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Toxicological information

Acute Toxicity: inhalation

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Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
other information
Study period:
06-10-1997 to 18-05-1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: 14 day Range-finding study for a 28 day subacute inhalation study, GLP
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
06-10-1997 to 18-05-1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Principles of method if other than guideline:
This study was designed to assess the potential inhalation toxicity of Methacrylamide, when administered by nose-only inhalation as a dust to Sprague-Dawley CD rats (5 males/group) for 6 hours per day, 7 days per week, for 2 weeks (a maximum of 14 exposures) at target concentrations of 12, 60 and 300 milligrams particulate per cubic meter of air (mg/m³). In addition, a control group (5 males) received air-only while in chamber exposures commenced 6 October 1997 and were completed on 19 October 1997.
Exposure levels were determined gravimetrically and by gas chromatography (GC) 4 times/chamber/day. Particle size distribution measurements were made once chamber/day/day using a TSI Aerodynamic Particle Sizer for the air control group and a Delron DCI-6 cascade impactor for the test material groups. Physical observations for abnormal signs were performed once during each exposure for all animals; detailed physical examinations were conducted on all animals twice pretest and weekly thereafter. Forelimb and hindlimb grip strength evaluations were performed on all animals pretest and on Day 14. Body weight measurements were recorded twice pretest, weekly thereafter and just prior to sacrifice. Food and water consumption measurements were conducted once pretest just prior to first exposure and weekly thereafter. Following 14 days of exposure, all animals were sacrificed, the brain and testes with epididymides) were weighed and organ/body and organ/brain weight ratios calculated.
Complete macroscopic postmortem examinations were conducted on all animals. Microscopic examination of peripheral nerves and gonads were conducted on all animals from the air control and high exposure level animals. Microscopic examination of respiratory tract, liver and kidneys were conducted on all animals from the air control, mid and high exposure level animals. The mean analytical exposure concentrations were determined to be 0.030, 12.8, 62.6 and 286 mg/m³, respectively, for the air control and the 3 test material exposed groups. The slight contamination of the air control samples was considered to have occurred during the analytical, not the exposure, process. Particle size distribution determinations showed the mass median aerodynamic diameter to be 2.20, 2.47 and 6.71 microns with a geometric standard deviation of 2.26, 2.28 and 2.50, respectively, for the 3 test material exposed groups. These results indicated that the aerosols for the groups II and III test material exposures were similar in size but that the Group IV aerosol was substantially larger in size. This disparity was probably the result of the electrostatic charge of the test material which was somewhat reduced by the use of a charge neutralizer.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Physical state: dust
- Supplier: Rohm Tech. Inc., Linden
- Analytical purity: > 99.2%
- Impurities (identity and concentrations): no data
- Purity test date: > 99.8% (measured by gas chromatography with FID-detector)
- Lot/batch No.: 111-601-10010
- Expiration date of the lot/batch: not available
- Storage condition of test material: refrigerated, no light.
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Kingston, New York, USA
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: mean: 186 g (range: 171 - 202 g)
- Fasting period before study: no data
- Housing: Animales were doubly housed in elevated, stainless steel, wire meash cages during the first week of the acclimation period and
individually housed during the remainder of the acclimation period and all other non-exposure periods.
- Diet: ad libitum; None was provided during exposure
- Water: ad libitum; None was provided during exposure
- Acclimation period: approximately 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24 °C (19 - 24 °C during exposure)
- Humidity (%): 31 - 62% (14 - 48% during exposure)
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light cycle via automatic timer
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
CHAMBER OPERATION:

The exposure chambers were operated dynamically under slight positive pressure at a calibrated airflow rate of 25.0 liters per minute (Lpm).
This flow rate was calculated to provide one complete air change every 1.6 minutes and a 99% equilibrium time (T99) of 7.4 minutes. This
chamber size and airflow rate was considered adequate to maintain the oxygen above 19%. At the end of each exposure, the animals remained
in the chamber for a minimum of 30 minutes during which clean air only flowed through the chamber. The nose-only chamber was grounded to
prevent a static charge from building. The chamber was exhausted through a system consisting of a coarse filter and a HEPA filter.
Recordings of airflow rate and static pressure were made every half-hour during exposure.

