Registration Dossier

Administrative data

Description of key information

For EMA, no repeated dose studies are available. However, EMA is rapidly hydrolyzed to Methacrylic acid (MAA) and Ethanol (see chapter Toxicokinetics or Category document chapter 5). There are reliable chronic or subchronic studies from Methyl methacrylate (as MAA donor substance) and Ethanol which allow the assessment of repeated dose toxicity of EMA after oral and inhalative administration:

 

Systemic (oral)

MMA, chronic, rat, drinking water (2 yrs) NOAEL 2000 ppm (124/ 164 mg/kg bw/d for males/ females; no adverse effects observed; Borcelleca 1964)

EtOH, subchronic, rat, feed (90 d) NOAEL 2% (2400 mg/kg bw/d due to histopathological liver findings; Holmberg 1986)

 

Systemic (inhalation)

MAA, 90 d rat: NOAEC 100 ppm (BASF 2008)

MMA, chronic, rat (2 yrs) NOAEC 500 ppm (2080 mg/m3; due to histopathological brain findings; NTP 1986)

For systemic effects, the NOAEC for EMA was not derived. Instead, the DNEL for EMA is derived by interpolation between the DNELs based upon the MMA 2-year oral and inhalation studies and the DNELs based upon the oral 90 d study and the 28 d inhalation study on n-BMA.

 

Local (inhalation)

MAA, 90 d rat: NOAEC 100 ppm (BASF 2008)

MMA, 2 yr rat: NOAEC 25 ppm (104 mg/m3; Lomax 1997); However, the EU ESR (2002) and SCOEL review (2005) recognised differences between rodents and humans regarding the physiology of the nasal passages, metabolic activity result in the greater susceptibility of rodents compared with humans to inhaled esters. Consequently, a value of 50 ppm (SCOEL, 2005) is regarded as being the NOAEC(=DNEL) in humans.

n-BMA 28 d rat; 310 ppm (1832 mg/m3; Rohm & Haas 1993)

Ethanol, occupational health assessment, human: 500 ppm (960 mg/m3; BAuA 2017)

For local effects, the NOAEC for EMA is interpolated between the human NOAEC (IOLV documentation) derived by SCOEL (2005) for MMA and the NOAEC in the 28 d inhalation study with n-BMA (Rohm & Haas, 1993)


Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Read across from the methacrylic metabolite donor substance
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see attached category document

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached category document, chapter 1.1

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached category document, chapter 1

3. ANALOGUE APPROACH JUSTIFICATION
see attached category document, chapter 5 (Toxikokinetics) and endpoint specific chapters

4. DATA MATRIX
see attached category document, endpoint specific chapters
Qualifier:
equivalent or similar to
Guideline:
other: not known
Principles of method if other than guideline:
Chronic, repeated dose study with exposure via drinking water
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: "young" (unspecified)
The  animals were individually housed and provided food (finely ground Purina Dog Chow Kibbled Meal; questionable information in the publication) ad libitum
Route of administration:
oral: drinking water
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
polarographic analysis of monomer content
Duration of treatment / exposure:
104 weeks (2 years)
Frequency of treatment:
Daily, ad libitum
Remarks:
Doses / Concentrations:
6, 60 and 2000 ppm at the start of the study; raised after 5 months to 7, 70 and 2000 ppm (limited by palatability)
Basis:

Remarks:
Doses / Concentrations:
ca. 14, 137, 3360 ppm
Basis:
other: for females based on dietary equivalents of fluid consumption
Remarks:
Doses / Concentrations:
ca. 12, 115, 3210 ppm
Basis:
other: for males based on dietary equivalents of fluid consumption
No. of animals per sex per dose:
25
Control animals:
yes, concurrent no treatment
Details on study design:
Twenty-five male and female albino (Wistar) rats were administered 6, 60 or 2000 ppm of methyl methacrylate in the drinking water. The concentrations of the low- and mid-dose groups were increased to 7 and 70 ppm at the beginning of the fifth month of the study.
Prior to the start of the study, it was apparent that methyl methacrylate was volatilizing at the tip of the water bottles. A special design was employed to reduce the volatilization and measurements showed that the methyl methacrylate concentrations remained within 15% of nominal for 72 hours.
The low and medium concentrations in the water were selected with the expectation that the diet equivalents would approximate 10 and 100 ppm. The high concentration was selected following preliminary tests that indicated that this level would significantly depress fluid consumption.
Observations and examinations performed and frequency:
Body weights were measured prior to study initiation, at weeks 1, 3, 6, 13, 26, 52, 78 and 104. Food and water consumption was measured over a three day period at the end of one and four weeks, monthly through month six and during even months thereafter. Hematological measurements, including hematocrit, hemoglobin, total white and differential white cell counts, were obtained from five rats from each sex in each treatment level at three month intervals. Pooled urine samples were collected from five rats per sex from each treatment group every three months to evaluate urinary concentrations of reducing substances and proteins.
Sacrifice and pathology:
Semiquantitative tests for urinary concentrations of reducing substances and protein were performed on urines pooled from 5 rats/sex per group at three month intervals. At two years, survivors were sacrificed and organ to body weight measurements were made for heart, spleen, kidney, liver and testes. Tissues preserved from all animals on study included heart, lung, kidney, liver, urinary bladder, spleen, gastrointeric, skeletal muscle, bone marrow, skin, brain, thyroid, adrenal, pancreas, pituitary and gonad. Histopathology was conducted on all tissues collected except from animals in the low dose group.
Details on results:
Body weight depression observed at 2000 ppm did not persist beyond the first few weeks of the study. Significant depression of fluid consumption was observed at 2000 ppm, although this tended to regress at the end of the study. Individual observations of depressed food consumption tended to parallel periods of depressed growth. These effects were considered as temporary non-adverse effects.
There were significantly increased kidney weight ratios for female rats at 2000 ppm. Since no substance-related effects were reported from histopathologic examinations in the kidneys, this effect is not considered as biologically relevant.
Dose descriptor:
NOAEL
Effect level:
>= 124.1 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: based on fluid consumption and body weight (see attached document)
Dose descriptor:
NOAEL
Effect level:
>= 164 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: based on fluid consumption and body weight (see attached document)
Dose descriptor:
NOAEL
Effect level:
>= 2 000 other: ppm nominal
Sex:
male/female
Basis for effect level:
other: corresponding to ca. 3300 ppm in the diet on the basis of fluid and food consumption observations
Critical effects observed:
not specified

Mortality: A summary of the mortality data for methyl methacrylate is presented below.

Dose group (ppm)            Male           Female
Negative              (0)          12/25           9/25
                           6/7           7/25           7/25
          60/70          10/25           7/25
           2000          12/25          10/25

No statistical differences were noted in the mortality of the animals exposed to methyl methacrylate and those in the control group. A statistically significant decrease in body weight was observed in the first week for the female rats and in weeks one through three in the male rats administered 2000 ppm methyl methacrylate. Water consumption was reduced in the animals from the high-dose group; however, it was reported that this finding tended to regress towards the end of the study. Food consumption was not affected by the administration of methyl methacrylate in the drinking water. 

  

Hematologic values varied within normal ranges in all groups of rats throughout the study, and urine concentrations of protein and reducing substances showed no trends that appeared relatable to treatment.

Organ to body weight ratios obtained at sacrifice of 2-year survivors differed from the controls only in significantly increased kidney ratios in female rats receiving 2000 ppm of methyl methacrylate (controls 0.0082 ± 0.0019; treated 0.0094 ± 0.0011).

