to systemic toxicity. Especially, any evidence for neurotoxicity, which is the known critical effect of methacrylamide when administered at higher doses in oral and dermal studies, was lacking.

 

Therefore, the no-observed-adverse-effect-level for systemic effects [NOAEL(systemic)] was set at the high dose level administered, 62.5 mg/m³.

This subchronic inhalation toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic inhalation study OECD 413 in the rat. 

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

22-Aug-2006 to 14-Aug-2008

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.

Justification for type of information:

Please refer to the CSR for the read-across rationale.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

adopted May 12, 1981

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
- Rat strain: HanRCC: WIST(SPF)
- Source: RCC Ltd., Füllingsdorf, Switzerland
- Age at study initiation: 8 weeks (males), 11 weeks (females)
- Weight at study initiation: males: 211.1 to 236.2 g
females: 184.0 to 211.4 g
- Fasting period before study:
- Housing: Groups of 5 in Makrolon type-4 cages with wire mesh tops and standard softwood bedding ("Lignocel", Schill AG, Muttenz, Switzerland).
- Diet (e.g. ad libitum): ad libitum, pelleted standard Kliba 3433, Batch 23/06
- Water (e.g. ad libitum): ad libitum access to tap water
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hr artificial flourescent light/ 12 hr dark, music/light period.

Route of administration:

inhalation

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: The particle size (expressed as MMAD) of the generated Methacrylamide aerosols was well within the ideal range of 1 to 3 µm in each treatment group. Details on particle size characteristics see below, Table 2 in section “Any other information on materials and methods incl. tables”

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:
Inhalation exposure was performed using a system similar to that originally described by Sachsse et al. (details see: K. Sachsse, L. Ullmann, G. Voss
and R. Hess: Measurements of Inhalation Toxicity of Aerosols in Small Laboratory Animals. In: Proceedings of the Europ. Soc. for the Study of Drug
Toxicity, Vol. XV, pp. 239-251, Zürich, June 1973.)
The design of this exposure system is based upon the fluid dynamic modelling of the aerosol flow. It ensures a uniform test item distribution,
provides a constant stream of "fresh" test item to each animal, and precludes re-breathing the exhaled air. The aerosol stream reaches the animal's
nose through ports situated at three different levels around the axis of the exposure chamber. Each level has 16 animal ports and can be rotated
allowing close observation of all the animals without interruption of exposure. In the present study the exposure chambers of groups 1 (air control)
and 3 (mid-dose) comprised three levels of animal ports. For the exposure of groups 2 (low-dose) and 4 (high-dose) chambers comprising five
levels of animal ports were utilized. All animals were placed on the top two levels during each exposure, thus minimising variation in exposure
conditions between the animals of any particular group.

- Method of holding animals in test chamber:
The animals were confined separately in Makrolon* restraint tubes which were positioned radially around the nose-only, flow-past exposure chamber (details see: Cannon, W.C., E.F. Blanton and K.E. McDonald: "The Flow-Past Chamber: An Improved Nose-Only Exposure System for Rodents", Am. Ind. Hyg. Assoc. J., 44 (12): 923-928, 1983.)

- Source and rate of air:

The design of this exposure system precludes re-breathing the exhaled air, details see above (Exposure apparatus).


- Method of conditioning air:
Oxygen Concentration
The oxygen concentration of the control and test atmospheres were monitored continuously during each 6-hour exposure period for each group
(Groups 1 to 4 inclusive using an oxygen sensor (VarioGard Transmitter 4...20 mA from Dräger AG, 8305 Dietlikon / Switzerland) connected to a data logger. In addition, the oxygen concentration was noted manually from the data logger for each group at appropriate intervals after the beginning of the daily exposure (three times during each 6-hour exposure period / group / day).


- System of generating particulates/aerosols:
Test Aerosol Generation
For each dose group (Groups 2, 3 and 4), the pre-milled test item was aerosolised using a piston feed/rotating brush aerosol generator followed by a micronising jet mill (see diagrams below). Then the aerosol output from the jet mill was discharged through a 63Ni charge neutraliser. For Groups 2
(Low Dose) and 4 (High Dose) the discharged aerosol was split, one portion of it being used for the exposure of Group 4 without further dilution, the other portion of it being further diluted with filtered dry air using an air vac device. For Group 3 (Mid Dose) the test aerosol was generated in a similar manner as for Group 4, but separately from that generated for Groups 2 and 4 without using an air vac device. In order to minimize the variation in
aerosol concentration over each exposure and remain close to the target aerosol concentrations, the piston advance speed was adjusted, as deemed necessary from the daily monitoring of aerosol concentration. The above generation and/or dilution system was chosen to achieve the required test
item concentrations. Animals of group 1 (Air Control) were exposed to compressed filtered air alone at conditions similar to those used for Groups 2 (Low Dose), 3 (Mid Dose) and 4 (High Dose).


- Temperature, humidity, pressure in air chamber:
Temperature / Relative Humidity
The temperature and relative humidity of the control and test atmospheres were monitored continuously during each 6-hour exposure period for
each group (Groups 1 to 4 inclusive) using a ROTRONIC Hygrometer (Series I-200, Rotronic AG, 8040 Zürich / Switzerland) connected to a data
logger. In addition, the temperature and relative humidity were noted manually from the data logger for each group at appropriate intervals after the
beginning of the daily exposure (three times during each 6-hour exposure period / group / day).