EXPOSURE PROCEDURE
Group I
Houseline air was delivered from a regulator and backpressure gauge via 1/4" plastic tubing into the inlet of a gas drying unit. The air exited the
drying unit via 1/4" plastic tubing and was directed into a plastic "Y" tube to split the airflow into a generation and dilution system.
For the generation system, air was delivered, via '/4" tubing , into a flowmeter. A metering valve was attached to the inlet of the flowmeter
to adjust the airflow rate. A backpressure gauge was attached to the outlet of the flowmeter. The air passed via '/4" plastic tubing, from the
gauge into the top of a nose-only exposure chamber. For the dilution system, air was directed into a flowmeter via 1/4" tubing. A metering valve was
attached to the inlet of the flowmeter to adjust the airflow rate. The dilution airflow was directed into the dilution port at the top of the nose-only
chamber via 1/4" tubing. The entire generation system and nose-only chamber was housed in a 10 m³ glass and stainless steel exposure chamber.
The total airflow was maintained at a rate of 25 Lpm for both the exposure and post-exposure to allow the chamber to clear.
Groups II - IV
Test material was jar milled, sieved and packed into labeled cups and mounted onto a dust feeder. The exposure was started with the dust
feeder at a selected gear ratio to produce a certain speed setting. Houseline air was delivered from a regulator and backpressure gauge via
1/4" plastic tubing into the inlet of a gas drying unit. The air exited the drying unit via 1/4" plastic tubing and was directed into a plastic "Y" tube
to split the airflow into a generation and dilution system. Speed settings were selected in order to achieve the target exposure levels. Specific initial
settings for each group are summarized as follows:

Group Speed settings (rpm)
--------------------------------
II 0.17
III 0.50
IV 2.0

For the generation system, air was delivered, via 1/4" tubing , into a flowmeter. A metering valve was attached to the inlet of the flowmeter to adjust
the airflow rate. A backpressure gauge was attached to the outlet of the flowmeter. The air passed via 1/4" plastic tubing, from the gauge to the inlet
of the dust feeder. A charge equilibrator was attached to the outlet of the dust feeder and into the nose-only exposure chamber. The test material
laden airstream was directed into the top of a nose-only exposure chamber. For the dilution system, air was directed into a flowmeter via 1/4" tubing.
A metering valve was attached to the inlet of the flowmeter to adjust the airflow rate. The dilution airflow was directed into the dilution port at the top
of the nose-only chamber via 1/4" tubing. The entire generation system and nose-only chamber was housed in a 10 m³ glass and stainless steel
exposure chamber. The total airflow was maintained at a rate of 25 Lpm for both the exposure and post-exposure to allow the chamber to clear.

EXPOSURE CHAMBER SAMPLING
CHAMBER SAMPLING
Chamber air samples were drawn four times per exposure at a rate of 10 Lpm through glass fiber filters mounted open-faced in a filter holder. A
flowmeter with a metering valve, a timer and a pump were used to pull the samples from the sampling ports, via 1/4" tubing. Unless otherwise
indicated, all samples were taken from the H-1 sampling port. The concentration of the test material in the chamber in mg/m³ was determined
gravimetrically by weighing the filter paper before and after sampling and dividing the difference in milligrams by the sample volume in liters and
multiplying by 1000 L/m³. The samples were stored in a refrigerator prior to being sent to Analytical Services for analysis.
The sample flowrates and durations are summarized below:

Sample Sample
Group Flowrate Duration
[Lpm] [min]
-------------------------------------
I 10 30
II 10 30
III 10 10
IV 10 2
-------------------------------------

The samples were also analyzed using a GC (Gas Chromatography). The analytical exposure concentrations in mg/m³ were calculated by dividing the quantity of test material detected in milligrams by volume of air sampled in liters and multiplied by 1000 L/m³.

PARTICLE SIZE DISTRIBUTION ANALYSIS
Group I
Particle size distribution samples were taken once per exposure using a TSI Aerodynamic Particle Sizer. A computer was used to program the system
to the appropriate settings. A printer was used to record the information. A sample was drawn once per exposure at a rate of 5.0 Lpm for 20 seconds.The samples were drawn from the H-1 sampling port of the chamber, using a stopper and '' 1/2" tubing connected to a 1/2" stainless steel tube,
which was extended from the chamber to the particle sizer. The particle size distributions were calculated by the computer and printed out.
Groups II - IV
Particle size distribution samples were taken once per exposure using a cascade impactor. Test atmosphere was drawn through plastic tubing
into the impactor by a pump. The airflow was regulated by a metering valve and a calibrated flowmeter. Plastic tubing connected the impactor
outlet, flowmeter and air pump. The flowrate was 12.6 Lpm. The pump was plugged into a timer which governed the sample duration. The
pump was exhausted via 1/4" tubing. The particle size distributions were calculated based in the amount of material collected on the greased
stainless steel slides or glass fiber filter of each stage as determined gravimetrically.

