Reaction mass of 2-hydroxy-1,3-propanediyl bismethacrylate and 101525-90-0

Endpoint summary

Description of key information

No data are available for GDMA. However, data from the metabolites (HPMA as analogous substance of the primary metabolites plus MAA/MMA and glycerol) allow the evaluation of repeated dose hazard of GDMA after oral and inhalative exposure.

Read-across to the metabolites is justified by the fact that carboxylesterases are ubiquitous in the body and half-lifes of the other category substances are only a few minutes (see category document, chapter 5.1). In the body, methyl methacrylate hydrolyses rapidly to methacrylic acid and thus serves as methacrylic acid donor in several test systems investigating systemic effects.

 

Following data were considered for read across:

Subacute (33-40 day) study; oral (gavage); rat, m/f (OECD guideline 422, GLP): NOAEL = 300 mg/kg bw/d due to mortality and clinical findings; read-across from the analogous substance of the primary metabolites, HPMA (Nihon Bioresearch 1996)

Chronic (2 yrs) study; oral (drinking water), rat, m/f (pre-guideline, pre-GLP): NOAEL ≥ 2000 ppm (=124 mg/kg bw/d in males and 164 mg/kg bw/d in females); read-across from the metabolite donor substance MMA (Borzelleca 1964)

Chronic/ Carcinogenicity (2 yrs) study; inhalation (vapour), rat, m/f (comp. to OECD guideline 453, non-GLP): LOAEClocal= 100 ppm (ca. 416 mg/m3) due to nasal lesions; NOAECsystemic= 400 ppm (ca. 1640 mg/m3) due to reduced bw gain read-across from the metabolite donor substance MMA (Lomax 1997)

Chronic/ Carcinogenicity (2 yrs) study; inhalation (vapour), rat, m/f (NTP protocol, GLP): LOAEClocal= 250 ppm for female rats and 500 ppm for male rats due to nasal lesions; NOAECsystemic≥ 500 ppm for female rats and ≥ 1000 ppm male rats; read-across from the metabolite donor substance MMA (NTP 1986)

Chronic (2 yrs) study; oral (feed), rat, m/f (pre-guideline, non-GLP); NOAEL = 5000 mg/kg bw/d; read across from the alcohol metabolite Glycerol (Hine 1953)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP, japanese study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Early version of guidance for OECD 422 not containing functional observation battery test 

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 8 weeks
- Weight at study initiation: male 315 ~ 359 g; females 210 ~ 243 g,
- Fasting period before study: yes
- Housing: During the quarantine: suspended using a stainless steel cage type 1 with 5 per cage; Breeding: divided into separate rearing cages. Moved to a separate plastic cage, having had a natural birth .
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: five-day quarantine period and then set up a six-day acclimation period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 deg C
- Humidity (%): 40-70%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Concentration of samples was prepared by dissolving the required water for injection. The concentrations of preparation is protectedc from light at room temperature for 7 days to ensure that there were no stability issues. Preparation of 0.6% solution concentration is below the threshold level of determination, because we could not confirm the stability during the preparation for the 6% solution diluted with water for injection prepared in concentration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 0.6, 0.8, 2.6 and 20%
- Amount of vehicle (if gavage): 5 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

Males :49 days
Females: from 14 days before mating to day 3 of lactation

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0 (vehicle), 30, 100, 300 and 1000 mg/kg/ day
Basis:
nominal in water

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: 2-week preliminary study
- Rationale for animal assignment (if not random): random by weight
- Terminal kill: Males, day 50; Females, day 4 of lactation

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day
- Cage side observations: general condition

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): 2 times per week
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:day after treatment
- Anaesthetic used for blood collection: Yes (identity) : sodium pentobarbital
- Animals fasted: No data
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day after treatment
- Animals fasted: No data
- Parameters checked in table [No.2] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: sexual cycle until confirmation; status of delivery