- Air flow rate:
Exposure Airflow Rate
The airflow rate through the exposure chambers was adjusted before inhalation exposure and monitored indirectly during each exposure through
the generation and/or dilution system using calibrated pressure gauges and/or flowmeters and were checked at least at the same intervals as the
temperature, relative humidity and oxygen concentration of the control and test atmospheres. In all dose groups, a proportion of the ports on each
exposure chamber were closed such that the airflow to each open port was approximately 1.0 L/minute/animal port during each exposure.


- Air change rate:
The design of this exposure system precludes re-breathing the exhaled air, details see above (Exposure apparatus).


- Method of particle size determination:
Particle Size Distributions and Mass Median Aerodynamic Diameters
The distribution of particle size in the generated aerosols was measured by gravimetry six times in Group 2 (Low Dose) and four times in each of
groups 3 (Mid Dose) and 4 (High Dose) during the 13-week treatment period, using a Mercer 7-stage cascade impactor (Model 02-130, In-Tox.
Products Inc. Albuquerque, New Mexico, USA). Representative samples of the test atmosphere were drawn through the impactor with a flow rate of 1.0 L/min and the particles deposited according to their aerodynamic size onto stainless steel slips and the final filter stage (Type HVLP, Polyvinylidene-
difluoride membrane, pore size 0.45 m), on each stage of the impactor. To obtain the mass deposited on each stage of the impactor, the steel slips
and the final filter stage were weighed before and after sampling using an appropriate analytical balance. For each measurement in Group 2, aerosol
was collected in the impactors over three to six consecutive treatment days, in order to accumulate sufficient particulate material in the impactor for
adequate aerodynamic particle size measurement. On the corresponding results table of gravimetric particle size data only the last sampling date is
given. For the measurements in Groups 3 and 4 sufficient test item accumulated within one treatment day. The total mass (g) deposited in the impactor was then calculated by adding together the mass deposited on each of the stainless steel slips and the final filter stage. As the Effective Cut-off
Diameters (ECD) represent the lower size limit of the particles collected on each stage, the percentages less than the indicated size were tabulated as a function of the ECD. The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) of the test aerosols were calculated on the basis of the gravimetric results from the impactors applying the built-in PROBIT function of Microsoft Excel Software. The target range for the mass median aerodynamic diameter was 1 to 3 μm. The gravimetric determination of particle size distribution is only indicative of the nonevaporated phase of the test item. Any vapour component or any fraction which might evaporate or sublimate are not accounted for.



- Treatment of exhaust air:

Re-breathing of exhaust air is precluded, deteis see above (exposure apparatus).


TEST ATMOSPHERE

- Brief description of analytical method used:
Analytical Concentrations
Sampling of test atmosphere for the determination of concentrations by chemical analysis was performed at least once weekly during most of a
6-hour inhalation exposure period in each of Groups 2 to 4 inclusive. The dust fraction of the test atmospheres was collected on Millipore® durapore filters, Type HVLP, the vapour fraction was trapped in phosphoric acid (0.033 molar). On each sampling occasion, the test atmosphere was drawn
through such a filter which was followed by two wash-bottles placed in series each containing approximately 80 mL of phosphoric acid (0.033 molar), cooled in water ice. Immediately after each atmosphere sampling and weighing, the filter was put into a light protected glass vial and covered with
10 g of phosphoric acid (0.033 molar) to minimise loss of test item by sublimation from the filter. The content of each wash-bottle was transferred
into appropriate glass flasks, the wash-bottles were rinsed with phosphoric acid (0.033 molar), and the flasks made up to 100 ml with the rinsing and tightly closed.
The filters and solutions were kept light protected at approximately +5 °C in a cool box until dispatch at this temperature to the analytical laboratory of RCC Ltd, for chemical analysis. The samples were analysed by high performance liquid chromatography (HPLC) with UV detection using the test
item, METHACRYLAMIDE (as received from the Sponsor), as reference item for the analytical calibration curves. The analytical method was based on
that which was provided by the Sponsor. An analytical phase report was provided for inclusion in the report of this study.


- Samples taken from breathing zone: yes
Test atmosphere samples were collected directly from the delivery tube in the breathing zone of the animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean chemically determined aerosol concentrations of METHACRYLAMIDE and corresponding mean gravimetrically determined aerosol
concentrations are summarised below, see Table 1 in section “Any other information on materials and methods incl. tables”


Exposure System Monitoring

The atmosphere concentrations of the test item determined gravimetrically (dust fraction, trapped on filters) and chemically (including dust fraction
trapped of filters and vapour fraction trapped in solvent), particle size distribution determined gravimetrically, relative humidity, temperature
and oxygen concentration, were measured on test atmosphere samples collected directly from the delivery tube in the breathing zone of the animals, at an empty sampling port of the exposure chamber, delivering "fresh" test item to the animal's nose. The position at which these test atmosphere
samples were taken has been considered to be representative for the breathing zone of the animals. This approach was chosen in order to obtain
representative samples of what was delivered to the animals. Airflow rates were measured during the collection of samples for the determination of
aerosol concentrations using dry-test meters and pressure gauges, calibrated with a reference dry-test meter. Airflow rates for particle size samples were calibrated using a Gilibrator bubble flow generator.