Group Sample duration [min]
--------------------------------------------
II 25
III 10
IV 5
--------------------------------------------


Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
14 days
Frequency of treatment:
6 hours per day, 7 days per week (14 exposures)
Dose / conc.:
0 mg/m³ air (nominal)
Remarks:
corresponding to 0.030 mg/m³ (analytical)
Dose / conc.:
12 mg/m³ air (nominal)
Remarks:
corresponding to 12.8 mg/m³ (analytical)
Dose / conc.:
60 mg/L air (nominal)
Remarks:
corresponding to 62.6 mg/m³ (analytical)
Dose / conc.:
300 mg/m³ air (nominal)
Remarks:
corresponding to 286 mg/m³ (analytical)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale:

EXPOSURE LEVEL JUSTIFICATION
A concentration of 300 mg/m³ would correlate to a dose of approximately 60 mg/kg/day and would therefore lie by a factor of 5 to 6 beneath the LD50 (i.v.) of 340 mg/kg. This measurement is based on a rat of 200 grams body weight, 6 hour exposure, a respiratory minute volume of
0.11 liters and deposition and resorption rate of 100%. According to experimental data published in the literature, the respiratory minute volume
of the rat, however, has to be considered as about 50% higher than that based on the Guyton-calculation (Guyton, 1947). Since consideration has
to be given to not only the dose deposited and absorbed in the lung but also the substance which is taken in by oral route, a 100% deposition rate, and for safety reasons, a 100% resorption rate, should be assumed. Therefore, a daily dose of 90 mg/kg/day would result from a concentration of 300 mg/m³ Even though this is only a factor of approximately 4 beneath the LD50 (i.v.) of 340 mg/kg/day, the Sponsor suggests the high
concentration to be 300 mg/m³, because by the assumption of a 100% deposition and retention rate, a safety-factor has been considered. The
medium and low concentrations should be 60 and 12 mg/m³.

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, once in the morning and once in the afternoon
- Cage side observations: mortality, general appearance and signs of severe toxic or pharmacologica effects, especially concerning the functional
indicators of a degeneration of peripheral nerves.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice pretest and weekly thereafter.

All visible animals were observed as a group at least once during each exposure.
Detailed Physical Examinations
Each animal was removed from its cage and examined. The results of the physical examinations were individually recorded. Examinations
included observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia as well as evaluations of
respiration, and palpation for tissue masses.


BODY WEIGHT: Yes
- Time schedule for examinations: : Twice pretest and weekly thereafter.


FOOD CONSUMPTION:
- Method
Feed was available without restriction 7 days/week. Animals were presented with full feeders weighing 570 grams (includes weight of feed, jar and lid). After the measurement period, feeders were reweighed using a Sartorius Universal Electronic Toploading Balance, Model U3600 (Sartorius
Corporation, Edgewood, New York) and the resulting weight was subtracted from the full feeder weight to obtain the grams consumed per animal.
- Frequency: Weekly, beginning one week prior to treatment.
- Calculation
grams of food consumed/kilogram of body weight/day (g/kg/ day)=

grams of food consumed
----------------------- divided # days (a)
body weight (kg) (b)

(a) Pretest measurement interval was 4 days; study period interval was 6 days.
(b) The average of the current and previous weight was used


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: No


WATER CONSUMPTION: Yes
- Time schedule for examinations: Pretest and weekly thereafter.


GRIP STRENGTH: Yes
- Time schedule for examinations: pretest and Test Day 14:


POSTMORTEM
NECROPSY INFORMATION
Method of Euthanasia

Exsanguination via the abdominal aorta following carbon dioxide
inhalation.

- Necropsy Interval
Termination: Test Day 15
- Number of Animals
Termination: 5 animals/group

Necropsy Order
One rat each was selected from the control, high-, mid- and low exposure level groups and necropsied in that order. This sequence was repeated
with additional rats until all animals had been
selected/necropsied.