Histopathologic findings showed no abnormalities or lesions, in kind or incidence, not explicable on the basis of naturally occurring ones in this strain of rat at this age.

  

Diet equivalents of the test materials were calculated from the fluid and food consumption data.

In these calculations, corrections were not made for evaporation losses of the test materials from the drinking water, the orders of magnitude of which are given under methods described above (maximum 15%). Allowing for such losses, it would appear that the concentrations of test materials in the drinking water were equivalent to approximately 10, 100, and 3000 ppm in the diet.

Conclusions:
No relevant effects were observed up to the highest dose tested (2000 ppm, limited by palatability)
Executive summary:

No relevant effects were observed after exposure of rats in drinking water up to the highest dose tested (2000 ppm, limited by palatability).

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
evaluated by OECD SIDS (2004) as good quality supporting study
Justification for type of information:
Read across from the alcohol metabolite
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see attached category document

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached category document, chapter 1.1

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached category document, chapter 1

3. ANALOGUE APPROACH JUSTIFICATION
see attached category document, chapter 5 (Toxikokinetics) and endpoint specific chapters

4. DATA MATRIX
see attached category document, endpoint specific chapters
Reason / purpose:
read-across source
Principles of method if other than guideline:
other: not specified in OECD SIDS
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Age at study start: 43 days
Route of administration:
oral: feed
Details on route of administration:
ethanol in liquid diet
Vehicle:
water
Details on oral exposure:
Ethanol supplied in nutritionally balanced liquid diet.
Duration of treatment / exposure:
90 d
Frequency of treatment:
daily
Dose / conc.:
1 other: %
Remarks:
w/w in liquid diet
Dose / conc.:
2 other: %
Remarks:
w/w in liquid diet; calculated to be equivalent to 2400 mg/kg bw/d
Dose / conc.:
3 other: %
Remarks:
w/w in liquid diet
Dose / conc.:
4 other: %
Remarks:
w/w in liquid diet
Dose / conc.:
5 other: %
Remarks:
w/w in liquid diet
Dose / conc.:
10 other: %
Remarks:
w/w in liquid diet
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Statistics:
No statistical tests for significance were used.
Details on results:
Bodyweight: All groups gained weight though final weights decreased with dose.
Food/water consumption: Consumption in the 10% group was reduced relative to controls (182 ml diet/kg-d versus 195 ml diet/kg-d).
Clinical signs: No adverse signs were observed
Ophthalmology, haematology: Not examined.
Clinical biochemistry: Serum liver enzymes were not affected by treatment and kidney findings were minimal.
Mortality and time to death: No deaths occurred.
Gross pathology: Liver yellowing, dosage-related.
Histopathology: Hepatic centrilobular steatosis increased in severity with dose as did the frequency and severity of Mallory bodies (hyaline) and acidophilic degeneration and necrosis. Most liver findings were absent or mild at 2% w/w ethanol but became more significant at 3% and higher dose. Reticulo-endothelial cell proliferation was slight at 1 and 2%. A few kidney casts were noted in animals from the 1-3% dose groups and there were a few calcifications in the 3-5% groups. Slight tubular fatty change occurred in all groups.
Dose descriptor:
NOAEL
Effect level:
2 400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
2 400 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Executive summary:

This study was selected from a larger set of repeated dose studies with ethanol, as this study was selected by OECD SIDS (2004) as good quality supporting study with the lowest reliable NOAEL.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Read across from the methacrylic metabolite
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see attached category document

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached category document, chapter 1.1

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached category document, chapter 1

3. ANALOGUE APPROACH JUSTIFICATION
see attached category document, chapter 5 (Toxikokinetics) and endpoint specific chapters

4. DATA MATRIX
see attached category document, endpoint specific chapters
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Age when supplied; sex: about 7 weeks, male and female
Supplier: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
During the period when the rats were not exposed they were housed singly in wire cages (type DK III, Becker & Co., Castrop-Rauxel, FRG (floor area about 800 cm²)). Underneath the cages, waste trays were fixed containing bedding material (Type Lignocel FS14 fibres,
dustfree bedding supplied by SSNIFF, Soest, Germany)
The motor activity measurements were conducted in Polycarbonate cages with wire covers from Ehret, Emmendingen, FRG (floor area about 800 cm²) and bedding.
The animals were kept in fully air-conditioned rooms in which a temperature in the range of 20 - 24°C and relative humidity in the range of 30 - 70% were ensured by means of a central air-conditioning system.
A light/dark rhythm of 12 hours was maintained.
The room was completely disinfected using a disinfector ("AUTEX", fully automatic, formalinammonia-based terminal disinfector) before the start of the study. Usually, each week the floor and the walls were cleaned with water containing about 1 % Mikroquat®.
The animals were maintained on milled mouse/rat laboratory diet “GLP”, (Provimi Kliba SA, Kaiseraugst, Basel Switzerland) and tap water ad libitum.
Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
For each concentration, the test substance was supplied to the two-component atomizer of a thermostated vaporizer at a constant rate by means of the piston metering pump. The vapor was generated by spraying the substance with compressed air into a counter current of conditioned supply air (about 50% ± 20% relative humidity, 22°C ± 2°C). Thereafter it was further mixed with conditioned supply air and passed into the inhalation system.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the inhalation atmospheres in test groups 1 - 4 were analyzed by HPLC.
Duration of treatment / exposure:
90 days
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
Doses / Concentrations:
0, 70 mg/m³ (20 ppm), 141 mg/m³ (40 ppm), 352 mg/m³ (100 ppm ), 1232 mg/m³ (350 ppm )
Basis:

No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, sham-exposed
Details on study design:
Ten male and ten female Sprague Dawley rats per test group were whole body exposed to a vapor of the test substance on 6 hours per working day for 90 days (65 exposures). The target concentrations were 20, 40, 100 and 350 ppm (corresponding to 70, 141, 352 and 1232 mg/m3). A concurrent control group was exposed to conditioned air.
Observations and examinations performed and frequency:
The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays. The clinical condition of the test animals was recorded once during the preflow period and on post-exposure observation days and at least 3 times (before, during and after exposure) on exposure days.
During exposure only a group wise examination was possible.The body weight of the animals was determined at the start of the preflow, at the start of the exposure period and then, as a rule, once a week as well as one day prior to gross necropsy. As a rule, the animals were weighed at the same time of the day.
Body weight change was calculated as the difference between body weight on the respective exposure day and body weight on the day of the first exposure. Group means were derived from the individual differences.
Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.Food efficiency (group means) was calculated based upon individual values for body weight and food consumption.
Before the start of the exposure period (day -6) the eyes of all animals, and at the end of the study (day 82) the eyes of the animals of test group 0 (control group) and test group 4 (high concentration) were examined with an ophthalmoscope (HEINE Optotechnik, Herrsching, FRG)) for any changes in the refracting media.
Functional observation battery (FOB) was carried out on the assigned animals once before the exposure period and once against the end of the exposure period.Motor activity was measured on the same day and with the same animals as FOB was performed.
Sacrifice and pathology:
In the morning, blood was taken from the retro-orbital venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH Munich, Germany). The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. The urine samples were evaluated in a randomized sequence. At necropsy specimen were sampled from fasted anesthetized male animals in a randomized sequence for sperm analyses.
Hematological parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Furthermore differential blood smears were prepared and stained according to Wright without being evaluated.
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters.
With the exception of volume, color, turbidity, sediment examination and the specific gravity, all the urine constituents were determined semi-quantitatively using test strips (Combur-9-test M, Roche, Mannheim, Germany) and a reflection photometer (Miditron M; Roche, Mannheim, Germany).
Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals.
Sperm motility examinations were carried out in a randomized sequence. Sperm morphology and sperm head count (cauda epididymis and testis) were evaluated for the control and highest dose group, only.
The animals were killed under Narcoren anesthesia by exsanguination from the abdominal aorta and vena cava. The animals were then be necropsied and subjected to a grosspathological assessment. Animals that died intercurrently or were killed in a moribund state were necropsied and assessed by gross-pathology as quickly as possible.
Statistics:
DUNNETT, C.W. (1955): A multiple comparison procedure for comparing several treatments with a control. JASA, Vol. 50, 1096– 1121
DUNNETT, C.W. (1964). New tables for multiple comparisons with a control. Biometrics, Vol. 20, 482 –491
SIEGEL, S. (1956): Non-parametric statistics for the behavioural sciences. McGraw-Hill New York
Nijenhuis, A.; Wilf, H.S.(1978): Combinatorial Algorithms. AcademicPress New York, 32-33
Hettmansperger, T.P. ( 1984); Statistical Inference based on Ranks. John Wiley & Sons New York, 132-142
International Mathematical and Statistical Libraries, Inc., 2500 Park West Tower One, Houston, Texas 77042-3020, USA, nakl-1 -nakl-3
MILLER, R.G. (1981): Simultaneous Statistical Inference Springer-Verlag New York Inc., 165-167
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
Subchronic vapor inhalation of the test substance led to the following treatment-related
adverse effects:
Test group 4 (350 ppm):
􀂾 Decreased body weight of the males (- 6.1% to - 12.8%) from study day 7 onward
􀂾 Decreased body weight change (gain) of the males (- 28.5% to - 64.8%) from study
day 7 onward
􀂾 Decreased food consumption in the male animals on study days 7 (- 13.5%),
14 (- 12.2%), and from study day 28 through to day 84 (from - 9.4% to - 13.7%)
􀂾 Decreased food efficiency in the male animals on study days 7, 28, 49 and 56
􀂾 Decrease of terminal body weights in both sexes
􀂾 Goblet cell hypertrophy/hyperplasia in the respiratory epithelium of the nasal cavity
(level I) of two females
Test group 1 (20 ppm), test group 2 (40 ppm) and test group 3 (100 ppm):
􀂾 No treatment-related findings
Dose descriptor:
NOAEC
Effect level:
100 ppm
Sex:
male/female
Basis for effect level:
other: = 352 mg/m³ for local effects and effects to body weight
Dose descriptor:
NOAEC
Effect level:
350 ppm
Sex:
male/female
Basis for effect level:
other: = 1232 mg/m³ for systemic effects in target organs other than body weight effects due to reduced food consumption
Dose descriptor:
LOAEC
Effect level:
350 ppm
Sex:
male/female
Basis for effect level:
other: = 1232 mg/m³ air; reduced food consumption and body weight gain
Dose descriptor:
LOAEC
Effect level:
350 ppm
Sex:
female
Basis for effect level:
other: = 1232 mg/m³ for local effects (goblet cell hypertrophy/hyperplasia) in the respiratory epithelium in 2/10 females
Critical effects observed:
not specified
Conclusions:
In a valid guideline study, the the no-observed adverse effect level (NOAEL) is 100 ppm for the male and female rats exposed by whole body inhalation for 90 days.
Executive summary:

In a valid guideline study acc OECD 413 ( Subchronic inhalation toxicity: 90 day exposure of rats) methacrylic acid induced signs of general toxicity as indicated by descreased body weight, body weight gain, food consumption and transiently food efficiency in the high concentration male animals. At a concentration as high as 350 ppm (1232 mg/m³), the local irritating effect was marginal, indicated by the hypertrophy/hyperplasia of the respiratory epithelium in the nasal cavity of two female animals. Substance-related changes of the sexual organs were not noted in any of the exposed animals, nor were there any changes of sperm mobility and sperm head counts. Under the current test conditions, the no-observed adverse effect level (NOAEL) in this study is 100 ppm (352 mg/m³) for the male and female rats.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint:
chronic toxicity: inhalation
Remarks:
combined repeated dose and carcinogenicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Remarks:
NTP standard protocol, cancer bioassay with limited dose range
Justification for type of information:
Read across from the methacrylic metabolite donor substance
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see attached category document

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached category document, chapter 1.1

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached category document, chapter 1

3. ANALOGUE APPROACH JUSTIFICATION
see attached category document, chapter 5 (Toxikokinetics) and endpoint specific chapters

4. DATA MATRIX
see attached category document, endpoint specific chapters
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: vapour
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
2 years
Frequency of treatment:
6 h / day, 5 d / wk
Remarks:
Doses / Concentrations:
0, 500, 1000 ppm for males
Basis:
other: analytically confirmed nominal concentrations
Remarks:
Doses / Concentrations:
0, 250, 500 ppm for females
Basis:
other: analytically confirmed nominal concentrations
No. of animals per sex per dose:
50
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: random (table)
- Dose selection rationale: Based on the results of several preliminary studies up to 90 d duration
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 2/d

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at weighing

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for the first 13 weeks, monthly afterwards

FOOD CONSUMPTION, FOOD EFFICIENCY and WATER CONSUMPTION: no data
HAEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS, NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY and HISTOPATHOLOGY: Yes
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
URT
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
no relevant effect in all investigated tissues
Dose descriptor:
LOAEC
Remarks:
for local effects in the URT (no NOAEC identified)
Effect level:
250 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Histopathology (Irritation)
Dose descriptor:
NOAEC
Remarks:
for systemic effects
Effect level:
500 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Gross pathology, Histopathology (organ effects)
Dose descriptor:
NOAEC
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Gross pathology, Histopathology (organs)
Critical effects observed:
not specified

Mortality: No difference in survival between treated and untreated groups.

Histopathology:

Site / Lesion

Males

Control

[0 ppm]

Low. Conc.

[500 ppm]

High Conc.

[1000 ppm]

Nasal Cavity /

           Serous inflammation

           Suppurative inflammation

 

 0/50

11/50

 

37/50

21/50

 

44/50

30/50

Olfactory sensory epithelium /

           Degeneration

 

 7/50

 

39/50

 

42/50

Lung /

           Alveolar macrophages

           Focal or multifocal fibrosis

 

 6/49

 6/49

 

20/49

 6/49

 

16/50

 5/50

 

 

 

 

Site / Lesion

Females

Control

[0 ppm]

Low. Conc.

[250 ppm]

High Conc.

[500 ppm]

Nasal Cavity /

           Serous inflammation

           Suppurative inflammation

 

4/50

7/50

 

17/50

12/50

 

32/50

12/50

Olfactory sensory epithelium /

           Degeneration

 

2/50

 

39/50

 

44/50

Lung /

           Alveolar macrophages

           Focal or multifocal fibrosis

 

9/50

1/50

 

14/50

 2/50

 

16/50

 7/50

 

No histopathological findings other than local findings in the respiratory tract. Systemic histopathological effects, as for example in the brain in females particularly at 2000 ppm and above in the subchronic range finding study (Batelle, 1980), are absent in this 104 week study.

Body weight: Mean body weight gain was reduced in females at 500 ppm resulting in 6 -11% lower body weights after week 73 and in males at 1000 ppm which were 5 -10 % lower than controls after week 81.