Nominal Determination of Aerosol Concentrations

Based on the proportion of primary (prior to further dilution) aerosol assigned to Group 2 (Low Dose) the quantity of test item assigned to this group was calculated and then divided by the sum of primary airflow volume assigned to Group 2 plus airflow used for further aerosol dilution to
give the nominal concentration of test item in the aerosol delivered to the animals of this group. For Group 3 (Mid Dose), the nominal concentration
was measured by weighing the generator cylinder containing the test item before and after each exposure to determine the quantity of test item used. The weight of test item used was then divided by the total airflow volume to give the nominal concentration. For Group 4 (High Dose), the nominal
concentration was measured by weighing the generator cylinder containing the test item before and after each exposure to determine the quantity of test item used for generation of the primary (prior to further dilution) aerosol. The weight of test item used was then divided by the total airflow
volume used for the jet mill, i.e. used for the primary aerosol (prior to further dilution), to give the nominal concentration of test item in the aerosol
delivered to the animals of this group.

Gravimetric Determination of Aerosol Concentrations

Gravimetric determinations of aerosol concentration (dust fraction) in the generated atmospheres were performed using a Millipore® durapore filter
(Type HVLP, Polyvinylidenedifluoride membrane, pore size 0.45 µm), loaded in a 47 mm in-line stainless steel filter sampling device.
The gravimetric sampling was performed once daily for each of groups 2 (Low Dose), 3 (Mid Dose) and 4 (High Dose) sampling aerosol during most
of the 6-hour exposure period. In addition, on a number of occasions during the study for each of these groups, a further three filter samples were
taken over shorter periods shortly after the beginning, at about the middle and towards the end of the 6-hour exposure period to give an indication
of the stability of the aerosol concentration over exposure time. Additional aerosol samples were taken for monitoring purposes as considered
necessary and the corresponding aerosol concentration data were retained in the raw data without reporting.
Technical atmosphere generation trials (not performed under GLP) led to the conclusion that in the present study the gravimetrically determined
concentrations were only approximate estimates of the true concentrations of the test atmosphere, because of sublimation losses of
METHACRYLAMIDE. Therefore, one purpose of the gravimetric determination of the aerosol concentrations was to monitor roughly the aerosol
concentration during the exposure period, as the test atmosphere concentrations (dust and vapour fraction) determined by chemical analysis were
only available post exposure. The gravimetric determination of aerosol concentration is only indicative of the non-evaporated phase of the test item. Any vapour component or any fraction which might evaporate or sublimate is not accounted for in the gravimetrically determined aerosol
concentration values.

Analytical Concentrations

Sampling of test atmosphere for the determination of concentrations by chemical analysis was performed at least once weekly during most of a
6-hour inhalation exposure period in each of Groups 2 to 4 inclusive. The dust fraction of the test atmospheres was collected on Millipore® durapore filters, Type HVLP, the vapour fraction was trapped in phosphoric acid (0.033 molar). On each sampling occasion, the test atmosphere was drawn
through such a filter which was followed by two wash-bottles placed in series each containing approximately 80 mL of phosphoric acid (0.033 molar), cooled in water ice. Immediately after each atmosphere sampling and weighing, the filter was put into a light protected glass vial and covered with
10 g of phosphoric acid (0.033 molar) to minimise loss of test item by sublimation from the filter. The content of each wash-bottle was transferred
into appropriate glass flasks, the wash-bottles were rinsed with phosphoric acid (0.033 molar), and the flasks made up to 100 ml with the rinsing and tightly closed. The filters and solutions were kept light protected at approximately +5 °C in a cool box until dispatch at this temperature to the
analytical laboratory of RCC Ltd, for chemical analysis. The samples were analysed by high performance liquid chromatography (HPLC) with
UV detection using the test item, METHACRYLAMIDE, as reference item for the analytical calibration curves.

Duration of treatment / exposure:

6 hours daily.

Frequency of treatment:

Once daily, 5 days per week for a total of 13 consecutive weeks.

Remarks:

Doses / Concentrations:
Test concentrations: 0 mg/m³; 10 mg/m³ ; 25 mg/m³; 62.5 mg/m³
Basis:
nominal conc.

No. of animals per sex per dose:

Group 1 – Air control: 10 Males and 10 Females
Group 2 – Low Dose: 10 Males and 10 Females
Group 3 – Mid Dose: 10 Males and 10 Females
Group 4 – High Dose: 10 Males and 10 Females

Control animals:

yes

Details on study design:

- Dose selection rationale:
The above target concentrations were chosen on the basis of the results attained in a 2-Week inhalation and neurotoxicity study in the rat via
nose-only exposures.

Rationale for animal assignment:
Selection and Randomization: The animals were allocated to groups by a body weight stratification procedure on the first day of acclimatisation
to ensure that differences in group mean body weights are minimized.