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; Males: Thymus, liver, kidney, testis and epididymis weight was measured after removal, adrenal gland, brain, heart and spleen and 10% neutral buffered formalin solution (However, testicular and epididymal fluid Buan) was fixed; Females: counting the number of corpora lutea and the number of implantation scars. Liver, kidney, ovary and thymus weight was measured after removal, adrenal gland, brain, heart and spleen with a fixed 10% neutral buffered formalin solution.
HISTOPATHOLOGY: Yes; Control group and 1000 mg / kg group of heart, liver, spleen, thymus, kidney, testis, epididymis, ovary, adrenal and brain for the Preparation HE staining of tissue was examined histologically.

Statistics:

In either test, significant risk factors were less than 5%.
1) multiple comparison test
Weight (the parent animals, babies), food consumption, number of estrus, days mating, pregnancy [Day delivery (feeding 0) - date confirmed mating, the number of implantation scars, the number of birth control mobilize (number of babies stillborn baby + ), the number of newborn, number of children born dead, birth rate [(number of birth control mobilize / number of implantation scars) × 100], rate of production of child [(number of infant feeding 0 days / number of implantation scars) × 100], corpus number, implantation rates [(number of implantation scars / number of corpora lutea) × 100], fertility [(number of infant feeding 0 day / mobilize all of birth control) × 100], feeding baby number four day, feeding 4 day survival rate [(number of newborn feeding 4 days / 0 Number of infant feeding day) × 100], unusual occurrence rate [(number of children with abnormal/ number of newborns) × 100], sex ratio (male / female), organ weights ( including the relative weight), results of blood tests, blood biochemistry test results for the mean and standard deviation were calculated for each group. Significant difference test, Bartlett's test and the homoscedasticity of Law, Law-way layout analysis of variance if the variance 1) and, if significant Dunnett method 2) or Scheff Method 3) were using. However, if the variance was not approved, the analysis method using centrally located position (Kruskal-Wallis test of 4)) and a significant if you use the ranking method or Dunnett Scheff Method was used.
2) χ ^ 2 test
Copulation rate [(number of established animal mating / number of live animals) × 100], fertility [(number of female fertility / Establishment of animal mating) × 100], the birth rate [(number of female newborns / number of female fertility) × 100] is, χ ^ 2 using the test.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

For the males, salivation, decrease in locomotor activity and ptosis were found in the 1000 mg/kg group, and 2 animals of the group died. Decrease in hematocrit, tendencies for decrease in RBC and hemoglobin, and increase in the relative liver weights were also found in the 1000 mg/kg group. 
For the females, salivation, decrease in locomotor activity and ptosis were found in the 1000 mg/kg group, and 1 animal died.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

clinical signs

haematology

mortality

organ weights and organ / body weight ratios

Critical effects observed:

not specified

Conclusions:

In an OECD 422 study in rats, the oral repeated dose NOAEL was determined to be 300 mg/kg based on body weights.

Executive summary:

According to the OECD test guidelines for combined repeated dose toxicity study with the reproductive/developmental toxicity screening test [OECD TG 422], SD (Crj: CD) rats (12 animals/dose/sex) were administrated with this substance by gavage at doses of 0 (vehicle; water for injection), 30, 100, 300, and 1,000 mg/kg bw/day (12 animal/dose/sex). Males were dosed for a total of 49 days beginning 14 days before mating, and females were dosed for total of 41 to 48 days starting from 14 days before mating to day 4 of lactation throughout the mating and pregnancy period.

At 1000 mg/kg bw/day for males, two of the 12 animals died. Salivation, decreases in locomotor activity, ptosis were observed. The decrease in hematocrit and the tendencies for decrease in erythrocyte (RBC) and hemoglobin were found in haematological examination. The statistically significant increase in the relative liver weights was observed in organ weight. At that for females, one of the 12 animals died. Salivation, decreases in locomotor activity, ptosis were observed. The other significant toxicological changes were not observed. 