MACROSCOPIC EXAMINATIONS
Complete macroscopic postmortem examinations were performed on all animals killed at a scheduled sacrifice interval immediately after death.
All abnormal observations were recorded. The macroscopic postmortem examination included examination of the external surface and all
orifices; the external surfaces of the brain and spinal cord; the organs and tissues of the cranial, thoracic, abdominal and pelvic cavities and neck;
and the remainder of the carcass.

ORGAN WEIGHTS
Brains and testes (with epididymides) were weighed for all animals at the scheduled sacrifice interval using a Mettler AK-160 (Mettler Instrument
Corporation, Hightstown, New Jersey). Prior to weighing, the organs were carefully dissected and properly trimmed to remove adipose and other
contiguous tissues in a uniform manner. Organs were weighed as soon as possible after dissection in order to avoid drying. Paired organs were
weighed together.

TISSUES PRESERVED AND EXAMINED HISTOPATHOLOGICALLY
The following tissues adrenal glands, brain, heart, kidneys, larynx, liver, lungs, nasopharygeal tissues, nerves, spleen, testes with epididymides and trachea were obtained at the scheduled sacrifice interval and preserved for all animals. In addition, slides of the indicated
tissues were prepared and examined microscopically for all animals in the control, mid and high-exposure groups. Any abnormalities not noted
during macroscopic examinations which were seen during microscopic examinations were recorded. Histopathological observations were recorded
as direct computer entry by the pathologist using the Xybion computer system. Microscopic data for individual animals were reviewed by the
pathologist for consistency following completion of slide reading, individual slides were reread when necessary and appropriate edits were made.
Statistics:
METHOD OF ANALYSIS
Mean values of all dose groups were compared to the mean value for the control group at each time interval.

Multiple Group Analysis
Statistical evaluation of equality of means was made by the appropriate one way analysis of variance technique, followed by a multiple comparison
procedure if needed. First, Bartlett's test (Snedecor and Cochran, 1967) was performed to determine if groups had equal variance. If the variances
were equal, parametric procedures were used; if not, nonparametric procedures were used. The parametric procedures were the standard one way
ANOVA (Snedecor and Cochran, 1967) using the F distribution to assess significance. If significant differences among the means were indicated,
Dunnett's test (Dunnett, 1955, 1964) was used to determine which means were significantly different from the control. If a nonparametric procedure
for testing equality of means was needed, the Kruskal-Wallis test (Hollander and Wolfe, 1973) was used, and if differences were indicated Dunn's
summed rank test (Hollander and Wolfe, 1973) was used to determine which treatments differed from control.
A statistical test for trend in the dose levels was also performed. In the parametric case (i.e., equal variance) standard regression techniques with a
test for trend and lack of fit were used (Snedecor, and Cochran,1967). In the nonparametric case Jonckheere's test (Hollander and Wolfe, 1973) for
monotonic trend was used.
The test for equal variance (Bartlett's) was conducted at the 1%, two-sided risk level. All other statistical tests were conducted at the 5% and 1%, two-
sided risk level.
Exceptions
Statistical evaluations were not performed when the standard deviation for the control group was 0 and/or N (number of animals) in the control
group was less than or equal to two. Dose groups were eliminated from statistical analysis if their standard deviation was 0 and/or N (number of
animals) in the group was less than or equal to two.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
All of the rats on test survived to the end of the study.
Body weight and weight changes:
no effects observed
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):
effects observed, treatment-related
Description (incidence and severity):
For water consumption, a significant increase was seen in the Group IV (300 mg/m³) animals compared to the Group I (0 mg/m³) animals in the second week of the study. The toxicological significance of this difference is unclear.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no adverse effects of the test material on brain or testes (with epididymides) weights after exposure to the test material for 14 days.
Values for the treated animals were comparable to those of the air control animals and there were no statistically significant differences.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic abnormalities in the rats examined postmortem.
Neuropathological findings:
no effects observed
Description (incidence and severity):
GRIP STRENGTH
There were no adverse effects of the test material on hindlimb or forelimb grip strength after exposure to the test material for 14 days. Values for the treated animals were comparable to those of the air control animals and there were no statistically significant differences.
SCATIC AND TIBLAL NERVES AND TESTES AND EPIDIDYMIDES.
There were no microscopic morphologic abnormalities in the sciatic and tibial nerves nor in the testes and epididymides of rats from the 0.0 and 300 mg/m³ exposure groups.

Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following tissues were examined by light microscopy: sciatic and tibial nerves, testes and epididymides, larynx, trachea, lungs, liver, kidneys and nasoturbinates including the nasopharynx. Findings related to the exposure to Methacrylamide were seen in the larynx and liver of rats from the 60 and 300 mg/m³ exposure levels.
LARYNX. Focal squamous/squamoid metaplasia (minimal) of the pseudostratified columnar epithelium covering the ventral seromucous gland was seen in 5/5 rats from the 300 mg/m³ and in 1/5 rats from the 60 mg/m³ exposure groups. The metaplastic epithelium was well differentiated with no evidence of atypia or dysplasia. This finding was considered to be a local, non-specific adaptive response and has been seen in rats of numerous studies following either nose-only or whole-body exposure to particulates, vapors and aerosols. Stratified squamous epithelium normally lines the upper portion of the larynx whereas the remainder is lined by pseudostratified columnar (ciliated/non-ciliated) epithelium. The pseudostratified columnar epithelium on the ventral floor of the larynx at the base of the epiglottis, cranial to the ventral diverticulum and overlying the seromucous glands, is especially sensitive to inhaled materials. Therefore, this is considered to be a target site for histopathological evaluations following inhalation exposure to particulates, vapors and aerosols ( John W. Sagartz, et. al., "Histological Sectioning of the Rodent Larynx for Inhalation Toxicity Testing", Toxicologic Pathology 20, No. 1 (1992), pp 118-121).

Other microscopic findings in the larynx occurred with comparable incidence and severity in the 0.0, 60 and 300 mg/m³ exposure groups or they occurred sporadically. These incidental findings have been seen in rats of this strain and age used in similar studies conducted in this facility; they were not considered to be related to the nose-only exposure to Methacrylamide.
LIVER: Hypertrophy of central lobular hepatocytes was seen in 4/5 rats from the 300 mg/m³ (slight to moderate) and in 2/5 rats from the 60 mg/m³ (minimal) exposure levels. Affected hepatocytes were enlarged and had an increased amount of granular eosinophilic cytoplasm. Although not performed in this study, the ultrastructural correlate to this finding is usually an increased amount and volume of smooth endoplasmic reticulum (SER) and possibly other subcellular organelles. Hypertrophy of central lobular hepatocytes may be considered to be an adaptive response of the liver to the presence of a number of xenobiotics and not a result of direct hepatotoxicity2. This change is generally considered to be reversible following cessation of treatment.
Other microscopic findings in the liver occurred with comparable incidence and severity in the 0.0, 60 and 300 mg/m³ exposure groups or they occurred sporadically. These incidental findings have been seen in rats of this strain and age used in similar studies conducted in this facility; they were not considered to be related to the nose-only exposure to Methacrylamide.

NASOTURBINAL TISSUES, TRACHEA, LUNGS AND KIDNEYS:
Microscopic findings in the nasoturbinal tissues (including the nasopharynx), trachea, lungs and kidneys occurred with comparable incidence and severity in rats from the 0.0, 60 and 300 mg/m³ exposure groups or they occurred sporadically. These incidental findings have been seen in rats of this strain and age used in similar studies conducted ose-only exposure to Methacrylamide.

Description (incidence and severity):
PHYSICAL OBSERVATIONS
IN-CHAMBER OBSERVATIONS
Test material exposed animals were comparable to the air control animals. A few observations of chromodacryorrhea and nasal discharge were seen but these were scattered amongst all of the exposure groups.
Dose descriptor:
NOAEL
Effect level:
12.8 mg/m³ air (analytical)
Sex:
male
Basis for effect level:
other: based on histopathological effects in the liver and larynx seen at higher levels of 62.6 and 286 mg/m³. However, these organs were not evaluated at the 12.8 mg/m³ level.
Critical effects observed:
not specified

All animals survived the duration of the study.

There were no test material effects seen with respect to clinical observations, body weights, food consumption, grip strength, organ weights, or macroscopic postmortem examinations. An increase in water consumption was seen in the high exposure level animals in the second week of exposure, although, the toxicological significance of this difference was unclear. Microscopic postmortem examinations revealed central lobular hepatocellular hypertrophy in the liver and squamous/squamoid metaplasia of the columnar epithelium covering the central seromucous gland in the larynx in the mid and high exposure level animals.

