Ethyl methacrylate

Repeated dose toxicity: other routes

Administrative data

Endpoint:

sub-chronic toxicity: other route

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Upon review findings of the original paper were due to artifacts. This paper and its findings have been reviewed. There is a difference of opinion about the relevance of some of the the test results - see also Garman, 2002.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

neurotoxicity

Remarks:

subchronic

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Upon review, findings of the original paper of Abou-Donia et al. were due to artifacts. This paper and its findings have been reviewed. There is a difference of opinion about the relevance of some of the the test results - see also Garman, 2002.

Qualifier:

according to guideline

Guideline:

other: not known

Principles of method if other than guideline:

Intraperitoneal injection (i.P.): 0, 100, 200, 400 or 800 mg/kg/d; Drinking water: 0, 0.1, 0.2, or 0.5 %
For the i.p. study, 4 groups of 10 rats were randomly assigned to the treatment groups [Note: the authors stated that 3 groups were assigned; this was assumed to be an error]. A control group also was evaluated. For the drinking water study, animals were randomly assigned to one of three treatment groups or a control group (8 animals/group).
Dosing Procedures: For the i.p. study, animals were injected daily with EMA at dose levels of 0, 100, 200, 400 or 800 mg/kg. Doses were administered 7 days a week for a total of 60 days. For the drinking water study, the animals received fresh drinking water or drinking water containing EMA each day for a total 60 days.

GLP compliance:

not specified

Specific details on test material used for the study:

99% pure obtained from Aldrich Chemical Company (Milwaukee, WI); MW = 114.14; colorless liquid with a boiling point of 118 - 119°C, specific gravity = 0.918 and water solubility of 0.5 g/100 mL.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

Animals were obtained from Charles River, Raleigh, North Carolina, USA, weighing between 250-285 g. Animals were housed at 21-23 deg C with a 12 h light/dark cycle. They were supplied Purina certified rodent chow and tap water ad libitum. Acclimated for one week.

Route of administration:

other: intraperitoneal injection and drinking water

Duration of treatment / exposure:

60 days

Frequency of treatment:

Daily, single i.p. injection; ad libitum drinking water

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

i.p/ 1st segment

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

i.p./1st segment

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

i.p./1st segment

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

i.p./1st segment

Dose / conc.:

0.1 other: %

Remarks:

drinking water/ 2nd segment

Dose / conc.:

0.2 other: %

Remarks:

drinking water/ 2nd segment

Dose / conc.:

0.5 other: %

Remarks:

drinking water/ 2nd segment

No. of animals per sex per dose:

i.p: 10 males per group
d.w.: 8 males per group

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: Not applicable

Observations and clinical examinations performed and frequency:

Clinical observations were performed daily throughout the testing period. Animals were assessed for overall activity, posture, balance, breathing rate and evidence of diarrhea. Bodyweights were measured weekly. Behavioral Testing: In the i.p. study, behavioral testing was evaluated in a quiet room to which the animals had been acclimated. These observations were conducted 'blind' by a single observer. Although unclear from the publication, behavioral testing was assumed to be limited to Morris water maze and motor activity testing. Performance in the Morris water maze and spontaneous motor activity were assessed in the animals dosed with 0, 100, 200, and 400 mg/kg EMA i.p.. The water maze tests were conducted by allowing the rat to swim for 60 seconds or until it located and climbed onto the escape platform. The rats were given 5 trials per day with a one minute rest between each trial. Escape latencies were summed across trials. Motor activity was measured using an automated system that measured horizontal and vertical photo beam breaks. Photo beam interruptions were summed over a one-hour period.

Sacrifice and (histo)pathology:

After 60 days of test substance administration in the drinking water, the animals were anesthetized with 50-mg/kg i.p. injection of sodium pentobarbital and were intracardially perfused with saline, followed by 4% phosphate buffered paraformaldehyde (pH 7.4). The brain, spinal cord and sciatic nerve were harvested and fixed in the same fixative solution. The brain and spinal cord were embedded in paraffin wax, select samples of the sciatic nerve were embedded in epoxy resin and histopathology was conducted on these select tissues.

