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Description of key information


Repeated dose toxicity of HEMA has been investigated in an OECD 422 combined repeated dose and reproductive toxicity study. A chronic study does not have to be performed, because, like MMA, the substances are rapidly metabolised, and by analogy to MMA ultimately metabolised to carbon dioxide and water. Therefore, as demonstrated in the case of MMA in carcinogenicity studies of up to 2 years duration, there is no concern for lesions due to accumulative toxicity. MMA data with different species and different application routes are used by read-across. For EG, the glycolic metabolite of HEMA, subchronic and chronic studies in rodents show toxicity with the kidneys as target organ.


 


Read across evaluation according to ECHA’s ReadAcrossAssessment Framework (RAAF)


The metabolism from HEMA to its primary metabolites is well understood. The same is true for the further metabolism pathways of MAA and the alcohol metabolite EG, respectively (see chapter 5.2, ATSDR 1997/ 2008/ 2010, NTP 2004a, 2004b). The endpoint specific “scientific assessment” of the read across is thus “acceptable with a high level of confidence”, see the attached Read Across Justification (2022) and Category document (2019).


Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
Deviations:
yes
Remarks:
A former version of OECD 422 (not containing functional observation battery test) had been  conducted.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Purity: 97.6%
0.2-0.3% Ethyleneglycol dimethacrylate
2.0-2.5% Diethyleneglycol-monomethacrylate
Supplier: Nippon-Shokubai
Batch No.: 5P05LA
Storage: regfrigerator, protected from light and air
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: 10 weeks
- Weight at study initiation: males: 341-380 g; females: 232-256 g.
- Housing: suspended, stainless steel cage; 5/cage until breeding, then divided into separate rearing cages.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days quarantine; 7 days acclimation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 ℃
- Humidity (%): 40 to 70%
- Photoperiod : 12 hours light / 12 hours dark

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: dissolved in water


VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 5 mL/kg
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Males, 49 days; Females, from 14 days before mating to day 3 of lactation
Frequency of treatment:
Once daily
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: Male, 50 days; Females, day 4 of lactation
- Dose selection rationale: based on range-finding
- Rationale for animal assignment (if not random): random
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked : general condition and mortality; estrus and abnormal labor conditions in females

BODY WEIGHT: Yes
- Time schedule for examinations: twice per week in males; before mating, twice a week during the mating period, 0, 7 ,14 and 21 days duirng pregnancy, during the feeding period was measured 0 and 4 days in females

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): one week prior to mating, then twice a week; additionally, in females, days 2,9,16 and 21 of pregnancy, four days over the feeding period.
- 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; sodium pentobarbital
- Animals fasted: No data
- How many animals: No data
- Parameters checked:
red blood cells
hemoglobin
hematocrit
platelet count
white blood cell count
prothrombin time
mean orpuscular volume
mean corpuscular hemoglobin
mean corpuscular hemoglobin concentration
reticulocyte count
white blood cell fraction
activated partial thromboplastin time
.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day after treatment
- Animals fasted: No data
- How many animals: No data
- Parameters checked:
GOT
GPT
ALP
γ-GTP
total protein
total bilirubin
urea nitrogen (BUN)
creatinine
glucose
total cholesterol
triglycerides
Ca
inorganic phosphorus
Na
K
Cl
protein fraction albumin
total protein content
A/G ratio

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Terminal kill: Males, day 50; Females, day 4 of lactation
GROSS PATHOLOGY: Yes; Thymus, liver, kidney, testis and epididymis weight in males and ovary in females was measured after removal, adrenal gland, brain, heart and spleen and 10% neutral buffered formalin solution (However, testicular and epididymal fluid Buan) was fixed. Post-mortem examination of feamles who did not give birth to Day 25 of pregnancy. Number of corpora lutea and the number of implantation scars in females.