At 0, 30, 100 and 300 mg/kg bw/day for both sexes, no animals died and no abnormal clinical changes were observed. Moreover, significant toxicological changes were not observed in body and organ weight, food consumption, haematological examination, blood chemical examination, necropsy, and histopathological examination. Therefore, no adverse effect was observed in both sexes. 

The NOAELs for the repeated dose toxicity are considered to be 300 mg/kg bw/day for both sexes.

Reference 2

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.

Qualifier:

equivalent or similar to guideline

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

Specific details on test material used for the study:

source: Rohm & Haas company, Philadelphia
stabilized with 10 ppm monomethylether of t-butylhydroquinone

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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
- rats were individually caged and weighed once a week

Route of administration:

oral: drinking water

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The rats were individually caged and weighed once a week. The diet consisted of finely ground Purina Dog Chow Kibbled Meal consumed ad libitum. Fluid consumption values were determined over a 3-day period at the end of 1 and 4 weeks,
monthly through 6 months, and on even months thereafter. Food consumptions were measuered over 3-day periods at the same intervals.
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.

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

Dose / conc.:

6 ppm

Remarks:

raised after 5 months to 7 ppm

Dose / conc.:

60 ppm

Remarks:

raised after 5 months to 70 ppm

Dose / conc.:

2 000 ppm

Dose / conc.:

12 other: ppm (on the basis of fluid and food consumption observations)

Remarks:

corresponding to roughly 0.6 mg/kg/bw (based on a conversion factor of 20 (Derelanko, M.J.,
2000)corresponding to roughly 0.6, 6 and 165 mg/kg/bw (based on a conversion factor of 20
(Derelanko, M.J., 2000)

Dose / conc.:

120 other: ppm (on the basis of fluid and food consumption observations)

Remarks:

corresponding to roughly 6 mg/kg/bw (based on a conversion factor of 20 (Derelanko, M.J., 2000)

Dose / conc.:

3 000 other: ppm (on the basis of fluid and food consumption observations)

Remarks:

corresponding to roughly 165 mg/kg/bw (based on a conversion factor of 20 (Derelanko, M.J., 2000)

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. The animals were individually housed and provided food ad libitum.  
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.

Haematological findings:

no effects observed

Description (incidence and severity):

Hematologic values varied within normal ranges in ali' groups of rats throughout the study

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urine concentrations of protein and reducing substances showed no trends that appeared relatable to treatment.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

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 methacrylatc (controls 0.0082 ± 0.0019; treated 0.0094 ± 0.0011).

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

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.

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) in a 2 years study in rats by oral administration in drinking water.

Executive summary:

A two years toxicity study was performend in 1964 to study the tolerance of animals to chronic ingestion of methyl methacrylate. No relevant effects were observed after exposure of rats in drinking water up to the highest dose tested (2000 ppm, limited by palatability)..

Reference 3

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study was conducted prior to GLP and test guidelines, but sufficient data is available for interpretation of results

Qualifier:

no guideline available

Principles of method if other than guideline:

Test material was administered in the diet for up to 2 years. Study design followed intent of OECD 452. The number of animals/dose level used was less than required and minimal histopathology was conducted.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

Natural glyerol, achieved from market stock, although purity was not indicated (impurities were fatty acids and esters) it was USP grade material.
Synthetic glycerol, purity 99.5% (rest mainly water with very small amounts of glycerol polymers and glyceraldehyde)

Species:

rat

Strain:

Long-Evans

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS
- Age: not indicated
- Weight at study initiation: 96-109 g (males), 92-108 g (females)
- Number of animals: 22/sex/treatment, 26/sex for controls
- Source: Institute of Experimental Biology of University of CaliforniaUpon arrival at the laboratory, each rat was assigned to an individual numbered cage. Distribution among the experimental groups was accomplished by reference to a standard random number table.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