Conclusions:
Under the conditions of this study, the exposure level of 12.8 mg/m³ of Methacrylamide was considered to be the NOAEL based on histopathological effects in the liver and larynx seen at higher levels of 62.6 and 286 mg/m³. However, these organs were not evaluated at the 12.8 mg/m³ level.
Executive summary:

In a 14 day range-finding inhalation toxicity study Methacrylamide (99.8%) was administered to male Spraque-Dawley CD rats by nose only exposure at concentrations of 0, 12, 60 and 300 mg/m3 for 6 hours per day, 7 days/week for a total of 14 days. In addition , a control group (5 males) received air-only while in chamber. The mean analytical exposure concentrations were determined to be 0.030, 12.8, 62.6 and 286 mg/m³, respectively, for the air control and the 3 test material exposed groups. The slight contamination of the air control samples was considered to have occured during the analytical, not the exposure, process. Particle size distribution determinations showed the mass medial aerodynamic diameter to be 2.20, 2.47 and 6.71 microns with a geometric standard deviation of 2.26, 2.28 and 2.50, respectively, for the 3 test material exposed groups. These results indicated that the aerosols for the groups II and III test material exposures were similar in size but that the group IV aerosol was substantially larger in size. This disparity was probably the result of the electrostatic charge of the test material which was somewhat reduced by the use of a charge neutralizer.

All animals survived the duration of the study.

There were no test material effects seen with respect to clinical observations, body weights, food consumption, grip strength, organ weights, or macroscopic postmortem examinations. An increase in water consumption was seen in the high exposure level animals in the second week of exposure, although, the toxicological significance of this difference was unclear. Microscopic postmortem examinations revealed central lobular hepatocellular hypertrophy in the liver and squamous/squamoid metaplasia of the columnar epithelium covering the central seromucous gland in the larynx in the mid and high exposure level animals.

Under the conditions of this study the NOAEL is 12.8 mg/m³ Methacrylamide based on histopathological effects in the liver and larynx seen at higher levels of 62.6 and 286 mg/m³. However, these organs were not evaluated at the 12.8 mg/m³ level.

 

This 14 day range-finding toxicity study in the rat is acceptable and satisfies the requirements for a 14 day rang-finding inhalation study  in the rat.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Principles of method if other than guideline:
This study was designed to assess the potential inhalation toxicity of Methacrylamide, when administered by nose-only inhalation as a dust to
Sprague-Dawley CD rats (5 males/group) for 6 hours per day, 7 days per week, for 2 weeks (a maximum of 14 exposures) at target concentrations of
12, 60 and 300 milligrams particulate per cubic meter of air (mg/m³). In addition, a control group (5 males) received air-only while in chamber
exposures commenced 6 October 1997 and were completed on 19 October 1997. Exposure levels were determined gravimetrically and by gas
chromatography (GC) 4 times/chamber/day. Particle size distribution measurements were made once chamber/day/day using a TSI Aerodynamic
Particle Sizer for the air control group and a Delron DCI-6 cascade impactor for the test material groups. Physical observations for abnormal signs
were performed once during each exposure for all animals; detailed physical examinations were conducted on all animals twice pretest and weekly
thereafter. Forelimb and hindlimb grip strength evaluations were performed on all animals pretest and on Day 14. Body weight measurements were
recorded twice pretest, weekly thereafter and just prior to sacrifice. Food and water consumption measurements were conducted once pretest just
prior to first exposure and weekly thereafter. Following 14 days of exposure, all animals were sacrificed, the brain and testes with epididymides) were weighed and organ/body and organ/brain weight ratios calculated.
Complete macroscopic postmortem examinations were conducted on all animals. Microscopic examination of peripheral nerves and gonads were
conducted on all animals from the air control and high exposure level animals. Microscopic examination of respiratory tract, liver and kidneys were
conducted on all animals from the air control, mid and high exposure level animals.
The mean analytical exposure concentrations were determined to be 0.030, 12.8, 62.6 and 286 mg/m³, respectively, for the air control and the 3 test
material exposed groups. The slight contamination of the air control samples was considered to have occurred during the analytical, not the
exposure, process. Particle size distribution determinations showed the mass median aerodynamic diameter to be 2.20, 2.47 and 6.71 microns with
a geometric standard deviation of 2.26, 2.28 and 2.50, respectively, for the 3 test material exposed groups. These results indicated that the aerosols
for the groups II and III test material exposures were similar in size but that the Group IV aerosol was substantially larger in size. This disparity was
probably the result of the electrostatic charge of the test material which was somewhat reduced by the use of a charge neutralizer.
GLP compliance:
yes
Test type:
other: 14 day tange finding study
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:
2-methylprop-2-enamide
Test material form:
solid
Specific details on test material used for the study:
Physical state: dust
- Supplier: Rohm Tech. Inc., Linden
- Analytical purity: > 99.2%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Kingston, New York, USA
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: mean: 186 g (range: 171 - 202 g)
- Fasting period before study: no data
- Housing: Animales were doubly housed in elevated, stainless steel, wire meash cages during the first week of the acclimation period and
individually housed during the remainder of the acclimation period and all other non-exposure periods.
- Diet: ad libitum; None was provided during exposure
- Water: ad libitum; None was provided during exposure
- Acclimation period: approximately 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24 °C (19 - 24 °C during exposure)
- Humidity (%): 31 - 62% (14 - 48% during exposure)
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light cycle via automatic timer