Rationale for special In-Life Observations:
Functional Observational Battery (FOB) and Motor Activity were part of the in-life observations as the critical toxic effect for methacrylamide is
neurotoxicity.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

Viability / Mortality

All animals were observed for mortality/moribundity once daily during the acclimatisation period (including the reserve animals), and twice daily,
before and after exposure, during the treatment period of the study. On weekend days of the treatment period, when there was no exposure of
animals, mortality was checked at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes

Clinical Signs – Time schedule

Clinical signs were recorded once during the acclimatisation period (including the reserve animals) and twice daily during the treatment period of the study (once pre-exposure and once post exposure outside the restraint tubes on all animals, except the reserve animals). On weekend
days of the treatment period, when there was no exposure of animals, clinical examinations were performed once daily.
During exposure, only grossly abnormal signs could be observed, as the animals were in restraint tubes. Observations were detailed and carefully
recorded using explicitly defined scales as appropriate. Observations included but were not limited to changes in behaviour, somatomotor
activity, body position, respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects, e.g. tremors or
convulsions, reactivity to handling or sensory stimuli, altered strength, alteration of the skin, fur, nose, eyes and mucous membranes.

BODY WEIGHT: Yes

Body Weights – Time schedule

Each animal was weighed at 7-day intervals (if appropriate), during the acclimatisation and treatment periods of the study. Reserve animals were also weighed during acclimatisation. During the treatment period the body weights were recorded before the exposure on the particular day.

FOOD CONSUMPTION:

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

Food consumption was recorded weekly per cage of five animals (reserve animals exclusive), over a 7-day period (if possible and/or appropriate),
during the acclimatisation and treatment periods of the study. The food consumption was calculated per cage and per food consumption interval. It expresses the average food consumed per animal and per day for each cage over the food consumption interval.

FC = C / AD

where
FC is food consumption (food fed - food left) in grams of food per animal and day,
C is measured food consumption in grams per cage over the consumption interval, and
AD is total consumption days over all animals in the cage during the consumption interval.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

The relative food consumption was calculated per cage at each time interval according to the
following formula:

RFC = [ FC/BW(i)] x 1000

where
RFC is relative food consumption, i.e. food consumption in grams of food per kg bodyweight and day,
FC is food consumption (food fed - food left) in grams of food per animal and day, and
BW(i) is the optimal (on day closest to the middle of food consumption period) body weight in grams.


WATER CONSUMPTION: Not recorded

OPHTHALMOSCOPIC EXAMINATION: Yes

Ophthalmoscopic Examinations – Time schedule – Dose Groups:

Ophthalmoscopic examinations were performed in all animals once during the acclimatisation period (reserve animals inclusive), and in all surviving animals once during treatment week 13. Method/Instrumentation: The observations were performed after instillation of a mydriaticum,
using a Heine Bifocal Type Miroflex ophthalmoscope (Eisenhut Vet. AG, 4123 Allschwil /Switzerland).
Dates: Acclimatisation: 24-Aug-2006
Treatment Period (Week 13): 24-Nov-2006
For unilateral findings unless otherwise indicated in the tables, the contralateral eye was without abnormalities.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 13 weeks of treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: All animals

Clinical Laboratory Investigations

Blood samples for hematology and clinical biochemistry were collected from the retro-orbital plexus from all animals (10 animals/sex/group) under
light isoflurane anaesthesia. This was followed by necropsy on the same day. The animals were fasted in metabolism cages for approximately 17 hours
before blood sampling, but water was provided ad libitum. The blood samples were collected early in the working day to reduce biological variation
caused by circadian rhythms. Date of blood sampling: After 13 weeks of treatment: 28-Nov-2006
In the summary and individual tables the names of some parameters have been abbreviated.
Clinical laboratory data are expressed, with a few exceptions, in general accordance with the International System of Units (SI).

Hematology parameters determined

Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Reticulocyte count
Reticulocyte maturity index
Total leukocyte count
Differential leukocyte count
Platelet count
Coagulation:
Prothrombin time
Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 13 weeks of treatment
- Animals fasted: Yes
- How many animals: All animals

Clinical Biochemistry parameters determined:

Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Phospholipids
Aspartate aminotransferase
Alanine aminotransferase
Lactate dehydrogenase
Glutamate dehydrogenase
Alkaline phosphatase
Gamma-glutamyl-transferase
Creatine kinase
Sodium
Potassium
Chloride
Calcium
Phosphorus inorganic
Protein, total
Albumin
Globulin
Albumin/Globulin ratio


URINALYSIS: Yes
- Time schedule for collection of urine: after 13 weeks of treatment

Date of urine sampling: After 13 weeks of treatment: 28-Nov-2006
Urine was collected during an approximately 16-hour fasting period into specimen vials using a metabolism cage.

- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

Urinalysis parameters determined:

Volume
Relative density
Colour
Appearance
pH
Protein
Nitrite
Glucose
Ketone
Urobilinogen
Bilirubin
Erythrocytes
Leukocytes


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: see below
- Dose groups that were examined: see below
- Battery of functions tested: sensory activity / grip strength / motor activity / other: see below

Functional Observation Battery (FOB)

The FOB observations were performed during treatment weeks 12 (male animals) and 13 (female animals) as from approximately two hours after the
end of the 6-hour exposure period and included hind- and forelimb grip strength, landing foot splay and Preyer’s reflex. Detailed clinical
observations were evaluated in the FOB. The FOB was conducted on all animals, as the animals were not considered to otherwise reveal signs of
toxicity to an extent that would significantly have interfered with the functional test performance. Relevant parameters from a modified Irwin screen
test were performed on all rats. Any abnormal findings were recorded and graded in severity. Hind- and forelimb grip strength measurements were
performed using a push-pull strain gauge.