ADMINISTRATION / EXPOSURE
- Exposure period: 2 year (1 year for the high dose group)
- Route of administration: oral in diet. Diet consisted of a standard dog-food meal with which the glycerol was thoroughly mixed. Feed was prepared once a week and stored in stoneware crocks. The crocks were weighed and refilled weekly, and the unconsumed feed was discarded.
- Doses: 5, 10 and 20% in diet; males 2000, 4000 and 8000 mg/kg bw, females 2500, 5000 and 10000 mg/kg bw
- Water: ad libitum

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

Not applicable

Duration of treatment / exposure:

continuous

Frequency of treatment:

daily, up to two years

Dose / conc.:

5 other: % nominal in diet

Remarks:

2000 mg/kg bw, females 2500 mg/kg bw

Dose / conc.:

10 other: % nominal in diet

Remarks:

4000 mg/kg bw, females 5000 mg/kg bw

Dose / conc.:

20 other: % nominal in diet

Remarks:

8000 mg/kg bw, females 10000 mg/kg bw

No. of animals per sex per dose:

22/sex/dose
26/sex for controls

Control animals:

yes, concurrent no treatment

Details on study design:

No additional information available.

Positive control:

no data

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND FREQUENCY:
- Clinical signs: daily in cage and weekly examination outside the home cage
- Mortality: daily- Body weight: weekly- Food consumption: weekly
- Haematology: erythrocyte and leucocyte count and haemoglobin after 3, 6, 12, 18 and 24 months
- Urinalysis: albumin, glucose, casts and red and white blood cells after 3, 6, 12, 18 and 24 months (24-48 hr urine collection)

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC):
- Organ weights: liver, kidneys, heart, spleen and lungs
- Macroscopic: no details provided
- Microscopic: liver, spleen, adrenals, kidney, small intestine, gonads and urinary bladder

Other examinations:

Glycogen and lipid content of the liver of surviving rats at 0 and 20% glycerol.

Statistics:

Chi-sqare test, student t-test, ANOVA (Fisher) were used as appropriate.

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

slightly increased (significant) in males at 5 and 10% natural glycerin

Food efficiency:

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Details on results:

TOXIC RESPONSE/EFFECTS BY DOSE LEVEL:
- Mortality and time to death: not indicated
- Clinical signs: not reported
- Body weight gain: no statistically significant differences between treated and control animals
- Food consumption: slightly increased (significant) in males at 5 and 10% natural glycerin
- Haematology: no treatment related effects
- Urinalysis: albumin: no significant treatment related effects (92% incidence in females at 20% natural glycerin compared to 54-64% in controls); glucose, casts, red and white blood cells: no treatment related effects
- Organ weights: incidental increases and decreases were reported without apparent relationship to treatment
- Gross pathology: no lesions related to treatment.
- Histopathology: Incidental bronchiectasis, pneumonia, pulmonary abcesses, taenia infestation of the liver, hydronephosis and pyelonephritis (total 27 rats were affected).
- Other: liver glycogen and lipid did not significantly differ between 0 and 20% glycerin (liver glycogen natural glycerin 4.2-4.3% and synthetic glycerin 3.7-4.2%)

Dose descriptor:

NOAEL

Effect level:

>= 8 000 - <= 10 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment related effects were observed

Critical effects observed:

no

Conclusions:

The NOAEL was 8000-10,000 mg/kg bw based on the absence of treatment related effects in high dose animals.