There was no treatment-related increase in tumour incidence.

Conclusions:
The primary finding in this study was inflammation of rat nasal cavity as well as olfactory epithelial degeneration at all exposure levels in male and female rats. For local effects the LOAEC was 250 ppm in this study while a NOAEC could not be found. 
In contrast to the 90 d range finding study with histopathological changes in females at exposures of 1000 ppm and above (Battelle, 1980), no other significant histopathological changes were reported in male and female rats after 104-week exposures to MMA vapour in this study. Based on this a NOEC for systemic effects of 500 ppm is derived.
Executive summary:

In this104-week study with groups of 50 animals each, male rats were treated with MMA vapour by whole-body exposure to 500 or 1000 ppm while female rats were exposed to 250 or 500 ppm.

 

The primary finding was inflammation of rat nasal cavity as well as olfactory epithelial degeneration at all exposure levels in male and female rats. For local effects the LOAEC was 250 ppm in this study while a NOAEC could not be found. 

In contrast to the 90 d range finding study with histopathological changes in females at exposures of 1000 ppm and above (Battelle, 1980), no other significant histopathological changes were reported in male and female rats after 104-week exposures to MMA vapour in this study. Based on this a NOEC for systemic effects of 500 ppm is derived.

 

Male and female rat body weights were lower at the 1000 ppm (5-10%) and 500 ppm (6-11%) exposure levels, respectively, presumably due to reduced food consumption due to nasal irritation and damage of olfactory epithelium. While food consumption was not recorded in this study this association is confirmed by two other studies, the developmental toxicity study with MMA with reduced food consumption and reduced body weight gain at concentrations higher than 99 ppm (Solomon, 1993) and a subchronic inhalation study with methacrylic acid where there was also an association of irritative effects in the nose and reduced food consumption and reduced body weight gain (BASF, 2008). Consequently, reduced body weight gain, while clearly treatment-related - is considered to be secondary to the local effects in the nose and not the result of true systemic toxicity.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Read across from the methacrylic metabolite
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see attached category document

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached category document, chapter 1.1

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached category document, chapter 1

3. ANALOGUE APPROACH JUSTIFICATION
see attached category document, chapter 5 (Toxikokinetics) and endpoint specific chapters

4. DATA MATRIX
see attached category document, endpoint specific chapters
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Age when supplied; sex: about 7 weeks, male and female
Supplier: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
During the period when the rats were not exposed they were housed singly in wire cages (type DK III, Becker & Co., Castrop-Rauxel, FRG (floor area about 800 cm²)). Underneath the cages, waste trays were fixed containing bedding material (Type Lignocel FS14 fibres,
dustfree bedding supplied by SSNIFF, Soest, Germany)
The motor activity measurements were conducted in Polycarbonate cages with wire covers from Ehret, Emmendingen, FRG (floor area about 800 cm²) and bedding.
The animals were kept in fully air-conditioned rooms in which a temperature in the range of 20 - 24°C and relative humidity in the range of 30 - 70% were ensured by means of a central air-conditioning system.
A light/dark rhythm of 12 hours was maintained.
The room was completely disinfected using a disinfector ("AUTEX", fully automatic, formalinammonia-based terminal disinfector) before the start of the study. Usually, each week the floor and the walls were cleaned with water containing about 1 % Mikroquat®.
The animals were maintained on milled mouse/rat laboratory diet “GLP”, (Provimi Kliba SA, Kaiseraugst, Basel Switzerland) and tap water ad libitum.
Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
For each concentration, the test substance was supplied to the two-component atomizer of a thermostated vaporizer at a constant rate by means of the piston metering pump. The vapor was generated by spraying the substance with compressed air into a counter current of conditioned supply air (about 50% ± 20% relative humidity, 22°C ± 2°C). Thereafter it was further mixed with conditioned supply air and passed into the inhalation system.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the inhalation atmospheres in test groups 1 - 4 were analyzed by HPLC.
Duration of treatment / exposure:
90 days
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
Doses / Concentrations:
0, 70 mg/m³ (20 ppm), 141 mg/m³ (40 ppm), 352 mg/m³ (100 ppm ), 1232 mg/m³ (350 ppm )
Basis:

No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, sham-exposed
Details on study design:
Ten male and ten female Sprague Dawley rats per test group were whole body exposed to a vapor of the test substance on 6 hours per working day for 90 days (65 exposures). The target concentrations were 20, 40, 100 and 350 ppm (corresponding to 70, 141, 352 and 1232 mg/m3). A concurrent control group was exposed to conditioned air.
Observations and examinations performed and frequency:
The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays. The clinical condition of the test animals was recorded once during the preflow period and on post-exposure observation days and at least 3 times (before, during and after exposure) on exposure days.
During exposure only a group wise examination was possible.The body weight of the animals was determined at the start of the preflow, at the start of the exposure period and then, as a rule, once a week as well as one day prior to gross necropsy. As a rule, the animals were weighed at the same time of the day.
Body weight change was calculated as the difference between body weight on the respective exposure day and body weight on the day of the first exposure. Group means were derived from the individual differences.
Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.Food efficiency (group means) was calculated based upon individual values for body weight and food consumption.
Before the start of the exposure period (day -6) the eyes of all animals, and at the end of the study (day 82) the eyes of the animals of test group 0 (control group) and test group 4 (high concentration) were examined with an ophthalmoscope (HEINE Optotechnik, Herrsching, FRG)) for any changes in the refracting media.
Functional observation battery (FOB) was carried out on the assigned animals once before the exposure period and once against the end of the exposure period.Motor activity was measured on the same day and with the same animals as FOB was performed.
Sacrifice and pathology:
In the morning, blood was taken from the retro-orbital venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH Munich, Germany). The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. The urine samples were evaluated in a randomized sequence. At necropsy specimen were sampled from fasted anesthetized male animals in a randomized sequence for sperm analyses.
Hematological parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Furthermore differential blood smears were prepared and stained according to Wright without being evaluated.
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters.
With the exception of volume, color, turbidity, sediment examination and the specific gravity, all the urine constituents were determined semi-quantitatively using test strips (Combur-9-test M, Roche, Mannheim, Germany) and a reflection photometer (Miditron M; Roche, Mannheim, Germany).
Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals.
Sperm motility examinations were carried out in a randomized sequence. Sperm morphology and sperm head count (cauda epididymis and testis) were evaluated for the control and highest dose group, only.
The animals were killed under Narcoren anesthesia by exsanguination from the abdominal aorta and vena cava. The animals were then be necropsied and subjected to a grosspathological assessment. Animals that died intercurrently or were killed in a moribund state were necropsied and assessed by gross-pathology as quickly as possible.
Statistics:
DUNNETT, C.W. (1955): A multiple comparison procedure for comparing several treatments with a control. JASA, Vol. 50, 1096– 1121
DUNNETT, C.W. (1964). New tables for multiple comparisons with a control. Biometrics, Vol. 20, 482 –491
SIEGEL, S. (1956): Non-parametric statistics for the behavioural sciences. McGraw-Hill New York
Nijenhuis, A.; Wilf, H.S.(1978): Combinatorial Algorithms. AcademicPress New York, 32-33
Hettmansperger, T.P. ( 1984); Statistical Inference based on Ranks. John Wiley & Sons New York, 132-142
International Mathematical and Statistical Libraries, Inc., 2500 Park West Tower One, Houston, Texas 77042-3020, USA, nakl-1 -nakl-3
MILLER, R.G. (1981): Simultaneous Statistical Inference Springer-Verlag New York Inc., 165-167
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
Subchronic vapor inhalation of the test substance led to the following treatment-related
adverse effects:
Test group 4 (350 ppm):
􀂾 Decreased body weight of the males (- 6.1% to - 12.8%) from study day 7 onward
􀂾 Decreased body weight change (gain) of the males (- 28.5% to - 64.8%) from study
day 7 onward
􀂾 Decreased food consumption in the male animals on study days 7 (- 13.5%),
14 (- 12.2%), and from study day 28 through to day 84 (from - 9.4% to - 13.7%)
􀂾 Decreased food efficiency in the male animals on study days 7, 28, 49 and 56
􀂾 Decrease of terminal body weights in both sexes
􀂾 Goblet cell hypertrophy/hyperplasia in the respiratory epithelium of the nasal cavity
(level I) of two females
Test group 1 (20 ppm), test group 2 (40 ppm) and test group 3 (100 ppm):
􀂾 No treatment-related findings
Dose descriptor:
NOAEC
Effect level:
100 ppm
Sex:
male/female
Basis for effect level:
other: = 352 mg/m³ for local effects and effects to body weight
Dose descriptor:
NOAEC
Effect level:
350 ppm
Sex:
male/female
Basis for effect level:
other: = 1232 mg/m³ for systemic effects in target organs other than body weight effects due to reduced food consumption
Dose descriptor:
LOAEC
Effect level:
350 ppm
Sex:
male/female
Basis for effect level:
other: = 1232 mg/m³ air; reduced food consumption and body weight gain
Dose descriptor:
LOAEC
Effect level:
350 ppm
Sex:
female
Basis for effect level:
other: = 1232 mg/m³ for local effects (goblet cell hypertrophy/hyperplasia) in the respiratory epithelium in 2/10 females
Critical effects observed:
not specified
Conclusions:
In a valid guideline study, the the no-observed adverse effect level (NOAEL) is 100 ppm for the male and female rats exposed by whole body inhalation for 90 days.
Executive summary:

In a valid guideline study acc OECD 413 ( Subchronic inhalation toxicity: 90 day exposure of rats) methacrylic acid induced signs of general toxicity as indicated by descreased body weight, body weight gain, food consumption and transiently food efficiency in the high concentration male animals. At a concentration as high as 350 ppm (1232 mg/m³), the local irritating effect was marginal, indicated by the hypertrophy/hyperplasia of the respiratory epithelium in the nasal cavity of two female animals. Substance-related changes of the sexual organs were not noted in any of the exposed animals, nor were there any changes of sperm mobility and sperm head counts. Under the current test conditions, the no-observed adverse effect level (NOAEL) in this study is 100 ppm (352 mg/m³) for the male and female rats.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Method and results sufficiently described, similar to OECD-guideline 453.
Justification for type of information:
Read across from the methacrylic metabolite donor substance
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see attached category document

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached category document, chapter 1.1

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see attached category document, chapter 1

3. ANALOGUE APPROACH JUSTIFICATION
see attached category document, chapter 5 (Toxikokinetics) and endpoint specific chapters

4. DATA MATRIX
see attached category document, endpoint specific chapters
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. (Wilmington, USA)
- Housing: group-housed by sex in wire mesh cages (seven per cage)
- Diet (e.g. ad libitum): Purina Laboratory Chow
- Water (e.g. ad libitum): water unspecified
- Acclimation period: 19 d


ENVIRONMENTAL CONDITIONS
not reported
Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
1. Original study (Reno FE, 1979):  The test substance was administered in 6000-liter inhalation chambers with pyramidal tops and bottoms, under  dynamic conditions of 1000 liters/minute of airflow. Control animals were exposed to filtered room air in the same manner as the treated animals. 
2. Re-Evaluation of the study (Lomax LG et al., 1997):  Animals were exposed to the test substance vapor in 6000 liter inhalation chambers under dynamic conditions of 1000  liters/minute of air flow. The control animals were exposed to filtered room air in a chamber with similar air-flow characteristics.  
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of MMA in each chamber was monitored, when possible, at hourly intervals during each 6-hr exposure using an Infrared 6-Station Analyzer (Wilks-Miran, Foxboro, MA, USA).
Duration of treatment / exposure:
2 years (104 weeks)
Frequency of treatment:
6 hr/day, 5 days / week
Remarks:
Doses / Concentrations:
25, 100 and 400 ppm (corresponding to ca. 0.10, 0.41 and 1.64 mg/L, respectively)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
25.0, 99.8 and 396.1 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
70 males and 70 females were assigned to each of the four exposure groups (including the  control). 
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: Not applicable
Observations and examinations performed and frequency:
1. Original study (Reno FE, 1979):
-NOTE: This study is also summarized with further evaluation of nasal passage microscopy in Lomax et al. (1997).
Methods: Groups of 70 male and 70 female (total animals = 280 males and 280 females) rats were randomly assigned to either a control or one of three test groups. The animals were group housed (7/cage) and provided food and water ad libitum, except during the exposure period. The animals were exposed to the test substance six hours a day five days per week for a total of 104 - 106 weeks. The test substance was administered in 6000-liter inhalation chambers with pyramidal tops and bottoms, under dynamic conditions of 1000 liters/minute of airflow. Control animals were exposed to filtered room air in the same manner as the treated animals. Concentrations of the test substance in each chamber were measured hourly during each exposure period using an infrared analyzer. Animals were observed daily for mortality and moribundity. Body weights and clinical observations were recorded prior to study initiation, weekly during weeks 1 though 12, biweekly from week 14 through 24, every fourth week from week 28 through 78 and biweekly from week 80 through 104. Ophthalmoscopic exams were performed on all rats prior to treatment using an indirect ophthalmoscope at weeks 13, 52 and 102.
2. Re-Evaluation of the study (Lomax LG et al., 1997): Animals: Three hundred male and 300 female rats were received from Charles River Breeding Laboratories, Inc. (Wilmington, MA, USA). Animals were maintained under quarantine for 19 days, during such time all rats were evaluated for clinical signs of disease and an ophthalmoscopic examination. Following the quarantine period, 70 males and 70 females were assigned to each of the four exposure groups (including the control). The animals were housed by sex in wire mesh cages (seven/cage). Animals were provided feed and water ad libitum, except during the exposure period. Exposure conditions: Animals were exposed to the test substance vapor in 6000 liter inhalation chambers under dynamic conditions of 1000 liters/minute of air flow. The control animals were exposed to filtered room air in a chamber with similar air-flow characteristics. Observations: All animals were observed for mortality and morbidity daily. Individual body weight data were collected at the start of the study, weekly for the first twelve weeks and biweekly to week 24, every fourth week to week 78 and biweekly until study termination (week 104). At such time, a more detailed evaluation of gross toxicity and tissue masses was performed. Ophthalmoscopic evaluations were conducted at weeks 13, 52 and 102.
Sacrifice and pathology:
1. Original study (Reno FE, 1979):
Blood samples were collected from ten males and ten females in each group at weeks 13, 52 and 102 and from ten males and ten females in the control and high-exposure group at weeks 26 and 78 for hematological evaluation via segmental amputation of the tail. The following parameters were examined: hematocrit, hemoglobin, erythrocyte count, erythrocyte morphology, total leukocyte count and differential leukocyte count. In addition, coagulation and prothrombin times were determined from all blood samples taken at 13, 52 and 104 weeks and from the samples collected from the control and high-exposure groups at week 78. Femoral bone samples were collected from all sacrificed animals at weeks 13 and 52 and from 10 animals/sex/group at termination. The number of myeloid and erythroid cells and the myeloid/erythroid ratios were determined. Blood samples were collected for clinical chemistry evaluations from the abdominal aorta of all animals sacrificed at weeks 13 and 51 and from ten males and ten females per group at study termination. The following parameters were evaluated: fasting glucose, blood urea nitrogen, serum glutamic pyruvic transaminase, alkaline phosphatase, total protein, total albumin, albumin/globulin ratio. In addition, total cholesterol and triglycerides were also determined from blood samples taken from all animals sacrificed at week 52 and from ten animals/sex/group at week 104. Twenty-four hour urine samples were collected from 10 animals/sex/group at weeks 13, 52 and 104 by individually housing the rats overnight in stainless steel cages. The following parameters were evaluated: appearance, pH, ketones, total protein, specific gravity, bilirubin, glucose and occult blood. Necropsy: Ten rats/sex/group were sacrificed by exsanguination after 13 and 52 weeks of exposure. Animals found moribund during the course of the study were sacrificed at the time of the observation. The remaining animals were sacrificed after 102-104 weeks of exposure to the test substance. A gross necropsy was performed on all animals that were sacrificed and most of the animals that died during the study. Brain, kidneys, lungs, spleen, thyroids, adrenals and testes/ovaries from each animal were weighed and the organ to body weight ratio was calculated. The following tissues were collected and preserved in formalin or Bouin's solution: brain, pituitary, thoracic spinal chord, esophagus, salivary glands, thyroids, lungs, thymus, heart, spleen, kidneys, adrenals, stomach, duodenum, ileum, jejunum, colon, skin, mesenteric lymph nodes, urinary bladder, ovaries, uterus, mammary gland costochondral junction, liver, sciatic nerve, skeletal muscle, pancreas, nasal turbinates, unusual lesions, eyes and the testes with epididymides. Intraperitoneal body fat was recorded without exception at week 52 and by exception at all subsequent intervals. Histopathology was performed on the brain, spinal cord, pituitary, thyroid, adrenal, heart, lung, spleen, liver kidney, and ovaries/testes from 10/animals/sex in the low- and mid-exposure groups; and the nasal turbinates of all low- and mid-level animals. Also, the adrenals, ovaries/testes, heart (with coronary vessels), kidneys, liver, lungs, nasal turbinates, pituitary and thyroid were evaluated from 10 animals/sex/group from the control and high-exposure groups sacrificed at weeks 13 and 52.