Functional examinations included tests for:

Sensorimotor functions: approach, touch, vision, audition, pain, vestibular
Autonomic functions: puppilary reflex, body temperature
Sensorimotor coordination: grip strength, landing foot splay

The observations included but were not limited to:

recumbency; ease of removal; chromorhinorrhea;
posture/gait; ease of handling; piloerection
gait abnormalities; vocalization; palpebral closure
paddling movements; Straub tail; eye prominence;
muscle tone; stereotypies; fecal consistency
activity; pupil size; urination
paralysis; click response; respiratory abnormalities
fasciculations; salivation; unkempt fur;
spasms; lacrimation; emaciation;
tremor; chromodacryorrhea; dehydration;
convulsions; rhinorrhea; distended abdomen.

Motor Activity

Motor activity was assessed after the conduct of the FOB in treatment week 12 (male animals) and 13 (female animals) as from approximately 3.5 to 4 hours after the end of the 6 h exposure period using an AMS Föhr Medical Instruments GmbH (FMI) activity measurement system.
Locomotor activity was measured quantitatively. Decreased or increased activity was recorded. Activity was recorded for 10-minute intervals over a period of 60 minutes. These data and the total activity over 60 minutes have been reported.


OTHER: no

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see below)
HISTOPATHOLOGY: Yes (see below)

Necropsy

After 13 Weeks of treatment: 28-Nov-2006 (40 males / 40 females)
All animals used during the treatment period of this study were transferred to the pathology unit on the scheduled day of necropsy (day 92), were
anaesthetised by an intraperitoneal injection of Eutha® 77 at a dose of at least 320 mg sodium pentobarbitone/kg body weight, weighed and
sacrificed by exsanguination. A complete examination was performed on all animals and all macroscopic abnormalities were described and reported.
Samples of the following tissues and organs were collected from all animals and fixed in neutral phosphate buffered 4 % formaldehyde solution,
unless otherwise stated (For paired organs, the left and the right one were examined). Lungs were instilled with this fixative at a hydrostatic pressure of 30 cm.

Adrenal glands; Aorta
Brain (cerebrum, cerebellum and brain stem);
Cecum; Colon; Duodenum
Epididymides (fixed in Bouin’s solution)
Esophagus; Extraorbital lacrimal glands
Eyes with optic nerves (fixed in Davidson’s solution)
Femur, including joint
Harderian glands (fixed in Davidson’s solution)
Heart
Ileum (including Peyer’s patches)
Jejunum (including Peyer’s patches)
Kidneys
Larynx (three transversal sections,at least levels II,
III and VI)
Liver
Lungs (infused with formalin; sections from 2 lobes)
Lymph nodes (mandibular, mesenteric, tracheobronchial)
Mammary gland area
Nasal cavities with ”paranasal sinuses”(Levels I to IV)
Nasopharyngeal duct and pharynx (1 longitudinal section)
Ovaries; Pancreas; Pituitary gland; Prostate; Rectum
Salivary glands – mandibular, sublingual
Sciatic nerve; Seminal vesicles; Skeletal muscle (thigh region)
Skin; Spinal cord - cervical, midthoracic, lumbar
Spleen; Sternum with bone marrow; Stomach
Testes (fixed in Bouin’s solution); Thymus
Thyroid gland with parathyroid gland; Tongue
Trachea (one transversal section below larynx)
Tracheal bifurcation, carina and mainstem bronchi
Urinary bladder (infused with formalin)
Uterus; Vagina
All gross lesions

Organ Weights

The following organ weights were recorded from all animals on the scheduled necropsy date and their ratios to terminal body weight and to brain
weight determined (The combined weight of paired organs was recorded):
Adrenal glands; Brain; Heart; Kidneys; Liver;
Lungs; Ovaries; Spleen; Testes; Thymus

Histotechnique / Histopathology

All organs and tissue samples collected at necropsy from all animals of Groups 1 (Air Control) and 4 (High Dose), and the lungs and nasal cavities
(Levels I to IV) of all animals of Groups 2 (Low Dose) and 3 (Mid Dose) were processed, embedded in paraffin, cut at a nominal thickness of
2-4 micrometers, stained with hematoxylin and eosin and histologically examined by use of a light microscope. Processiong and examination of the
nasal cavities was extended to the intermediate dose groups, because of treatment-related changes seen in these tissues in the high dose group. In
addition, all organs and tissues with gross lesions were processed and histologically examined. Attempts were made to correlate gross observations with microscopic findings.

Other examinations:

No

Statistics:

Statistics

The following statistical methods were used to analyze food consumption, body weight, clinical laboratory data, organ weights and ratios as well as macroscopic findings:

The Dunnett-test
[Dunnett 1955, details see Dunnett, C.W.: A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).] (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups with the air control group for each sex.