Executive summary:

The chronic toxicity of glycerin was examined in rats fed the test material in the diet at concentrations of 5, 10 and 20% for up to two years. The NOAEL was 8000-10,000 mg/kg bw based on the absence of treatment related effects in high dose animals.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

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 or test system 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

Dose / conc.:

20 ppm

Remarks:

= 70 mg/m3

Dose / conc.:

40 ppm

Remarks:

= 141 mg/m³

Dose / conc.:

100 ppm

Remarks:

= 352 mg/m³

Dose / conc.:

350 ppm

Remarks:

= 1232 mg/m³

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

Remarks:

systemic effects

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

food efficiency

Remarks on result:

other: = 352 mg/3

Dose descriptor:

NOAEC

Remarks:

local effects

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

= 352 mg/3

Dose descriptor:

LOAEC

Effect level:

350 ppm

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

food efficiency

Remarks on result:

other: = 1232 mg/m3

Dose descriptor:

LOAEC

Remarks:

local effets

Effect level:

350 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: = 1232 mg/m3

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.

Reference 2

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Method and results sufficiently described, similar to OECD-guideline 453.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system 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.

Reference 3

Endpoint:

chronic toxicity: inhalation

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: NTP standard protocol, cancer bioassay with limited dose range

Qualifier:

equivalent or similar to guideline

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.

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

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 or test system 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

Dose / conc.:

20 ppm

Remarks:

= 70 mg/m3

Dose / conc.:

40 ppm

Remarks:

= 141 mg/m³

Dose / conc.:

100 ppm

Remarks:

= 352 mg/m³

Dose / conc.:

350 ppm

Remarks:

= 1232 mg/m³

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

Remarks:

systemic effects

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

food efficiency

Remarks on result:

other: = 352 mg/3

Dose descriptor:

NOAEC

Remarks:

local effects

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

= 352 mg/3

Dose descriptor:

LOAEC

Effect level:

350 ppm

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

food efficiency

Remarks on result:

other: = 1232 mg/m3

Dose descriptor:

LOAEC

Remarks:

local effets

Effect level:

350 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: = 1232 mg/m3

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.

Reference 2

Endpoint:

chronic toxicity: inhalation

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: 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 1.1ff

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, table in chapter 1.2 and endpoint specific chapters

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system 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.

Reference 3

Endpoint:

chronic toxicity: inhalation

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: NTP standard protocol, cancer bioassay with limited dose range

Qualifier:

equivalent or similar to guideline

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.

Endpoint conclusion

Endpoint conclusion:

no study available

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

No study is available for GDMA. Reliable, relevant and adequate studies are however available on repeated dose toxicity conducted with the analogous substance of the primary metabolites HPMA, and the secondary metabolites MAA/MMA and Glycerol as follows. Like other investigated substances from this category, GDMA is expected to be rapidly hydrolysed by carboxylesterases in two subsequent steps to Methacrylic Acid (MAA) and the respective alcohol Glycerol, with Hydropropyl Methacrylate (HPMA) as analogue of the primary metabolites after the first hydrolysis step. The read across is justified in detail in the attached category document.

 

HPMA (analogous substance of the primary metabolites of GDMA)

As a part of the Japanese HPV program HPMA has been evaluated for repeat-dose toxicity in Sprague-Dawley rats in the OECD 422 combined repeat-dose developmental/ reproductive toxicity screening test in 1996.

In the study for HPMA, male rats (12/group) were given daily gavage doses of 0 (vehicle), 30, 100, 300 or 1000 mg/kg for 50 days including pre-mating, mating and post-mating intervals. Females (12/group) were administered the same doses for two weeks prior to mating, during mating and gestation up until day 4 of lactation. (approximately 54 days depending upon time to conception). Animals were observed for clinical symptoms of intoxication daily and food consumption and body weight were monitored throughout the study. Blood samples were taken for hematological and clinical chemistry analysis at study termination. Thymus, liver, kidney, testes, epididymes, and ovaries were weighed. In addition to these tissues, adrenal gland, brain, heart and spleen were fixed in 10% neutral buffered formalin solution for subsequent staining and histopathological evaluation.