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
CHAMBER OPERATION:

The exposure chambers were operated dynamically under slight positive pressure at a calibrated airflow rate of 25.0 liters per minute (Lpm).
This flow rate was calculated to provide one complete air change every 1.6 minutes and a 99% equilibrium time (T99) of 7.4 minutes. This
chamber size and airflow rate was considered adequate to maintain the oxygen above 19%. At the end of each exposure, the animals remained
in the chamber for a minimum of 30 minutes during which clean air only flowed through the chamber. The nose-only chamber was grounded to
prevent a static charge from building. The chamber was exhausted through a system consisting of a coarse filter and a HEPA filter.
Recordings of airflow rate and static pressure were made every half-hour during exposure.

EXPOSURE PROCEDURE
Group I
Houseline air was delivered from a regulator and backpressure gauge via 1/4" plastic tubing into the inlet of a gas drying unit. The air exited the
drying unit via 1/4" plastic tubing and was directed into a plastic "Y" tube to split the airflow into a generation and dilution system.
For the generation system, air was delivered, via '/4" tubing , into a flowmeter. A metering valve was attached to the inlet of the flowmeter
to adjust the airflow rate. A backpressure gauge was attached to the outlet of the flowmeter. The air passed via '/4" plastic tubing, from the
gauge into the top of a nose-only exposure chamber. For the dilution system, air was directed into a flowmeter via 1/4" tubing. A metering valve was
attached to the inlet of the flowmeter to adjust the airflow rate. The dilution airflow was directed into the dilution port at the top of the nose-only
chamber via 1/4" tubing. The entire generation system and nose-only chamber was housed in a 10 m³ glass and stainless steel exposure chamber.
The total airflow was maintained at a rate of 25 Lpm for both the exposure and post-exposure to allow the chamber to clear.
Groups II - IV
Test material was jar milled, sieved and packed into labeled cups and mounted onto a dust feeder. The exposure was started with the dust
feeder at a selected gear ratio to produce a certain speed setting. Houseline air was delivered from a regulator and backpressure gauge via
1/4" plastic tubing into the inlet of a gas drying unit. The air exited the drying unit via 1/4" plastic tubing and was directed into a plastic "Y" tube
to split the airflow into a generation and dilution system. Speed settings were selected in order to achieve the target exposure levels. Specific initial
settings for each group are summarized as follows:

Group Speed settings (rpm)
--------------------------------
II 0.17
III 0.50
IV 2.0

For the generation system, air was delivered, via 1/4" tubing , into a flowmeter. A metering valve was attached to the inlet of the flowmeter to adjust
the airflow rate. A backpressure gauge was attached to the outlet of the flowmeter. The air passed via 1/4" plastic tubing, from the gauge to the inlet
of the dust feeder. A charge equilibrator was attached to the outlet of the dust feeder and into the nose-only exposure chamber. The test material
laden airstream was directed into the top of a nose-only exposure chamber. For the dilution system, air was directed into a flowmeter via 1/4" tubing.
A metering valve was attached to the inlet of the flowmeter to adjust the airflow rate. The dilution airflow was directed into the dilution port at the top
of the nose-only chamber via 1/4" tubing. The entire generation system and nose-only chamber was housed in a 10 m³ glass and stainless steel
exposure chamber. The total airflow was maintained at a rate of 25 Lpm for both the exposure and post-exposure to allow the chamber to clear.