2. Re-Evaluation of the study (Lomax LG et al., 1997):
Blood samples were collected from 10 males and 10 females per dose group at weeks 13, 52 and 104 and 10 males and 10 females in the control and the high-dose group at weeks 26 and 78. The following hematological parameters were evaluated at each sampling interval: hematocrit, hemoglobin, red blood cells counts, erythrocyte counts, total white blood cell counts, erythrocyte morphology and prothrombin time. Bone marrow samples were collected from the femurs of all rats killed at week 13 and 52 and from 10 males and 10 females from each group at study termination. Blood also was obtained from the abdominal aorta of all rats killed at week 13, 52 and study termination. The following clinical chemistry parameters were evaluated: glucose, blood urea nitrogen, serum glutamic-pyruvic transaminase, alkaline phosphatase, total protein, total albumin, total cholesterol (except for week 13) and triglycerides (except for week 13). Twenty-four hour urine samples were collected from 10 animals per sex per group at weeks 13, 52 and 104. The following parameters were evaluated: appearance, pH, specific gravity, glucose, ketones, total protein, bilirubin, and occult blood. Necropsy: Ten rats per sex per group were sacrificed after 13 and 52 weeks of exposure. The remaining animals were sacrificed after 104 - 106 weeks of exposure. Necropsies were performed on all decedents. The brain, kidneys, lungs spleen, adrenal and thyroid glands and the testis or ovaries were weighed and the organ to body weight ratios were calculated. The following tissues were preserved in buffered 10% formalin: brain, pituitary, spinal cord, esophagus, salivary glands, thyroid glands with parathyroid, lungs, mediastinal lymph nodes, thymus, heart, aorta, larynx, spleen, kidneys, adrenals, stomach, duodenum, ileum, jejunum, colon, skin, mesenteric lymph nodes, urinary bladder, uterus, mammary gland, prostate, seminal vesicles, costochondral junction, liver, sciatic nerve, skeletal muscle, pancreas, nasal cavity and gross lesions. The eyes from all rats and the testes with epididymides were preserved in Bouin's fixative. Microscopic evaluations were made using the tissue listed above in the control and the high-dose groups at study termination. The brain, spinal cord, pituitary, thyroid, adrenal, heart, lung, liver, spleen, kidney, and ovaries/testes from 10 animals per sex in the low- and mid-dose groups and the nasal cavities from all animals in the low- and mid-dose groups were evaluated microscopically. Sections from the adrenals, testes or ovaries, heart, kidneys, pituitary, thyroids, liver, nasal cavities and lungs of control and high-dose animals were examined microscopically after the week 13 and 52 interim sacrifices. Following the issuance of the original report, tissue blocks of the nasal cavities from the animals killed at the terminal sacrifice and the control and high-dose group at week 13, were obtained and a composite cross-sectional map of representative nasal cavity lesions with the approximate distribution was prepared for the mid- and high-dose groups.
Statistics:
1. Original study (Reno FE, 1979): Data Analysis: Pairwise comparisons of the mean body weights from weeks 12, 24, 36, 48, 52, 60, 72, 78, 90 and 104 were conducted using the F test for equality of two variances and Student's t-test. Clinical laboratory data (except urinalysis and leukocyte differentials), terminal body weights and absolute and relative organ weights (organ/body weight) of all animals sacrificed at weeks 13, 52 and term were subjected to a preliminary test for equality of variance followed by one-way analysis using Bartlett's test for homogeneity and Snedecor and Cochran, respectively. When statistical significances were observed, an additional set of analyses was conducted using Scheffe's method for judging all contrast in analysis of variance.
2. Re-Evaluation of the study (Lomax LG et al., 1997): Data analysis: Pair-wise comparisons of the mean body weights were performed. Clinical laboratory data, with the exception of urinalysis and leukocyte differentials, terminal body weights and absolute and relative organ weights of all animals killed at weeks 13 and 52 and at study termination were subjected to a preliminary test for the equality of variance. To evaluate tumor incidence, Fisher's one-sided exact test was conducted between the control and high-dose groups. For all analyses, statistical significance was determined by a p value < 0.05.
Dose descriptor:
NOAEC
Remarks:
systemic (gross pathology histopathology, clinical effects)
Effect level:
ca. 1 640 mg/m³ air
Sex:
male/female
Basis for effect level:
other: corresponding to 400 ppm
Dose descriptor:
LOAEC
Remarks:
local effects (Histopathology, olfactory epithelium)
Effect level:
ca. 416 mg/m³ air
Sex:
male/female
Basis for effect level:
other: nasal lesions; corresponding to 100 ppm
Dose descriptor:
NOAEC
Remarks:
local effects (Histopathology, olfactory epithelium)
Effect level:
ca. 104 mg/m³ air
Sex:
male/female
Basis for effect level:
other: corresponding to 25 ppm
Critical effects observed:
not specified

1. Original study (Reno FE, 1979):

-----------------------------------

The mean analytical concentration was evaluated. The overall mean concentrations of MMA vapour were 25.0, 99.8 and 396.1 ppm for the 25, 100 and 400 ppm exposure groups, respectively.