Two male and one female of 12 exposed animals died on study at the 1000 mg/kg dose level. Clinical symptoms of intoxication observed at 1000 mg/kg [the highest dose tested] included: salivation, decrease in locomotor activity and ptosis for both sexes. Liver weight was increased in males only at 1000 mg/kg/d, with minimal hepatocyte vacuolation as a histopathological correlate. Also in males, a decrease in RBCs was observed at 1000 mg/kg only. The NOAEL for these effects was considered to be 300 mg/kg for both males and females.

 

MAA (common primary methacrylic metabolite of all category substances)

In an OECD 413, 90-day vapour inhalation study in Sprague Dawley rats, MAA revealed general toxicity at 350 ppm (1253 mg/m3, the highest tested dose) in male animals (BASF, 2008). Local, marginal irritation of the respiratory epithelium in the nasal cavity was observed in two female animals. No changes in sexual organs or sperm mobility and sperm head counts were noted. The NOAEC was 100 ppm (352 mg/m3) for local irritation effects in male and females The NOAEC for systemic effects based upon reduced body weight gain in the presence of reduced feed intake but no other systemic effects was also 100 ppm (352 mg/m3) in male and females.

 

MMA (donor substance for the common primary metabolite MAA)

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 and 164 mg/kg bw/d) in male and female rats.

--

EU ESR on MMA (2002) concluded: “The combined chronic toxicity/carcinogenicity study of methyl methacrylate in F344 rats (Rohm and Haas, 1979a; the nasal tissues were reevaluated by Lomax, 1992; Lomax et al., 1997) was assessed for the requirements of the regulation 793/93/EEC as a valid study with restrictions. In comparison to the minimal requirements of a 28-day inhalation study (OECD 412), the list of organs to be weighted did not include the liver and the heart. 70 male and 70 female f 344 rats were exposed to vapor concentrations of 0, 25, 100 or 400 ppm methyl methacrylate for two years. Ten male and ten female rats from all groups were sacrificed after 13 and 52 weeks of exposure and all surviving rats were killed during week 104-106. Histological examination was conducted on more than 35 tissues including 3-4 cross-sections of the nasal cavity. Tissues from the trachea and the pharynx/larynx were not preserved for histopathologic examination. Mortality rates of treatment and control groups did not show significant differences. No compound-induced clinical signs were observed. After week 52, mean body weight of high dose females was generally lower than controls gaining intermittently significance. Reduced growth represented the only adverse effect outside the respiratory ‘tract. Evaluation of hematology, clinical chemistry and urinalysis data did not reveal any methyl methacrylate associated effect.

At the end of the study, there were weight changes of some organs in males or females without any consistent relationship to the treatment. Similarly, no treatment-related macroscopic findings were observed in any of the dose groups. No histomorphological lesions other than nasal lesions were attributable to methyl methacrylate exposure of any exposed group. The examination of nasal cavities from male and female rats exposed to 400 ppm for 13 weeks or 52 weeks revealed a degeneration of the neuroepithelial olfactory cells lining the dorsal meatus of the anterior portions of the nasal cavities in conjunction with atrophy of Bowman’s glands and focal basal cell hyperplasia. Chronic active inflammation, respiratory epithelial hyperplasia and squamous metaplasia characterized the lesions on the tips of the maxilloturbinate and nasoturbinats and focally along the nasal septum in more anterior regions of the nose. At the final sacrifice, nasal lesions were evident in males and females of the 100 ppm and 400 ppm exposure groups characterised by inflammatory degeneration of nasal epithelium. The primary target tissue was the olfactory epithelium with degeneration and/or atrophy of neurogenic epithelium and submucosal (Bowman’s) glands lining the dorsal meatus, hyperplasia of basal cells, replacement of olfactory epithelium with ciliate (respiratory like) epithelium (metaplasia), and inflammation of the mucosa and/or submucosa. The severity of the lesions varied from minimal to slight at 0.4 mg/l (equivalent to 100 ppm) to moderate at 1.7 mg/l (equivalent to 400 ppm). At 0.1 mg/l (equivalent to 25 ppm) no pathological effects on the olfactory epithelium were reported, representing the NOAEC for local effects on the respiratory tract. Slight to moderate changes in respiratory epithelium occurred at 1.7 mg/l (equivalent to 400 ppm) and were characterized as hyperplasia of submucosal glands and/or goblet cells in the anterior regions of the nasal cavity. In the respiratory epithelium, there was inflammation of the mucosa and/or submucosa in males and females exposed to 400 ppm.... The NOAEC for systemic effects was considered to be 100 ppm for female rats and 400 ppm for male rats.

