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Repeated dose toxicity: inhalation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Methacrylamide
EC Number:
201-202-3
EC Name:
Methacrylamide
Cas Number:
79-39-0
Molecular formula:
C4H7NO
IUPAC Name:
methacrylamide
Test material form:
solid
Specific details on test material used for the study:
- Name of test material (as cited in study report): Methacrylamide
-Supplier: Röhm GmbH, Darmstadt, Germany
- Physical state: solid (powder)
- Analytical purity: 99.7 %
- Impurities (identity and concentrations): -
- Purity test date: 99.7 %
- Lot/batch No.: 1160109
- Expiration date of the lot/batch: 28.02.2007
- Stability under test conditions: yes
- Storage condition of test material: refrigerator (+4 °C), away from direct sunlight

Test animals

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.

Administration / exposure

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.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/m³ air (nominal)
Dose / conc.:
10 mg/m³ air (nominal)
Remarks:
corresponding to a mean aerosol concentration of 10.24 µg/L/ mean chemical aerosol concentration of 9.45 µg/L
Dose / conc.:
25 mg/m³ air (nominal)
Remarks:
corresponding to a mean aerosol concentration of 26.85 µg/L/ mean chemical aerosol concentration of 25.67 µg/L
Dose / conc.:
62.5 mg/m³ air (nominal)
Remarks:
corresponding to a mean aerosol concentration of 69.95 µg/L/ mean chemical aerosol concentration of 68.39 µg/L
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

Examinations

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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
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.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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”

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.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
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 effects observed
Description (incidence and severity):
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 and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ophthalmic findings attributable to treatment with the test item.
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
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 findings:
no effects observed
Description (incidence and severity):
No adverse effects, details see clinical chemistry.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
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 pathological findings:
no effects observed
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):

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.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see details on results
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

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
system
Effect level:
62.5 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects
Remarks on result:
other: stand-alone bw effects only in males not considered as adverse
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
10 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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 [NOAEC(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