HISTOPATHOLOGY: Yes; Paraffin-embedded specimens were prepared. Control group and 1000 mg / kg group of heart, liver, spleen, thymus, kidney, testis and epididymis in males ovary in females, adrenal and brain for the Preparation HE staining of tissue was examined histologically. In males, 1000 mg / kg in the kidney was considered to indicate a difference in the number of abnormal animals in the test group compared with the control group; 30, 100 and 300 mg / kg group were similarly examined. In females, 1000 mg / kg differences in the brain was considered to indicate an abnormal number of animals in the test group than the control group and changes in adrenal cases and 30, 100, 300 mg / kg group were similarly examined.
Other examinations:
Fetal examinations
(1) number of preterm birth and sex, number of stillborn children, the presence of abnormalities observed and the number of newborn.
(2) general condition and mortality
(3) measurement of body weight
(4) autopsy
Statistics:
Sex ratio (male / female), organ weight (including relative weight) and
weight) as well as the results of the haematological examination and the biochemical
biochemical blood tests were calculated as average values for each group and the
and the standard deviation was calculated. The significance test
according to Bartlett's equidistribution method was performed, where we used the ANOVA method, and in the case of significance, the Dunnett significance method was used. If, on the other hand, there was no equal dispersion, according to the rank-based ANOVA method was analyesd (i.e. according to the Kruskal-Wallis test3)). In the case of significance, the rank-based Dunnett test was used.


Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
MALES:
General condition: With the survival animals, no death and no moribund were found for 30,
100 and 300 mg/kg/day groups. At 1000 mg/kg/day, one death on day 20 of dosing was seen a
nd abnormality wasn't seen except for salivation until the previous day. With the dead animals, no
abnormality was found for 30, 100 and 300 mg/kg/day groups. At 1000 mg/kg/day, salivat
ion was seen in about 1 to 30-minutes after dosing from day 3.

FEMALES:
General condition: With the existence animales, no death and no moribund were seen for 30, 100
and 300 mg/kg/day groups. At 1000 mg/kg/day, three death on day 6 of dosing, one death on
day 12 of dosing and one death on day 17 of dosing were seen. Salivation, decrease in locomotor a
ctivity, adoption of a prone position, acrimation, soiled fur, hypothermia, bradypnea were seen at
1000 mg/kg. With the death animals, no abnormality was found for 30, 100 and300 mg/kg/day
groups. At 1000 mg/kg/day, salivation was seen in about 1 to 30-minutes after dosing from day 3.

Mortality:
mortality observed, treatment-related
Description (incidence):
MALES:
General condition: With the survival animals, no death and no moribund were found for 30,
100 and 300 mg/kg/day groups. At 1000 mg/kg/day, one death on day 20 of dosing was seen a
nd abnormality wasn't seen except for salivation until the previous day. With the dead animals, no
abnormality was found for 30, 100 and 300 mg/kg/day groups. At 1000 mg/kg/day, salivat
ion was seen in about 1 to 30-minutes after dosing from day 3.

FEMALES:
General condition: With the existence animales, no death and no moribund were seen for 30, 100
and 300 mg/kg/day groups. At 1000 mg/kg/day, three death on day 6 of dosing, one death on
day 12 of dosing and one death on day 17 of dosing were seen. Salivation, decrease in locomotor a
ctivity, adoption of a prone position, acrimation, soiled fur, hypothermia, bradypnea were seen at
1000 mg/kg. With the death animals, no abnormality was found for 30, 100 and300 mg/kg/day
groups. At 1000 mg/kg/day, salivation was seen in about 1 to 30-minutes after dosing from day 3.