The Steel-test (many-one rank test)
[Miller 1981, details see Miller, R.G.: Simultaneous Statistical Inference, Springer Verlag, New York (1981).] was applied instead of the Dunnett-test
when the data could not be assumed to follow a normal distribution.

Fisher's exact-test
[Fisher 1950, details see Fisher, R.A.: Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950)]. Group means and standard
deviations were calculated for continuous data and medians for discrete data (scores), as appropriate.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY

Viability / Mortality
There were no premature deaths.

Clinical Signs

There were no clinical signs in test item treated animals during the present study. The only finding occasionally noted was hair loss in various body
regions in three females of the control group. This finding is occasionally seen in rats of this strain and age regardless of their treatment.

BODY WEIGHT AND WEIGHT GAIN

Body Weights

There were no statistically significant differences from controls in body weight and body weight gain at the low dose level (Group 2) throughout the
13-week treatment period. At the mid and high dose levels (Groups 3 and 4, respectively), male body weights and body weight gain were statistically
significantly lower than concurrent controls during most of the treatment period. Female body weights and, with one exception on Day 22, female
body weight gain were not statistically significantly affected by treatment with the test item. By the end of the 13-week treatment period, group mean
body weights differed from concurrent controls, see Table 3 below in “remarks on results including tables and figures”



FOOD CONSUMPTION

Food Consumption

Food Consumption and relative food consumption data were very limited, since they were recorded per cage of 5 animals and there were only 2
cages/group/sex. Therefore, and since spillage of food by individual animals cannot be ruled out, conclusions drawn from these data can only be
vague and tentative. Overall mean food consumption (g/animal/day) over the 13-week treatment period is demonstrated in Table 4, see below
(“remarks on results including tables and figures”)

Although a trend to reduced food consumption in treated animals might be concluded from these data, the change was considered to be minor in
degree.

FOOD EFFICIENCY

No clear pattern was evident in the relative food consumption data (relative to body weight; g / kg body weight / day), details see Table 5 in “remarks on results including tables and figures”

WATER CONSUMPTION

not examined

OPHTHALMOSCOPIC EXAMINATION

Ophthalmoscopic Examinations

There were no ophthalmic findings attributable to treatment with the test item.

HAEMATOLOGY

No adverse effects, details see clinical chemistry.

CLINICAL CHEMISTRY

Clinical Laboratory Investigations

There were no adverse effects on haematology, clinical biochemistry and urinalysis parameters.

A number of haematology and clinical biochemistry parameters differed statistically significantly from concurrent controls in one or three test item
treated groups. However, these differences were not considered to be toxicologically relevant, because they lacked dose relationship, were restricted to one sex and/or the respective group mean values were within the historical reference range.
The only differences from concurrent controls statistically significant in all three treated groups were slightly reduced globulin levels and slightly
increased albumin to globulin ratios. However, these changes were restricted to males and also for these parameters, all group mean values were
within the historical reference range. In addition, group mean urine volumes were lower in treated animals than in concurrent controls, but did not
attain statistical significance.

URINALYSIS

No adverse effects, details see clinical chemistry.

NEUROBEHAVIOUR

Functional Observational Battery

Functional observational battery (FOB) parameters did not appear to be affected by treatment with the test item, although the finding of Straub tail was seen in one mail and one female animal of Group 4 (High Dose). The toxicological relevance of this, rather unusual, finding remained unclear. Apart
from this, the findings noted were low in incidence, lacked dose relationship and/or were considered to be within the normal range of biological
variation.

Locomotor Activity

Intergroup comparison of the motor activity of treated animals with concurrent controls over a period of 60 minutes (analysed for 10-minute intervals) revealed statistically significantly (Steel test significant at 5%) increased motor activity in mid dose males at approximately 60 minutes,
in mid dose females at 50 and 60 minutes and for the data pooled over the total 60 minute period, and in high dose females at 50 minutes. These
increases were not attributed to treatment with the test item, because they lacked dose relationship and were evident only towards the end of the 60
minute recording period. At the low dose level, the motor activity data did not differ statistically significantly from concurrent controls throughout
the 60 minute recording period.

ORGAN WEIGHTS

After 13 weeks of treatment, terminal body weights did not differ statistically significantly from concurrent controls in males of the low dose group
(-4.7%), but were significantly lower than concurrent controls in males of the mid (-11.0%) and high dose (-12.7%) groups. Female terminal body
weights were unaffected by treatment with the test item at all dose levels. These findings were consistent with the effects on live body weight and body weight gain recorded during the treatment period. Effects on organ weight were evaluated by intergroup comparison of absolute organ weights and
their ratios to terminal body weight and to brain weight with concurrent controls. The following changes were noted:
- Absolute brain weights reduced in mid dose males (-4.3%),
- Absolute kidney weights reduced in mid dose males (-12.1%),
- Absolute lung weights reduced in mid dose males (-7.7%) and high dose males (-8.5%),
- Heart to body weight ratios increased in mid dose males (+9.3%) and high dose males (+11.7%),
- Testes to body weight ratios increased in mid dose males (+16.6%) and high dose males (+19.8%).
The reductions in absolute brain weight and kidney weight in mid dose males were considered to be incidental, because they were minor in degree,
were not dose-related and there were no confirmatory histopathology findings in the brain or kidneys in high dose animals.
The reductions in absolute lung weight and increases in heart to body weight and testes to body weight ratios in mid and high dose males were
considered be a reflection of the effects on body weight in these groups, rather than representing a primary effect of the test item on these organs.
Female absolute and relative organ weights were not affected by treatment with the test item.