 

The cancer studies of the National Institutes of Health (NTP, 1986) revealed toxicological effects regarding the respiratory tract in male and female rats and mice. Groups of 50 male F344 rats and 50 B6C3Fl mice of each sex were exposed 6 hours per day, 5 days per week to air containing methyl methacrylate at target concentrations of 0, 2.1 or 4.2 mg/l (equivalent to 500 or 1,000 ppm) for 102 weeks. Groups of 50 female rats were exposed at concentrations of 0, 1.0 or 2.1 mg/l (equivalent to 250 or 500 ppm) on the same schedule. Increased incidences of serous and suppurative inflammation of the nasal cavity were observed in male and female rats.

Degeneration of the olfactory sensory epithelium characterized by loss of neuroepithelial cells was also observed in male and female rats. In exposed male and female mice inflammation of the nasal cavity, epithelial hyperplasia in the nasal mucosa and degenerative changes of the olfactory sensory epithelium were reported. In addition methyl methacrylate caused interstitial inflammation of the lung in high dose male mice. The trachea and larynx were also included in the histopathology, but no compound-related effects on these tissues were reported for rats and mice of each dose and sex. A NOAEC could not be established either from rats or mice.” This is true for local effects. For systemic effects, the NOAEC is the highest tested dose, i.e. 1000 ppm for mice and male rats and 500 ppm for female rats.

 

Glycerol (specific metabolite of GDMA)

Glycerol is an endogeneous metabolite in mammals (OECD 2002, EFSA 2017) for which several older studies are available. OECD (2002) stated for the best available dietary study with limitations mentioned below, that groups of 22 rats/sex/treatment received glycerol in their diet in concentrations of 5 and 10% (i.e. males 2000 and 4000 mg/kg bw; females 2500 and 5000 mg/kg bw) for 2 years and 20% (i.e. males 8000 mg/kg bw; females 10000 mg/kg bw) for 1 year. For all treatments, routine clinical observations were made, and bodyweight and food consumption was determined weekly. No clinical chemistry investigation and a limited range of haematological and urinary analyses were performed. Only liver, spleen, adrenals, small intestine, gonads and urinary bladder were examined microscopically. Glycogen and fat content of the liver was determined in surviving rats from the control and high dose groups. No individual data were reported. The high dose treatment was discontinued after 1 year. No data on mortality and clinical observations were reported. Food consumption was slightly increased in males treated with 5 and 10% glycerol. Incidental observations considered by the report-authors to be without relationship to treatment Based on this limited dietary study it can be concluded that no adverse effects were observed at up to 10,000 mg/kg bw for one year or up to 5,000 mg/kg bw for two years (Hine 1953). This assessment is consistent with the re-evaluation of EFSA (2017).

Compliance to REACh requirements

The requirements are covered with an OECD 422 oral rat study with HPMA as analogous substance of the the primary metabolites and with chronic studies with the methacrylic metabolite donor substance MMA (2 yr oral rat, 2 yr inhalation rat) and the alcohol metabolite Glycerol (2 yr oral rat). All mentioned studies are reliable (Reliability 1 or 2) and the read across is done with a medium level of confidence as a rapid hydrolysis is expected but not confirmed experimentally (see chapter toxicokinetics and category document, respectively).

Justification for classification or non-classification

No classification for systemic target organ toxicity based on the discussed studies from primary and secondary metabolites.