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
MALES:
No significant difference from control group was seen in 30, 100, and 300 mg/kg/day groups. At 1000 mg/kg/day, the significant low value was recorded during day 18 to day 25 of dosing andduring day 32 to day 50 of dosing

FEMALES:
Before mating period, no significant difference from control group was seen at 30,
100 and 300 mg/kg/day. At 1000 mg/kg/day, the significant lower values were recorded on
day 4 and 5 of dosing. During gestation period, no significant difference from control groups was s
een in 30, 300 and 1000 mg/kg/day groups. At 100 mg/kg/day, the significant high values were recor
ded on day 21 of gestation, but no dose-related changes were observed. During lactation period, no
significant difference from control groups was seen in 300 and 1000 mg/kg/day groups. At 30 and 1
00 mg/kg/day, the significant high values were recorded on day 4 of lactation, but no dose-related
changes were obserbed.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):

MALES:
At 30 and 300 mg/kg/day, no significant difference from control was seen.
At 100 mg/kg/day, the significant high values were seen on day 31. but no dose-related changes wer
e obserbed.At 1000 mg/kg/day, the statistically significant low values were recorded on day 13, 31
and during day 38 to day 45.

FEMALES:
Before mating period, no significant difference from control group was see
n at 30, 100 and 300 mg/kg/day. At 1000 mg/kg/day, the significant low value from control group was
recorded on day 3, 6 and 13 of dosing. During gestation period, no significant difference from con
trol groups was seen in 30 and 300 mg/kg/day groups. At 100 and 1000 mg/kg/day, the sign
ificant high value from control group was recorded on day 16 of gestation, but no dose-related chan
ges were observed. During lactation period, no significant difference from control groups was seen.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
MALES:
No significant difference from control group was seen for all group s up to 1000 mg/kg/day dose.
Details in Table below under "Overall remarks, attachments"
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):

MALES:
Blood chemical examination: At 30 and 300 mg/kg/day,the significant high value in BUN
were seen. As the difference was very small, this was not considered as the adverse effect of HEMA
dosing. At 100 mg/kg/day, a higher value of BUN but not statistically signifficant difference from
control was recorded. At 1000 mg/kg/day, the significant high values were recorded in BUN, K, Cl,Iphosphorous and Triglyceride.
Details in Table below under "Overall remarks, attachments"
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
MALES:
At 30 mg/kg/day, no significant difference from control group in absolute and re
lative weight was seen for all organs. At 100 and 300 mg/kg/day, the significant high value was rec
orded in the absolute weight of kidneys. At 1000 mg/kg/day, the statistically significant high val
ues were recorded in the relative weight of liver and kidneys.

FEMALES:
At 30 mg/kg/day, no significant difference from control group in absolute an
d relative weight was seen for all organs. At 100 mg/kg/day,the significant high value was recorded
in the absolute weight of kidneys. At 1000 mg/kg/day, the significant high values were recorded i
n the relative and absolute weight of kidneys.
Details in table below under "Overall remarks, attachments"
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
MALES:
No abnormality was found for 30 and 100 mg/kg groups. In the 300 mg/kg group, the al
bedo spot in the kidney of the unilateral in the 1 animal and, the atrophy of the testiculus of the
bilaterality and softening were observed in the 1 animal. In the 1000 mg/kg group, the dark-red o
f the thymus gland in the 1 animal and the hypertrophy of the kidney of bilaterality in the 1 anima
l were observed.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
MALES:
At 1000 mg/kg/day in the survival animals, the dilatation of
renal tubule in 3 animals in the kidney and the dilatation of collecting tubules in 2 animals were
obserbed. But, all these changes were just slight. And the dilatation of renal tubule has a signi
ficant difference but no dose-related changes. As for the dilatation of collecting tubules, it has
no significant difference but increase tendency. In the other group, there were hemorrhage of thym
us gland, microgranuloma of the heart, microgranuloma of the liver and hepatocyte vacuolar degener
ation of the centrilobular, renal basophilic tubules, eosinophilic corpuscle in proximal tubule, cy
st, diffusive mineral deposition and neutrophilic infiltration. But it was judged with the inciden
tal change, because they were whether it equivalently seems even in the control group or small numb
er animals. And no abnormality was observed in spleen, adrenal, testiculus and brain in the control
and 1000 mg/kg group. In animal of death of the 1000 mg/kg group, there were hemorrhage
of the thymus gland, edema of the lung, autolysis of adrenal and lung and thymus gland with the de
adanimal of 1000 mg/kg group. As for those degrees, all were just slight. In the adrenal with the a
bnormality in the autopsy, no change which suggested hypertrophy was seen.