GROSS PATHOLOGY

Macroscopic Findings

A number of macroscopic pathology findings were seen in male animals but none in females. These findings were considered to be within the range
of natural background findings occasionally seen in rats of this strain and age. They did not distinguish test item treated animals from concurrent
controls.


HISTOPATHOLOGY: NON-NEOPLASTIC

Microscopic Findings

The following microscopic findings in the nasal cavities were considered to distinguish test item treated rats from concurrent controls:

Nasal Cavities Level III
Intracytoplasmic hyaline droplets in olfactory epithelium,
minimal in degree in 2 low dose animals and 2 mid dose animals, and
minimal to slight in degree in 4 high dose animals.

Nasal Cavities Level IV
Intracytoplasmic hyaline droplets in olfactory and/or respiratory epithelium,
minimal to slight in degree in 5 control animals,
minimal in degree in 7 low dose animals
minimal to slight in degree in 9 mid dose animals, and
minimal to slight in degree in 14 high dose animals.
Unilateral focal degeneration of olfactory mucosa,
minimal in degree in 1 mid dose animal.
Squamous metaplasia of olfactory mucosa,
slight in degree in 1 mid dose animal, and
minimal in degree in 2 high dose animals.
Respiratory metaplasia of olfactory mucosa,
minimal in degree in 1 high dose animal.
The findings of degeneration, squamous metaplasia and respiratory metaplasia of olfactory mucosa in Level IV of the nasal cavities were considered
to represent local lesions of adverse character and were attributed to treatment with the test item.
A number of other microscopic findings noted in the present study were considered to represent findings commonly seen in rats of this strain and
age. Their incidence, distribution ormorphology did not provide any evidence for a relationship to treatment with the test item.


HISTOPATHOLOGY: NEOPLASTIC (if applicable)

not examined

HISTORICAL CONTROL DATA (if applicable)

no data

OTHER FINDINGS

No

Dose descriptor:

NOAEL

Remarks:

system

Effect level:

62.5 mg/m³ air

Sex:

male/female

Basis for effect level:

other: no adverse effects

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

10 mg/m³ air

Sex:

male/female

Basis for effect level:

other: histopathology; (organ weights)

Critical effects observed:

not specified

Table 3 - Body weight by the end of the 13-week treatment period

	Group 2 (Low Dose)	Group 3 (Mid Dose)	Group 4 (High Dose)
Males	-3.8% ns	-9.3%**	-9.3%**
Females	+1.3% ns	-0.9% ns	-4.3% ns

ns: No statistically significant difference; */** Dunnet-Test based on pooled variance significant at 5% (*) or 1% (**) level

Table 4 - Overall mean food consumption (g/animal/day) over the 13-week treatment period

	Group 1 (Air Control)	Group 2 (Low Dose)	Group 3 (Mid Dose)	Group 4 (High Dose)
Males	21.8	20.7	19.7	20.0
Females	16.2	15.4	15.0	15.1

Table 5 - Relative food consumption data (relative to body weight; g / kg body weight / day)

	Group 1 (Air Control)	Group 2 (Low Dose)	Group 3 (Mid Dose)	Group 4 (High Dose)
Males	63.1	62.1	62.5	63.4
Females	68.7	64.7	64.1	66.7

Conclusions:

The NOAEL (local) in rats exposed to METHACRYLAMIDE dust for 13 weeks is set at the lowest dose of 10 mg/m³. At higher concentrations
administered, local adverse effects as degeneration, sqamous metaplasia and respiratory metaplasia were found in nasal tissues (level IV).
In addition, a statistical significant decrease of body weight gains was observed in male animals of the mid- and high dose group.

There are no observations in this study which are assigned to systemic toxicity, especially any signs of neurotoxicity, which is a known toxic effect of METHACRYLAMIDE, are lacking. Therefore, the NOAEL (systemic) is set at the highest dose administered, 62.5 mg/m³.

Executive summary:

In a subchronic inhalation toxicity study [OECD TG 413],   METHACRYLAMIDE dust (purity 99.7 %) was administered to 40 male and 40 female wistar rats by nose only inhalation at nominal concentrations of 0, 10, 25, and 62.5  mg/m³ which is equivalent to 0, 0.01, 0.025, and 0.0625 mg/l for 6 hours per day, 5 days / week for a total of  13  weeks (65 exposures).

 The chemically determined mean aerosol concentrations were 10.24 (Group 2, Low Dose), 26.85 (Group 3, Mid Dose) or 69.95 (Group 4, High Dose) mg of METHACRYLAMIDE/m³. These aerosol concentrations were equivalent to mean aerosol concentrations of  9.45, 25.67 and 68.39 mg of METHACRYLAMIDE/m³ air in Groups 2, 3 and 4, respectively, when the attained ratios between the mean chemically determined aerosol concentrations and the respective mean gravimetric concentrations were applied to the overall mean gravimetric aerosol concentrations attained from 64 or 65 exposure days.