Statistics:

Data analysis: The Mann-Whitney U-test was used to determine treatment-related mortality and the Kruskal-Wallis test was used to determine dose dependence. For all parametric measures ANOVA, analysis of variance, was used to determine the treatment related effects. Effects were considered significant at p<0.05.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

Abou-Donia
--
Histopathology (non-neoplastic): yes

I.P.:
Clinical Conditions: No differences from the control group were observed in the 100 mg/kg-dose group; however, the author reported that the animals receiving 200 and 400 mg/kg appeared sluggish and lethargic, respectively. Animals receiving 800 mg/kg EMA exhibited severe lethargy, hunched posture, irregular breathing and slight diarrhea. At 200 mg/kg, bodyweight gain was significantly reduced, except during week 7. A transient decrease in body weight was observed in the third week and the first two weeks of test substance administration in the 400 and 800 mg/kg dose group, respectively. Significant mortality ['significant', statistics not mentioned] was observed in the animals dosed with 200, 400 or 800 mg/kg; however, this was not considered dose-dependent, probably because the earliest effects were seen in the 200 mg/kg/d dose group [no mortality reported in controls and at 100 mg/kg, 5/10 at 200 mg/kg, 6/10 at 400 mg/kg and all animals - 10/10 at, 800 mg/kg/d. Water maze, i.p. study: A significant decrease in the escape latency across a 5-day testing period is anticipated due to the learning of the platform location. The author reported that the rate of acquisition was dose-dependent with longer latency periods between the 400 mg/kg/day and the other dosages on days 3 and 5. Statistical evaluation showed that the 400-mg/kg dose group was different from both the low dose and the control group on day 3. On day 5, the 400-mg/kg dose group was different from that of the controls.

Motor activity: EMA treatment significantly reduced horizontal activity and rearing (vertical motion). The decrease in rearing was dose-dependent. A significant effect of trial and treatment was observed in horizontal and vertical activity; however, there was no significant interaction between trial and treatment.

Drinking Water:
There were no effects in the animals given 0.1% in the drinking water. Lethargy and alterations in gait were noted in the animals that received 0.2 and 0.5% of the test substance in drinking water, respectively. Data regarding the consumption of drinking water throughout the study were not reported. The author stated that the signs appeared dose related; however, it is unclear whether both signs were observed in both groups.

Bodyweights of the treated rats were similar to those of the controls.

No mortality was noted in any of the groups during the course of the study.

Histopathological observations [only drinking water study]: In the drinking water study, the authors reported the following findings: morphological variations in rats from each of the treatment groups; clusters of enlarged axons scattered throughout the dorsal, ventral and lateral columns of the spinal cord; and axonal enlargements clustered at internodal segments in the longitudinal sections of the spinal cord that appeared to be distributed along the length of individual axons (17 at 0.1 %, 14 at 0.2 % and 22 at 0.5 %). Although the number of clusters was statistically greater than that of the control group, they were not considered dose-dependent. An influence of the test material on water consumption has not been discussed in the paper. Such an influence cannot be excluded, but no evidence has been presented. A reduction of neurons in the sections of the ventral horn of the spinal cord was observed; however, these reductions were not dose-dependent (control: 22; 11 at 0.1 %, 15 at 0.2 % and 13 at 0.5 %). For some lesions the relationship to dose was not provided. Spongiform alterations in myelin and clusters of enlarged axons were found in the white matter tracts in the brainstems and forebrain. Two types of alterations were reported dispersed throughout the sciatic nerve of the treated animals; shrunken axons with separated myelin lamellae and large axons with thinner than normal myelin sheathes. The findings were observed at all three doses in a non-dose-related fashion.
--
Garman

The reviewing pathologist concluded that the empty spaces within the spinal cord sections that were originally reported to represent axonal enlargement actually represent foci of myelin artifact. In addition, these foci of myelin artifact were thought to be qualitatively and quantitatively similar between the control and EMA-treated groups. No treatment-related microscopic findings were found within the sections of sciatic nerve. Some evidence of myelin sheath separation was thought to represent handling artifact and/or normal Schmidt-Lanterman incisures. There was no evidence of any neuropathologic process within the sections
of brain examined (although only a representative number of the brain sections were examined microscopically).

Dose descriptor:

NOEL

Remarks:

i.p.

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

behaviour (functional findings)

Dose descriptor:

LOEL

Remarks:

drinking water

Effect level:

0.1 other: %

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: The OECD SIAR reported potential neurotoxicity with EMA citing this study but noted that a peer-review of the nervous system tissues collected in this study concluded that the reported effects were most consistent with foci of myelin artifacts.

Critical effects observed:

no

The following are the results from the original author: Histological examination: Spongiform alterations in fiber tracts of the forebrain, brainstem, and spinal cord. Clusters of axonal swellings were scattered throughout the dorsal, ventral, and lateral columns of the spinal cord, and typically involved internodal segments of two or three neighboring axons. Shrunken axons with separated myelin lamellae and large axons with thinner than normal myelin sheaths were apparent in the sciatic nerve. The patterns of alterations in the white matter of the spinal cord and the sciatic nerve are consistent with myelinopathy, but additional experiments are necessary to confirm whether oligodendroglia and Schwann cells are the primary sites of injury. In addition to the alterations associated with myelin, there was a decrease in the density of neurons in the ventral horn of the spinal cord.