FEMALES:
Though at 1000 mg/kg/day survival groups, neutrophilic infiltratio
n (unilateral ) to medulla and papilla mammae part in the kidney were observed in the 1 animal, the
degree was slight. Though extensive softening of the medulla oblongata in the brain was observed
in the 1 example at 1000 mg/kg group, the degree was slight. In dead 6 animals of the 1000 mg/kg g
roup, there were the edema in 1 animal in the lung, the atrophy in 1 animal in the thymus gland, th
e atrophy in 5 animals and the atrophy of a Malpighian body in 1 animals in the spleen, the hyperp
lasia of zona fasciculata in 3 animals and the autolysis in 1 animal in the the adrenal and the ero
sion in 1 animal in the small intestinal mucosa. The degrees of the atrophy in the thymus gland an
d the atrophy of a Malpighian body were moderate, but the others were slight. All the changes are
noted related agonism. No changes which suggested, though the hypertrophy of the adrenal
in 2 animals, dark-red of the glandular stomach mucosa in 2 animals and dark-red of the intestinum
tenue were observed as abnormal in the autopsy of the 1000 mg/kg group.

Histopathological findings: neoplastic:
no effects observed
Details on results:
In order to address the issue of biological relevance of the elevated BUNvalues
a substantial number of datasets on the GINC
database (methacrylates and other chemicals) has been analysed by the registrant. In this analysis, control
values for BUN ranged between 16.1 and 24.3 (only 3 test results reported)
for female rats and between 11.4 and 19.96 for males. Looking at the individual dose response relationships it is apparent that a variability of about 10 to 15 %
is normal for the absolute value of this parameter. This indicates that for the
HEMA test all BUN values between 0 and 100 mg/kg/d are well within the
control range. The value at 300 mg/kg/d (20.82) is suspicious and may
already be a true effect of HEMA exposure. Furthermore, the value at
1000 mg/kg/d (25.06) is clearly elevated and well beyond the range of
the observed control values and should be regarded as clearly related
to HEMA exposure. As it appears, the statistical evaluation has been
mislead by an accidentally low control value which lead to a statistical overestimation
of the significance especially at 30 mg/kg/d. This is corroborated
by the fact that the BUN value at 100 mg/kg/d is statistically not
significantly elevated and by an absence of a dose-effect relationship between
30 and 100 mg/kg/d (the value at 100 mg/kg/d being lower).
In conclusion, there is a very high probability that the statistical significance
of the BUN value at 30 mg/kg/d is a chance finding of no biological relevance.
If one assumes that the BUN value at 30 mg/kg/d was a relevant, substance related
finding, what could be the physiological basis? Generally, changes
in BUN are regarded as an indicator for a disturbance of nitrogen/urea
excretion in the kidneys. There is, however, no indication for histopathological
changes in the kidneys of all test concentrations up to 300 mg/kg/d except
for some findings at the highest test concentration (1000 mg/kg/d). A dose related
increase of kidney weights (relative and/or absolute) was observed
beginning with slight effects at 100 mg/kg/d and higher. As a consequence,
in the conclusions of the repeated dose part of the study at the end of the
summary which is available online, an overall NOAEL of 30 mg/kg
has been defined by Furuhashi et al. for effects of HEMA upon subacute
administration in male and female rats in the combined OECD 422 study.
In the absence of other, e.g. histopathological findings, this is still a very
conservative assumption. Based on histopathological and other, more
substantial findings the NOAEL of the study could very well be defined
as 100 or 300 mg/kg/d.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Critical effects observed:
no