 

Mortality: Mortality rates were relatively low through week 78. High mortality was observed through week 104. The author indicates that the increase in mortality was probably due to aging, not related to test substance exposure. The mortality rates for treated groups were comparable to the control group. A summary of the mortality rates (%) is provided below.

 

 

Dose group (ppm)

Week 0-13

Week 0-52

Week 0-104

MALES

 

 

 

Negative Control (0)

0

0

16

25

0

1.7

20

100

0

1.7

16

400

0

0

20

FEMALES

 

 

 

Negative Control (0)

0

0

24

25

0

3.3

36

100

1.4

3.3

26

400

0

5.0

30

 

Clinical signs

No signs of test substance-related toxicity were observed in any of the treated animals throughout the 104-week exposure period. The most frequent observations included cloudy eye(s) and bloody crust around one or both eyes. The author reported that these findings occurred with approximately the same frequencies in treated and control groups.

Body weight

Male body weights were significantly higher in the mid-level exposure group at week 24, lower weights in the low-level exposure group at week 104, and lower weights of the high-level exposure group at weeks 28 and 78. In the females, the low-level exposure groups showed a significant decrease in body weight at weeks 60, 72 and 78 and an increase at weeks 12 and 24. The females in the mid-level exposure group showed a significant decrease at weeks 52, 60 and 78 and in the high-level exposure group at weeks 28, 36, 52, 60, 72, 78 and 90. The author concluded, the body weight reduction observed in the females exposed to ca. 1.64 mg/L (400 ppm) MMA was test substance-related.

 

Ophthalmoscopy

Ophthalmoscopic observations were noted at weeks 13, 52 and 102. The author reports that no consistent ocular abnormalities were noted at weeks 13 and 52. Ocular findings noted at week 102 included cataracts, pale coloration, corneal cloudiness and red discharge. The cataract findings were considered to be caused by aging.

Haematology/ Clinical chemistry

Evaluation of the haematology and clinical chemistry data did not reveal any remarkable trends. Statistical analyses showed numerous significant differences between the treated and the control groups; however, these differences were considered sporadic and were considered by the author a reflection of sampling and biological variability. A transitory appearance of occult blood was observed in all groups at week 52. All remaining intervals were generally unremarkable.

 

Organ weights

A statistically significant increase in absolute and relative organ weights of the females exposed to ca. 1.64 mg/L (400 ppm) MMA was observed in the lungs, liver, kidneys, and ovaries at week 13. A statistically significant decrease in absolute and relative thyroid and adrenal weights were observed in both males and females in the high-level exposure group at week 52. Absolute thyroid and adrenal weights were significantly higher in the males exposed to ca. 0.41 mg/L (100 ppm), MMA for 52 weeks. Other significant differences were noted at weeks 52 and 104; however, the author concluded that no consistent dose-related pattern was established.

 

Gross pathology

Findings noted in animals that were sacrificed at weeks 13 and 52 were mainly discolorations of the lung and liver. None of the findings were considered treatment-related. Tissue mass findings for animals sacrificed at week 104 were typical for the age and the species of rats. No treatment-related differences with respect to the frequency were observed.

 

Histopathology 

Week 13

No treatment-related histopathological findings were noted in the rats exposed to ca. 1.64 mg/L (400 ppm) MMA for 13 weeks. Findings were consistent among groups and were typical for rats of this age and strain.

Week 52

No treatment-related histopathological findings were noted in the rats exposed to ca. 1.64 mg/L (400 ppm) MMA for 13 weeks. Findings were consistent among groups and were typical for rats of this age and strain.

Week 104

Treatment-related histopathological findings were limited to a very slight increase in the lesions of mild rhinitis observed in the mucosal lining of the nasal turbinates. A summary of the lesions is provided below.

 

Incidence of Lesions in Nasal Mucosa

 

 

Males

 

 

 

Females

 

 

 

Group No.*

1

2

3

4

1

2

3

4

No. of Nasal Turbinates Examined

48

49

49

48

44

48

45

46

Serous Exudate

3

11

12

16

15

8

17

23

Purulent Exudate

2

6

4

8

2

9

6

6

Pleocellular Infiltrate

1

4

6

19

3

14

9

11

Distended Submucosal Glands

5

21

21

12

3

14

12

9

Squamous Metaplasia (focal)

2

3

1

5

0

5

1

2

Inflammatory Polyp

0

0

1

2

0

0

0

0

*Groups 1, 2, 3 and 4 were exposed to ca. 0, 0.10, 0.41 and 1.64 mg/L (0, 25, 100 and 400 ppm) MMA, respectively.

 

No clear treatment-related effect could be established. Although lesions of mild rhinitis occurred more often in treated rats than control rats, it could not be determined if the rhinitis was a result of direct chemical insult to the turbinate area or whether the presence of MMA vapors predisposed the rats to an increase in spontaneous disease. [NOTE - Subsequent evaluation of the nasal lesions (Lomax et al., 1997) indicated that there were exposure related nasal lesions at ca. 0.10 and 0.41 mg/L (100 and 400 ppm)]. Neoplasms and spontaneous disease lesions were observed with comparable frequency in control and treated rats. Chronic nephritis was observed in most rats; however, it was more pronounced in males.

 

2. Re-Evaluation of the study (Lomax LG et al. (1997)):

-------------------------------------------------------

 

The mean analytical concentrations of the test substance in the exposure chambers were 25.0, 99.8 and 396.1 ppm less than 10% per dose level.

 

Mortality rates for the treated animals were similar to those of the controls. No signs of treatment-related toxicity were observed. At the 13, 52 and 104-week observation intervals, cloudy eyes and bloody crusts around one or both eyes were noted in all of the treatment groups, as well as the control animals. Body weights for males were lower than the control at various intervals but overall were considered equivalent over the 104-week period. Mean body weights for females were lower than the controls at ca. 1.64 mg/L (400 ppm) after week 52. Haematology, clinical chemistry and urinalyses did not indicate any treatment-related effects in any of the parameters evaluated.

 

Gross necropsy of the rats sacrificed at weeks 13 and 52 did not show any treatment-related effects.

 

The following information was obtained from the reevaluation of the nasal tissues from this study originally conducted by Reno et al.(1979) - see also summary for this study in this Dossier. Microscopic evaluation of the nasal cavity sections obtained from the animals exposed to the test substance for 13 weeks showed degeneration of the neuroepithelial cell lining of the dorsal meatus in conjunction with atrophy of Bowman's glands and focal basal cell hyperplasia. Lesions were identified on the tips of the maxilloturbinates and nasoturbinates and focally along the nasal septum in the more anterior regions of the nose. These lesions were characterized by chronic active inflammation, respiratory epithelial hyperplasia and squamous metaplasia. No microscopic findings were identified in the ocular tissue or the lungs or other tissues. Blocks of the nasal cavities of animals from the 52-week sacrifice were unable to be located and, therefore, were not evaluated. No new findings were identified in the tissues that were available for animals exposed to the test substance for 52 weeks. Spontaneous disease lesions included early respiratory disease in both the control animals and the animals exposed to 400 ppm of the test substance. Also focal areas of pneumonitis were observed in two females in the control group.