EXPOSURE CHAMBER SAMPLING
CHAMBER SAMPLING
Chamber air samples were drawn four times per exposure at a rate of 10 Lpm through glass fiber filters mounted open-faced in a filter holder. A
flowmeter with a metering valve, a timer and a pump were used to pull the samples from the sampling ports, via 1/4" tubing. Unless otherwise
indicated, all samples were taken from the H-1 sampling port. The concentration of the test material in the chamber in mg/m³ was determined
gravimetrically by weighing the filter paper before and after sampling and dividing the difference in milligrams by the sample volume in liters and
multiplying by 1000 L/m³. The samples were stored in a refrigerator prior to being sent to Analytical Services for analysis.
The sample flowrates and durations are summarized below:

Sample Sample
Group Flowrate Duration
[Lpm] [min]
-------------------------------------
I 10 30
II 10 30
III 10 10
IV 10 2
-------------------------------------

The samples were also analyzed using a GC (Gas Chromatography). The analytical exposure concentrations in mg/m³ were calculated by dividing the quantity of test material detected in milligrams by volume of air sampled in liters and multiplied by 1000 L/m³.

PARTICLE SIZE DISTRIBUTION ANALYSIS
Group I
Particle size distribution samples were taken once per exposure using a TSI Aerodynamic Particle Sizer. A computer was used to program the system
to the appropriate settings. A printer was used to record the information. A sample was drawn once per exposure at a rate of 5.0 Lpm for 20 seconds.The samples were drawn from the H-1 sampling port of the chamber, using a stopper and '' 1/2" tubing connected to a 1/2" stainless steel tube,
which was extended from the chamber to the particle sizer. The particle size distributions were calculated by the computer and printed out.
Groups II - IV
Particle size distribution samples were taken once per exposure using a cascade impactor. Test atmosphere was drawn through plastic tubing
into the impactor by a pump. The airflow was regulated by a metering valve and a calibrated flowmeter. Plastic tubing connected the impactor
outlet, flowmeter and air pump. The flowrate was 12.6 Lpm. The pump was plugged into a timer which governed the sample duration. The
pump was exhausted via 1/4" tubing. The particle size distributions were calculated based in the amount of material collected on the greased
stainless steel slides or glass fiber filter of each stage as determined gravimetrically.

Group Sample duration [min]
--------------------------------------------
II 25
III 10
IV 5
--------------------------------------------
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
14 d
Concentrations:
0, 12, 60 and 300 mg/m³ nominal concentration (0.030, 12.8, 62.6 and 286 mg/m³ mean analytical exposure)
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
For further information see chapter 7.5.3
Statistics:
For further information see chapter 7.5.3

Results and discussion

Effect levels
Sex:
male
Dose descriptor:
LCLo
Effect level:
286 mg/m³ air (analytical)
Exp. duration:
14 d
Remarks on result:
other: Range-finding study for a 28 day subacute toxicity study
Mortality:
no mortality observed
Clinical signs:
other: no effects ovserved
Body weight:
no effects observed
Gross pathology:
no effects observed
Other findings:
Microscopic postmortem examinations revealed central lobular hepatocellular hypertrophy in the liver and squamous/squamoid metaplasia of the columnar epithelium covering the central seromucous gland in the larynx in the mid and high exposure level animals.
An increase in water consumption was seen in the high exposure level animals in the second week of exposure, although, the toxicological significance of this difference was unclear.


NASOTURBINAL TISSUES
Microscopic findings in the nasoturbinal tissues (including the nasopharynx), trachea, lungs and kidneys occurred with comparable incidence and severity in rats from the 0.0, 60 and 300 mg/m³ exposure groups or they occurred sporadically. These findings have been seen in rats of this strain and age used in similar studies conducted ose-only exposure to Methacrylamide and were considered as signs of respiratory irritation.

Any other information on results incl. tables

There were no test material effects seen with respect to clinical observations, body weights, food consumption, grip strength, organ weights, or macroscopic postmortem examinations.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
For further information see chapter 7.5.3
Executive summary:

In a 14-d inhalation range-finding study for a 28-day subacute study 5 male Sprague Dawley rats per dose group were exposed to 0.030, 12.8, 62.6 and 286 mg/m³ dusty Methacrylamide (> 99,2 %)by inhalation (nose-only).

LCL0 (14d) males = 286 mg/m³