Heamatology and blood chemistry data, organ weights and histopathological observations under "Overall remarks, attachements"

Conclusions:
An OECD 422 combined repeated dose and reproduction developmental toxicity study study was conducted with rats by oral gavage at doses of 0, 30, 100, 300 and 1000 mg/kg. The NOAELs for repeated dose toxicity are considered to be 100 mg/kg bw/day for males, and 300 mg/kg bw/day for females.
Executive summary:

2-Hydroxyethyl methacrylate was studied for oral toxicity in rats in an OECD 422, combined repeated dose and reproduction/developmental toxicity screening test at doses of 0, 30, 100, 300 and 1000 mg/kg/day. One male and 6 females of the 1000 mg/kg group (12 animals of each sex) died during the treatment period. Male rats in 300 mg/kg and 1000 mg/kg dose groups had elevated kidney weights and also elevated levels of BUN. Histopathological changes in the kidney of male rats were confined to minimal (+/-) grade severity observations in 1000 mg/kg group animals that included: renal tubule dilatation (3 rats); collecting duct dilatation (2 rats); unilateral cyst (1 rat); diffuse mineralization (1 rat) and neutrophilic cellular infiltration (1 rat). In addition, basophilic tubules and eosinophilic bodies in proximal tubules were described in similar numbers of animals for both control and 1000 mg/kg groups. The single histological observation in 300 mg/kg group males was focal renal tubule degeneration of minimal (+/-) grade in one male rat.


Female rats had elevated kidney weight at 1000 mg/kg and at 100 mg/kg, but not at 300 mg/kg. Histopathological changes in kidney tissue described in one female rat as grade 1 (+) unilateral neutrophilic cellular infiltration into the medulla and papilla were observed in female rats only at the 1000 mg/kg dose level. Clinical chemistry measurements were not reported for female rats.


Overall, these results for repeated dose toxicity testing of HEMA indicate that the kidney is a target organ for toxicity in both male and female rats. Male rats had elevated kidney weights and BUN levels at 300 and 1000 mg/kg dose levels. However, histopathology was described as minimal and occurred only in a few animals. The 100 mg/kg dose was the No Observed Adverse Effect Level (NOAEL) for these effects on kidney in males. Female animals had elevated kidney weights and histopathological changes in one animal and only at the 1000 mg/kg dose level; 300 mg/kg was the NOAEL for lethality and effects on kidney in females.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat

Additional information



Repeated dose toxicity: oral


There are reliable data available to assess the toxic potential of HEMA after repeated oral exposure. HEMA has been evaluated for repeat-dose toxicity in Sprague-Dawley rats in the OECD 422 combined repeat-dose developmental/reproductive toxicity screening test. This study was conducted by the Japan Ministry of Health and Welfare and assigned a K-2, reliable with restriction reliability rating.


For the primary metabolites data are available for subchronic to chronic exposures. The eight studies used are reliable with or without restrictions (K-1 or K-2; partially presented under the chapter “carcinogenicity”). The metabolism from HEMA to the primary metabolites is well understood and the same is true for the further metabolism pathways of MAA and EG, respectively (see category document, chapters 5.2 and 5.7). The endpoint specific “scientific assessment” of the read across is thus acceptable with at least medium confidence.


 


HEMA


In the study for HEMA, 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.


Six animals of the 1000 mg/kg dose groups died (one male, five females). Clinical symptoms of intoxication observed at 1000 mg/kg [the highest dose tested] included: salivation, suppression of body weight gain, decrease in food consumption, increased kidney weights and renal histopathological changes as well as decrease in locomotor activity, adoption of a prone position and lacrimation (females only). Liver weight was increased in males only, but there was no histopathological correlate. Further, females exposed to 1000 mg/kg HEMA had an elevated incidence of malacia of the medulla oblongata, but this effect was not observed at lower doses and was not observed in males at any dose level.