Review of Garman:

Garman

The reviewing pathologist concluded that the empty spaces within the spinal cord sections that were originally reported to represent axonal enlargement actually represent foci of myelin artifact. In addition, these foci of myelin artifact were thought to be qualitatively and quantitatively similar between the control and EMA-treated groups. No treatment-related microscopic findings were found within the sections of sciatic nerve. Some evidence of myelin sheath separation was thought to represent handling artifact and/or normal Schmidt-Lanterman incisures. There was no evidence of any neuropathologic process within the sections of brain examined (although only a representative number of the brain sections were examined microscopically).

Conclusions:

The authors concluded that daily administration of the test substance can result in neurotoxicity in male rats. The results of the histological evaluations where morphological (histological) alterrations had been found in sections of brain, spinal cord and sciatic nerve at all dose levels in the drinking water study (0.1-0.5% in dw.) were subsequently deemed incorrect after further independent scientific review (Garman, 2002).

Executive summary:

The authors concluded that daily administration of the test substance can result in neurotoxicity in male rats. The results of the histological evaluations where morphological (histological) alterrations had been found in sections of brain, spinal cord and sciatic nerve at all dose levels in the drinking water study (0.1-0.5% in dw.) were subsequently deemed incorrect after further independent scientific review (Garman, 2002).

The OECD SIAR reported potential neurotoxicity with EMA citing this study but noted that a peer-review of the nervous system tissues collected in this study concluded that the reported effects were most consistent with foci of myelin artifacts.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

2000

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: not known

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl methacrylate

EC Number:

202-597-5

EC Name:

Ethyl methacrylate

Cas Number:

97-63-2

Molecular formula:

C6H10O2

IUPAC Name:

ethyl methacrylate

Details on test material:

99% pure obtained from Aldrich Chemical Company (Milwaukee, WI); MW = 114.14; colorless liquid with a
boiling point of 118 - 119°C, specific gravity = 0.918 and water solubility of 0.5 g/100 mL.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

Male Sprague-Dawley rats were obtained from Charles River  Laboratories. The animals were housed at 21 - 23°C  with a 12-hour  light/dark cycle. The animals were provided Purina certified rodent chow  ad libitum and the animals 
were acclimated to their surroundings for a  week prior to test substance administration. 

Administration / exposure

Route of administration:

other: Intraperitoneal injection and Drinking water

Duration of treatment / exposure:

60 days

Frequency of treatment:

Daily, single i.p. injection; ad libitum drinking water

Doses / concentrations

Remarks:

Doses / Concentrations:
Intraperitoneal injection (i.P.): 0, 100, 200, 400 or 800 mg/kg/d; Drinking water: 0, 0.1, 0.2, or 0.5 %

For the i.p. study, 4 groups  of 10 rats were randomly assigned to the treatment groups [Note: the  authors stated that 
3 groups were assigned; this was assumed to be an  error]. A control group also was evaluated. For the drinking water 
study,  animals were randomly assigned to one of three treatment groups or a  control group (8 animals/group).  

Dosing Procedures: For the i.p. study, animals were injected daily with  EMA at dose levels of 0, 100, 200, 400 
or 800 mg/kg. Doses were  administered 7 days a week for a total of 60 days. For the drinking water  study, the 
animals received fresh drinking water or drinking water  containing EMA each day for a total 60 days.  

No. of animals per sex per dose:

10 males per group

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: Not applicable

Examinations

Observations and examinations performed and frequency:

Clinical observations were performed daily  throughout the testing period. Animals were assessed for overall  
activity, posture, balance, breathing rate and evidence of diarrhea.  Bodyweights were measured weekly.  

Behavioral Testing: In the i.p. study, behavioral testing was evaluated  in a quiet room to which the animals 
had been acclimated. These  observations were conducted 'blind' by a single observer. Although  unclear 
from the publication, behavioral testing was assumed to be  limited to Morris water maze and motor activity 
testing. Performance in  the Morris water maze and spontaneous motor activity were assessed in the  animals 
dosed with 0, 100, 200, and 400 mg/kg EMA i.p.. The water maze  tests were conducted by allowing the rat to 
swim for 60 seconds or until  it located and climbed onto the escape platform. The rats were given 5  trials 
per day with a one minute rest between each trial.  Escape  latencies were summed across trials. Motor activity 
was measured using an  automated system that measured horizontal and vertical photo beam breaks.  Photo 
beam interruptions were summed over a one-hour period.

Sacrifice and pathology:

After 60 days of test substance administration in the drinking  water, the animals were anesthetized with 
50-mg/kg i.p. injection of  sodium pentobarbital and were intracardially perfused with saline,  followed by 
4% phosphate buffered paraformaldehyde (pH 7.4). The brain,  spinal cord and sciatic nerve were harvested 
and fixed in the same  fixative solution. The brain and spinal cord were embedded in paraffin  wax, select 
samples of the sciatic nerve were embedded in epoxy resin and  histopathology was conducted on these 
select tissues.