There were no premature deaths. Clinical signs attributable to treatment with the test item were not evident and functional observational battery (FOB) parameters did not appear to be affected by treatment with the test item. The toxicological relevance of the finding of Straub tail seen in one mail and one female animal of Group 4 (High Dose) remained unclear. Motor activity was not affected by treatment with the test item. Motor activity values on some occasions statistically significantly higher in Groups 3 (Mid Dose) and/or 4 (High Dose) than in concurrent controls were not attributed to treatment with the test item, because they lacked dose relationship and were evident only towards the end of the 60 minute recording period.

In males of Group 2 (Low Dose) and females of all treated groups relevant adverse effects on body weight and body weight gain were not evident. In males of Groups 3 (Mid Dose) and 4 (High Dose) body weight and body weight gain statistically significantly lower than concurrent controls during most of the treatment period were attributed to treatment with the test item. Absolute lung weight significantly lower and heart to body weight and testes to body weight ratios significantly higher in males of Groups 3 and 4 than in concurrent controls were considered to be a reflection of the effects on body weight in these groups, rather than representing a primary effect of the test item on these organs.

The findings of degeneration, squamous metaplasia and/or respiratory metaplasia of olfactory mucosa in Level IV of the nasal cavities affecting one female animal in Group 3 (Mid Dose) and two females in Group 4 (High Dose) were considered to represent local lesions of adverse character and were attributed to treatment with the test item. The minor increases in incidence and/or severity of intracytoplasmic hyaline droplets in nasal cavity levels III and IV were also attributed to treatment with the test item, but were not considered to be of adverse character, as this finding was also present in nasal cavity Level IV in Group 1 (Air Control).

The no-observed-adverse-effect-level for local effects [NOAEL(local)] was set at the low dose level administered, 10 mg/m³, because at the mid and high dose levels the findings of degeneration, squamous metaplasia and/or respiratory metaplasia of olfactory mucosa were evident in nasal cavity level IV and male body weight and body weight gain were statistically significantly lower than concurrent controls during most of the treatment period. However, the effects on body weight at the mid and high dose levels were considered to be only moderate in degree, as they were restricted to one gender, relevant body weight loss was not evident during the study, and by the end of the 13 week treatment period the group mean body weights of mid and high dose male animals were only 9.3% lower than that of concurrent control animals.

There were no changes observed in organs, tissues or other parameters investigated which were attributed to systemic toxicity. Especially, any evidence for neurotoxicity, which is the known critical effect of methacrylamide when administered at higher doses in oral and dermal studies, was lacking.

 

Therefore, the no-observed-adverse-effect-level for systemic effects [NOAEL(systemic)] was set at the high dose level administered, 62.5 mg/m³.

This subchronic inhalation toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic inhalation study OECD 413 in the rat. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

10 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

The subchronic inhalation toxicity study in the rat, on a read-across substance (methacrylamide) is acceptable and satisfies the guideline requirement for a subchronic inhalation study OECD 413 in the rat.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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

The oral administration of N-Isopropylmethacrylamide (NIPMAA) to rats for a period of up to eight weeks (including two weeks pre-pairing, gestation and early lactation period for females) at dose levels of up to 350 mg/kg bw/day A.I. resulted in treatment-related finding in animals of either sex from all treatment groups.

Clinical observations were confined to increased salivation detected in animals of either sex treated with 350 mg/kg bw/day A.I. from Day 14 to Day 38. One male from this treatment group also had pilo erection and hunched posture on Day 29. A reduction in

body weight gain was evident in males treated with 350 mg/kg bw/day A.I. during the first week of treatment and a reduction in overall body weight gain was also evident in these males. Food consumption for treated animals was comparable to controls however food efficiency was reduced during the first week of treatment in males treated with 350 mg/kg bw/day A.I.

Microscopic examinations revealed minimal axonal swelling with mineralisation in the sciatic nerve in animals of either sex from all treatment groups. Although a true dose related response was not evident, observations of this nature are not observed in controlanimals therefore this effect is related to treatment with the test item.

There were no toxicologically significant effects observed during the weekly open field arena observations or the haematological or blood chemical parameters measured.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The available OECD 422 study is assigned as reliability study 1 and is the only oral repeat dose toxicity study available on the substance itself.

Justification for classification or non-classification

The substance has been classified for:

Specific target organ toxicity - repeated exposure (STOT-RE) (Category 2):

H373: May cause damage to organs (Nervous system) through prolonged or repeated exposure.

This classification has been based on the following findings in the OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) conducted on the test substance.

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 (100, 190 and 350 mg/kg bw/day).

Although a true dose related response was not evident, observations of this nature were not observed in control animals therefore this effect was considered to be related to treatment with the test item.

A NOAEL for systemic toxicity was not established.

However, the findings observed at the dose level of 100 mg/kg bw/day and above were of minimal grade. There was no manifestation on the level of functional or behavioural parameters observed during the study up to the highest administered dose of 350 mg/kg bw/day.

On evaluation of all data, it is considered that classification for STOT-RE is appropriate, in Category 2, on the basis of observations from appropriate studies in experimental animals in which significant toxic effects, of relevance to human health, were produced at generally moderate exposure concentrations.