Male rats in 300 mg/kg and 1000 mg/kg dose groups had elevated kidney weights and also elevated levels of BUN. Histopathological changes in the kidney of male rats were confined to minimal (+/-) grade severity observations in 1000 mg/kg group animals that included: renal tubule dilatation (3 rats); collecting duct dilatation (2 rats); unilateral cyst (1 rat); diffuse mineralization (1 rat) and neutrophilic cellular infiltration (1 rat). In addition, basophilic tubules and eosinophilic bodies in proximal tubules were described in similar numbers of animals for both control and 1000 mg/kg groups. The single histological observation in 300 mg/kg group males was focal renal tubule degeneration of minimal (+/-) grade in one male rat.


Female rats had elevated kidney weight at 1000 mg/kg and at 100 mg/kg, but not at 300 mg/kg. Histopathological changes in kidney tissue described in one female rat as grade 1 (+) unilateral neutrophilic cellular infiltration into the medulla and papilla were observed in female rats only at the 1000 mg/kg dose level. Clinical chemistry measurements were not reported for female rats.


Overall, these results for repeat-dose toxicity testing of HEMA indicate that the kidney is a target organ for toxicity in both male and female rats. Male rats had elevated kidney weights and BUN levels at 300 and 1000 mg/kg dose levels. However, histopathology was described as minimal and occurred only in a few animals. The 100 mg/kg dose was the No Observed Adverse Effect Level (NOAEL) for these effects on kidney in males. Female animals had elevated kidney weights and histopathological changes in one animal and only at the 1000 mg/kg dose level; 300 mg/kg was the NOAEL for lethality and effects on kidney in females.


 


 



The short half-life of HEMA (as desribed in the metabolism chapter) implies that, under normal physiological conditions, the systemic exposure to HEMA as parent ester is irrelevant for the hazard assessment. This is especially true when the low level of electrophilic reactivity as potential mode of action of the parent esters is considered. The combination of the short half-life and the weak electrophilic reactivity of the parent esters means that toxicity profile upon repeated dosing is determined by the profiles of the primary metabolites (EG and MAA).



Primary Metabolites


Primary Metabolite MAA (donor substance MMA)


Due to the corrosivity of MAA, a repeated dose study by the oral route has not been performed. In a guideline inhalation study, ten male and ten female Sprague Dawley rats per test group were whole body exposed to MAA vapour on 6 hours per working day for 90 days (65 exposures). The target concentrations were 20, 40, 100 and 350 ppm (corresponding to 72, 143, 358 and 1252 mg/m³). At the high dose of 350 ppm, equivalent to a body burden of 308 mg/kg/d in males and 377 mg/kg/d in females[1], the 90-day inhalation exposure of rats to MAA induced signs of general toxicity as indicated by decreased body weight, body weight gain, food consumption and transiently food efficiency in the high concentration male animals. At 350 ppm, the local irritating effect was marginal, indicated by the hypertrophy/hyperplasia of the respiratory epithelium in the nasal cavity of two female animals. Under the current test conditions, the no-observed adverse effect level (NOAEL) in this study is 100 ppm (equivalent to a body burden of 88 mg/kg/d in males and 96 mg/kg/d in females) for the male and female rats.


 


Primary metabolite EG


There are several rodent studies available with orally applied Ethylene glycol (EG). From those, subchronic and chronic feeding studies mainly performed by NTP were selected for the IUCLID dataset of HEMA for the reason of sufficient reliability and high relevance of the continuous uptake mode of a metabolite. In general, “the kidney is clearly identified as the most sensitive target organ in rats and mice after intermediate-duration oral exposure ” of high doses > 1000 mg/kg/d”. Typical renal effects included oxalate crystal deposition and renal tubular dilation, vacuolation, and degeneration. Oxalate, a metabolite of glycolic acid, forms a precipitate in the presence of calcium, and the deposition of these crystals in the renal tubules are hallmarks of ethylene glycol toxicity. Glycolic acid accumulation and metabolic acidosis do not contribute to renal toxicity, which is solely caused by oxalate crystal accumulation. Males were more sensitive than females, and rats were more sensitive than mice” (ATSDR 2010).