Gross necropsy after two years of exposure to the test substance showed no treatment-related effects. The nasal cavity was the target organ for chronic toxicity. Rats exposed to the 100 and 400 ppm dose group had dose-dependent lesions in the anterior portions of the nasal cavity. The olfactory epithelium lining the dorsal meatus in the anterior region of the nasal cavity was affected by exposure to higher concentrations of the test substance. The microscopic changes consisted of degeneration of the olfactory epithelium and underlying Bowman's glands, hyperplasia of basal cells, replacement of olfactory epithelium by ciliated epithelium and inflammation of

the mucosa and/or submucosa. Lesions tended to be bilateral in distribution. The olfactory lesions in rats exposed to 100 ppm were localized in the more posterior (level 3) portion of the dorsal meatus, while those in animals exposed to ca. 1.64 mg/L (400 ppm) were found in levels 2 and 3. Hyperplasia of glands in the lamina propria and/or goblet cells and inflammation of the mucosa/lamina propria were observed in the respiratory epithelium in the high exposure group animals. No effects were seen in nasal epithelium of rats exposed to ca. 0.10 mg/L (25 ppm) MMA. No statistically significant differences were observed in the frequency of tumours between the rats exposed to ca. 1.64 mg/L (400 ppm) of the test substance and that of the controls. In female rats exposed to ca. 1.64 mg/L (400 ppm) of the test substance, a statistically significant decrease in pituitary adenoma/carcinomas and mammary gland fibroadenomas was recorded. In male rats, a decreased incidence of pheochromocytoma was observed.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

For EMA, no repeated dose studies are available. However, EMA is rapidly hydrolyzed to Methacrylic acid (MAA) and Ethanol (see chapter Toxicokinetics or Category document chapter 5). There are reliable chronic or subchronic studies from Methyl methacrylate (as MAA donor substance) and Ethanol which allow the assessment of repeated dose toxicity of EMA after oral and inhalative administration:

 

Metabolites

Oral

For the assessment by the oral route reference is made to the 2-year study on MMA and the 90 d study on n-BMA.

In an early 2-year chronic drinking water study with 25 male and 25 female rats administered with 6, 60 and 2000 ppm MMA no adverse effect were observed other than elevated kidney weights without corresponding histopathology in female rats at 2000 ppm (Borzelleca et al., 1964). The NOAEC was reported as 2000 ppm (124/164 mg/kg bw/d) in male and female rats.

In an OECD guideline 408 90-day gavage study in Wistar rats with n-BMA, with a recovery period of 28 days, revealed general signs of liver and kidney toxicity at 360 mg/kgbw/d, with a NOAEL of 120 mg/kg body weight/day for systemic effects in both males and females. Local degenerative and regenerative effects in the olfactory epithelium of the nasal cavity were observed in the 360 mg/kg bw/day and 120 mg/kg bw/day animals. The NOAEL for these effects was 60 mg/kg body weight/day in both males and females. This substance-related effect was completely reversible by 28 days after cessation of exposure. Therefore, the no observed adverse effect level (NOAEL), under the conditions of the present study, was 120 mg/kg body weight/day for systemic effects and 60 mg/kg body weight/day for local effects in the nose in both sexes.

Most (repeat dose) data available on ethanol is via the oral route of exposure. Much is at high doses which limits its value to risk assessment of ethanol as a chemical substance. From the data available, it is possible to surmise that ethanol is of repeat dose low toxicity by the oral route, with a lowest reported NOAEL in a 90 d feeding study of 2400 mg/kg for rats (Holmberg et al. 1986, in OECD SIDS, 2004).This rather continuous administration type is considered as more suitable than bolus dosing per gavage for both, occupational exposure considerations and the interpretation of ethanol as a metabolite of EMA.

 

Inhalation

For the assessment for local effects by the inhalation route reference is made to the Human NOAEL derived in the SCOEL review and the 28d inhalation study on n-BMA. For systemic effects by the inhalation route reference is made to the 2-year NTP study on MMA and the 28d inhalation study on n-BMA.

 

Local effects

MAA: In an OECD 413, 90-day vapour inhalation study in Sprague Dawley rats with MAA revealed general toxicity at 350 ppm (1253 mg/m3) in male animals. Local, marginal irritation of the respiratory epithelium in the nasal cavity was observed in two female animals. The NOAEL was 100 ppm (358 mg/m3) for local irritation effects in male and females (BASF 2008).

MMA: From the chronic inhalation studies on MMA in rodents the NOAEC for local effects in the nose was 25ppm and the LOEC was 100 ppm (Lomax et al. 1997). However, the recognised differences between rodents and humans regarding the physiology of the nasal passages, metabolic activity result in the greater susceptibility of rodents compared with humans to inhaled esters. In the EuRA (2002) these differences were disregarded and rodents were regarded being of comparable sensitivity to humans. Subsequently, in the SCOEL review (SCOEL, 2005) greater emphasis was placed on human data, showing the absence of adverse respiratory effects up to at least 50 ppm and this was considered to be consistent with rodents being at least three times more sensitive than humans based on PBPK considerations (Andersen et al. 2002, Mainwaring et al. 2001).

Ethanol: There are no robust chronic inhalation studies for ethanol. Major health-based occupational exposure standards are in excess of 500 ppm (960 mg/m³) indicating that ethanol is of low inhalation toxicity for both, local and systemic effects (BAuA 2017).

nBMA: In an OECD Guideline 412 Repeated Dose 28-day inhalation study with n-BMA treatment-related effects included lacrimation, eye squinting, and laboured breathing in the 952 and 1891 ppm (5626 and 11175 mg/m3) concentration groups throughout the study. Local effects of inflammation of the olfactory region of the nasal cavity at 952 and 1891 ppm (5626 and 11175 mg/m³) were observed in both sexes. The NOAEC was 310 ppm (1832 mg/m3; Hagan et al., 1993).

 

Systemic

MAA: In an OECD 413, 90-day vapour inhalation study in Sprague Dawley rats with MAA revealed general toxicity at 350 ppm (1253 mg/m3) in male animals. The NOAEL for systemic effects based upon reduced body weight gain in the presence of reduced feed intake but no other systemic effects was 100 ppm (358 mg/m3) in male and females.

MMA: In a two-year chronic inhalation studies of MMA the only systemic effects observed was reduced mean body weights (6-11 % in week 73) in female rats at 500 ppm (2080 mg/m3) and male rats at 1000 ppm (NTP, 1986). As this effect was likely the result of reduced food consumption and therefore not a true adverse effect the observation of malacia and gliosis of the brain in the 14 week range finder to the NTP study (Battelle, 1980) is considered being the relevant systemic effect. This was observed in 5/9 female rats exposed at 2000 ppm and 1/8 females at 1000 ppm. Therefore, the absence of this effect at 500 ppm (2028 mg/m³) in the corresponding 2-year study (NTP) is considered representing the NOAEC for chronic systemic effects of MMA.

Ethanol: There are no robust chronic inhalation studies for ethanol. Major health-based occupational exposure standards are in excess of 500 ppm (960 mg/m³) indicating that ethanol is of low inhalation toxicity for both, local and systemic effects (BAuA 2017).

 

 Dermal

There are no relevant dermal repeated dose studies. For assessment purposes the oral data are used with a route-to-route extrapolation factor of 1.


Repeated dose toxicity: inhalation - systemic effects (target organ) respiratory: nose

Justification for classification or non-classification

Based on the available information, the potential of ethyl methacrylate for systemic toxicity after repeated dosing is low. Hazards based on local effects were covered by the classification for the irritation potential on skin and respiratory tract (see chapter 7.3). Therefore, no additional classification is considered as justified.