Statistics:

Data analysis: The Mann-Whitney U-test was used to determine  treatment-related mortality and the 
Kruskal-Wallis test was used to  determine dose dependence.  For all parametric measures ANOVA, 
analysis  of variance, was used to determine the treatment related effects.   Effects were considered 
significant at p<0.05.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

I.P.: 
Clinical Conditions:  No differences from the control group were observed  in the 100 mg/kg-dose group; however, 
the author reported that the  animals receiving 200 and 400 mg/kg appeared sluggish and lethargic,  respectively. 
Animals receiving 800 mg/kg EMA exhibited severe lethargy,  hunched posture, irregular breathing and slight diarrhea. 
At 200 mg/kg,  bodyweight gain was significantly reduced, except during week 7. A  transient decrease in body weight 
was observed in the third week and the  first two weeks of test substance administration in the 400 and 800 mg/kg  
dose group, respectively. Significant mortality ['significant',  statistics not mentioned] was observed in the animals 
dosed with 200, 400  or 800 mg/kg; however, this was not considered dose-dependent, probably  because the earliest 
effects were seen in the 200 mg/kg/d dose group [no  mortality reported in controls and at 100 mg/kg, 5/10 at 200 
mg/kg, 6/10  at 400 mg/kg and all animals - 10/10 at, 800 mg/kg/d.  

Water maze, i.p. study: A significant decrease in the escape latency  across a 5-day testing period is anticipated due 
to the learning of the  platform location. The author reported that the rate of acquisition was  dose-dependent with 
longer latency periods between the 400 mg/kg/day and  the other dosages on days 3 and 5. Statistical evaluation 
showed that the  400-mg/kg dose group was different from both the low dose and the control  group on day 3. 
On day 5, the 400-mg/kg dose group was different from  that of the controls.  

Motor activity, i.p. study: EMA treatment significantly reduced  horizontal activity and rearing (vertical motion). 
The decrease in  rearing was dose-dependent. A significant effect of trial and treatment  was observed in horizontal 
and vertical activity; however, there was no  significant interaction between trial and treatment.

Drinking Water: There were no effects in the animals given 0.1% in the  drinking water. Lethargy and alterations 
in gait were noted in the  animals that received 0.2 and 0.5% of the test substance in drinking  water, respectively. 
Data regarding the consumption of drinking water  throughout the study were not reported. The author stated 
that the signs  appeared dose related; however, it is unclear whether both signs were  observed in both groups. 

Bodyweights of the treated rats were similar to  those of the controls. 

No mortality was noted in any of the groups during  the course of the study. 

Histopathological observations [only drinking water study]: In the  drinking water study, the authors 
reported the following findings:  morphological variations in rats from each of the treatment groups;  
clusters of enlarged axons scattered throughout the dorsal, ventral and  lateral columns of the spinal 
cord; and axonal enlargements clustered at  internodal segments in the longitudinal sections of the 
spinal cord that  appeared to be distributed along the length of individual axons (17 at  0.1 %, 14 at 
0.2 % and 22 at 0.5 %). Although the number of clusters was  statistically greater than that of the 
control group, they were not  considered dose-dependent. An influence of the test material on water  
consumption has not been discussed in the paper. Such an influence cannot  be excluded, but no 
evidence has been presented. A reduction of neurons  in the sections of the ventral horn of the spinal 
cord was observed;  however, these reductions were not dose-dependent (control: 22; 11 at 0.1  %, 15
 at 0.2 % and 13 at 0.5 %). For some lesions the relationship to  dose was not provided. Spongiform 
alterations in myelin and clusters of  enlarged axons were found in the white matter tracts in the 
brainstems  and forebrain. Two types of alterations were reported dispersed  throughout the sciatic 
nerve of the treated animals; shrunken axons with  separated myelin lamellae and large axons with 
thinner than normal myelin  sheathes. The findings were observed at all three doses in a  
non-dose-related fashion.

Effect levels

1

Dose descriptor:

NOEL

Remarks:

i.p.

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

behaviour (functional findings)

2

Dose descriptor:

LOEL

Remarks:

d.w.

Effect level:

0.1 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: The OECD SIAR reported potential neurotoxicity with EMA citing this study but noted that a peer-review of the nervous system tissues collected in this study concluded that the reported effects were most consistent with foci of myelin artifacts.

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The authors concluded that daily administration of the test substance can result in neurotoxicity in male rats. The results of the histological evaluations were subsequently deemed incorrect after further scientific review
(Garman, 2002).

Executive summary:

In a reliable publsihed study, daily administration of the test substance can result in neurotoxicity in male rats. The results of the histological evaluations were subsequently deemed incorrect after further scientific review (Garman 2002).