After subchronic feeding, the NOAEL in male mice was found to be 3230 mg/kg/d based on liver and kidney effects, while no adverse effects were found in female mice up to 16,000 mg/kg/d (NTP 1993). After subchronic feeding, the NOAEL in male rats was found to be 600-1000 mg/kg/d, while the NOAEL in female rats was found in female rats to be 1000-1500 mg/kg/d; both based on kidney effects (Melnick et al. 1984). After chronic feeding, the NOAEL in male mice was found to be 1500 mg/kg/d based on liver effects, while the NOAEL in female mice was found to be 3,000 mg/kg/d based on blood effects (all NTP 1993).


Repeated dose toxicity: inhalation


There are no relevant inhalation studies available on HEMA. A limited three-week inhalation study in rats at saturated VP (approx. 0.5 mg/L) showed no toxic effects.


See also discussion regarding “Route” below


Repeated dose toxicity: dermal


There are no relevant dermal studies available on HEMA.


Repeated dose toxicity: other routes


No relevant studies available.


Human information


There are no relevant data available


Summary and discussion of repeated dose toxicity


Subacute (~50 d) repeated dose studies are available for both, HEMA and HPMA. For the primary metabolites methacrylic acid/MMA, ethylene glycol and propylene glycol subchronic or chronic repeated dose data are available. For comparison reasons, results from rat studies are shown below.




 


Table11: Repeated dose toxicity - NOAEC/NOAEL summary

















































































Route & effects type



HEMA


MW 130.14



HPMA


MW 144.17



MAA      MW 86.09



MMA


MW 100.12



EG


MW 62.07



PG


MW 76.09



Inhalation


Local effects



---



---



100 ppm


358 mg/m³



25 ppm


208 mg/m³
*
   **



---



---



Inhalation


Systemic effects



---



---



100 ppm


358 mg/m³



500 ppm


1040 mg/m³



---



---



 



 



 



 



 



 



 



Oral


Systemic effects



100 mg/kg/d


(gavage)



300 mg/kg/d


(gavage)



---



164 mg/kg/d


(drinking water)



600 mg/kg/d


(feeding)



≥2500 mg/kg/d


(feeding)



0.77 mmol/ kg/d



2.08 mmol/ kg/d



 



1.64 mmol/ kg/d



9.67 mmol/ kg/d



32.9 mmol/ kg/d



*



The departure point for the risk assessment is the SCOEL IOLV of 50 ppm, taking into account observations in humans



 



**



No time-dependency of the lesion was found in several studies between 6 hours and 2 years



 


         


 


HEMA and HPMA have been evaluated for repeat-dose toxicity by the oral route in the OECD 422 protocol. In this protocol, both males and females were given daily oral gavage doses of HEMA and HPMA up to the limit dose of 1000 mg/kg/day for approximately 50 consecutive days. Toxicity was achieved in these studies as demonstrated by reduced food consumption and body weight gain at 1000 mg/kg/d. In addition, for HEMA only (not found for HPMA), kidney toxicity particularly in male rats as indicated by elevated BUN levels in serum, elevated kidney weights and minimal histopathological changes were described in 1000 mg/kg group animals. For males dosed with HEMA, the NOAEL was 100 mg/kg/d while the NOAEL for females administered HEMA was 300 mg/kg/d. No target organ was identified for HPMA even at doses which depressed body weight gain and food consumption.


These results indicate a mild effect of HEMA on the kidney which is considered a target organ for HEMA at very high repeat oral doses that depressed body weight gain and food consumption. These mild changes in kidney after approximately 50 days exposure indicate a small likelihood that effects of greater severity at the same effect doses or effects at lower doses would be observed in 90 day studies. The male rat (and Sprague-Dawley rat in particular) is prone to the spontaneous development of kidney toxicity beginning as early as 90 days of age (Goldstein 1988; Masoro, 1989). Thus, the mild effects described upon administration of limit doses of HEMA to male Sprague-Dawley rats may represent the interaction of the material with initial stages of development of spontaneous kidney toxicity in these animals.


 


Route


As outlined earlier, the saturated vapour density is approx. 80 ppm (427 mg/m³) for HEMA equivalent to a maximum body burden of approx. 100 mg/kg/d for a standard inhalation study in rat. In practice, the saturated vapour density is a theoretical value that cannot be achieved in full because under the conditions of an inhalation study the equilibrium is not reached fast enough. In consequence, that means that a study could only be performed below the expected NOAEL. The existing inhalation screening study confirms that.  


 


Discussion


As described in the toxicokinetics chapter, the short half-life of the parent ester combined with their weak electrophilic reactivity mean that toxicity profile upon repeated dosing is determined by the primary metabolites (EG and MAA). In comparison of the two hydroxyalkyl methacrylates HEMA has a lower NOAEL based on repeated dosing as compared to HPMA.


In the case of HEMA and EG after repeated exposure both substances demonstrate general signs of toxicity and body weight loss at high doses with specific target organ toxicity in the kidney (Furuhashi et al. 1997; ATSDR 2010). It is well understood that the kidney effects of EG are related to its metabolites, glycolic and oxalic acids. The relevant NOAEL of HEMA for the derivation of the DNEL is 100 mg/kg bw/d. So, the two predominant modes of action which have to be considered are general toxicity of the methacrylates as expressed by body weight effects and kidney effects as a specific MoA of the glycol metabolite.


Body weight: The common primary metabolite MAA is metabolised via the TCA cycle predominantly being eliminated as CO2 and water. After repeated exposure (350 ppm/-477 mg/kg/d, 90 days) there are no apparent adverse systemic findings other than reduced food consumption and transiently food efficiency in the high concentration male animals resulting in decreased terminal body weight and body weight gain in these animals. The dose range is consistent between the MAA study and HEMA. Bearing in mind that the study duration is different, and consistent findings have been observed e.g. in the 2-year carcinogenicity study with MMA (NTP, 1986) there is apparently not much progression of this lesion with an extended duration of the study,


Kidney effects: As mentioned earlier, the kidney effects of EG are related to its metabolites, glycolic and oxalic acids. Also, the markedly lower NOAELs in studies with bolus dosing (e.g. gavage studies) as compared to continuous dosing (e.g. feed studies) highlight the effect of transiently high serum concentrations. A typical exposure situation at the workplace would be more similar to the continuous dosing scheme so that kidney effects seen in gavage studies are probably less relevant in practice. In addition, the EG studies indicate, that a further progression with extended duration of the study to even lower NOAELs is not expected


As outlined earlier, the inhalation route is not an appropriate route of exposure for the hydroxyalkyl methacrylates HEMA and HPMA, because it is very unlikely that toxicologically relevant concentrations could be reached using the inhalation route.


Taken together, results of repeat-dose studies in HEMA and results from studies on MAA/MMA as representative of the chemical class of short-chain alkyl esters of methacrylic acid and on EG, the alcohol metabolite, indicate that additional studies of longer duration (90 days) are not critical for the assessment of HEMA and may therefore be waived. 


 


[1]Calculated using R8 guidance defaults for respiration / body weight, assuming 100 % absorption



Justification for classification or non-classification

HEMA is not classified for repeated dose toxicity. No severe or irreversible effects were identified. With a LOAEL of 300 mg/kg for male rats in a screening study with a study period between 28d and 90d, neither CLP nor UN GHS criteria are met.