2-hydroxyethyl methacrylate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-04-25 to 2007-07-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: European Economic Community (EEC) Annex to the commission directive 87/302/EEC, "Sub-Chronic Inhalation Toxicity Study", published in the Official Journal of the European Communities L133, 30 May 1988.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methacrylic acid

EC Number:

201-204-4

EC Name:

Methacrylic acid

Cas Number:

79-41-4

Molecular formula:

C4H6O2

IUPAC Name:

2-methylprop-2-enoic acid

Test material form:

liquid

Specific details on test material used for the study:

Test substance No.: 06/0581-2
Batch-Identification: 011835eda0
Purity: 99.8 area-%
Homogeneity: Homogeneous
Storage stability: Expiry day: 13 Apr 2008Date of production: 12 Apr 2007
Physical state/Appearance:Liquid / colorless, clear
Storage conditions: Room temperature, no direct sunlight, under light exclusion,
protect against heat, storage between 18-25°C, under atmospheric oxygen

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD(SD)

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
Arrival in the testing facility: 11 Apr 2007
The female animals were nulliparous and non-pregnant.
Only animals free from clinical signs of disease were used for the study.

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.

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 ppm

Remarks:

= 70 mg/m3 (Targeted conc.)

= 19.9 ppm ± 2.2 (Measured concentration)

Doses / concentrations 2

Dose / conc.:

40 ppm

Remarks:

= 141 mg/m³ (Targeted conc.)

= 39.6 ± 2.4 (Measured concentration)

Doses / concentrations 3

Dose / conc.:

100 ppm

Remarks:

= 352 mg/m³ (Targeted conc.)

= 100.5 ± 6.0 (Measured concentration)

Doses / concentrations 4

Dose / conc.:

350 ppm

Remarks:

= 1232 mg/m³ (Targeted conc.)

= 357.0 ± 11.4 (Measured concentration)

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.

Examinations

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

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Alopecia was noted in one female animal of the high concentration group (350 ppm). Despite
of this, the detailed clinical observations did not reveal any other abnormalities in the low
concentration (20 ppm), low intermediate concentration (40 ppm), intermediate concentration
(100 ppm) and high concentration (350 ppm).

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male animal exposed to the low intermediate concentration (40 ppm) was found dead on
study day 55 after the exposure.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight during the exposure period:
The mean body weight of the male animals of the low concentration (20 ppm), low
intermediate concentration (40 ppm) and intermediate concentration (100 ppm) were not
statistically significantly different from the control group 0.
The mean body weight of the male animals of the high concentration (350 ppm) was
significantly decreased about - 6.1% to - 12.8% from study day 7 onward.
The mean body weight of the female animals were not significantly different from the control
group 0. The decrease of - 7.3 % in the high concentration group on study 70 was considered
to be incidencial because this change did not continue with the time of continuous treatment.
Body weight change during the exposure period:
The body weight change of the male animals of the high concentration (350 ppm) was
significantly decreased from study day 7 onward (- 28.5% to - 64.8%).
In all other test groups, the main body weight change of the male and female animals were
not different from those of the controls during the whole study period. An increase of 41.3 %
in the male animals of test group 2 (40 ppm) on day 7 was considered to be incidential
because there were no concentration-response relation and the effect did not continue with
the continuous exposure.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption was significantly decreased in the male animals of the high
concentration (350 ppm):
- On study day 7 (- 13.5%) and day 14 (- 12.2%)
- From study day 28 through to day 84 (from - 9.4% to - 13.7%)
The food consumption of the male and female animals of the other test groups was not
significantly different from the control group 0.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

In general, the food efficiency was decreased only in the male animals exposed to 350 ppm
test substance, indicating by significant decreases on study days 7, 28, 49, 56. Other
changes are considered not to be substance-ralated and are described and discussed in
detail in the following section.
Male animals:
There were slight decrease of the food efficiency in the male animals exposed to 40 ppm test
substance on study day 7 and a slight increase in the male animals exposed to 100 ppm test
substance on study day 21. These changes were considered to be incidential, because there
were no concentration-response relation and did not continue with the ongoing exposure. At
the end of the study (study day 91), in comparison with the previous values of the same test
group, a high food efficiency was observed in all groups, including control. This high food
efficiency was considered to be a compensation reaction after the overnight fasting.
Significant increase of the food efficiency of all the test groups are due to the lower control
value. Because of the missing concentration-response relation, all these increased are
considered to be not substance-related.
Female animals:
In the female animals on study day 35, the food efficiency was with - 0.9 g/kg/day (standard
deviation of 13.1 g/kg/day) much lower than the control values on the other study days.
Therefore, the increased food efficiency of test group 1 (20 ppm) and test group 4 (350 ppm)
are considered to be not substance-related. Similarly, decreased food efficiency was
observed in group 1 and 4 female animals on study day 42. On this day, the control value
was high. Due to the missing concentration-response relation and the lack of timely
correlation, we considered these two changes as not substance-related

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmologic examinations did not show any impairment of the refracting media.
Spontaneous findings such as remainders of the pupillary membrane, corneal stippling or
blood vessel in the vitreous body (one group 2 male animal only) were observed in several
animals of all test groups and the control group without any concentration-response
relationship.

Haematological findings:

no effects observed

Description (incidence and severity):

Concerning hematology and blood coagulation parameters no treatment-related deviations
were found.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

After 3 months of substance inhalation, there was a decrease of the albumine concentration
in the females of the 350 ppm dose group. Consequently, there was also a decrease in total
protein levels in this group.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the third study months there was found a decrease of the urine volume combined with an
increase of the specific gravity of the urine in the 350 ppm dose group of both sexes (not
statistically significant in the females).

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
The terminal body weights in the high concentration group are significantly decreased, which
is regarded a treatment-related, systemic effect. The significant decrease of the epididymides
weights is considered to be related to the body weight decrease and not a direct substance induced effect.

The significant changes in the brain and heart the high concentration males are regarded to
be related to the decreased terminal body weights and are not regarded a direct substancerelated
effect.

Females:
The terminal body weights in the high concentration group are significantly decreased, which
is regarded as treatment-related, systemic effect. The weak but significant decrease of the
liver weights is considered to be related to the body weight decrease and not a direct
substance-induced effect.

The significant changes in the brain and lungs of the high concentration females and the
brain of the concentration group 2 females are regarded to be related to the decreased
terminal body weights or incidental and are not regarded a direct substance-related effect.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All gross lesions noted are considered to be incidental or spontaneous in nature and not
related to treatment

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Deviations from "zero values" were obtained in several animals. However, as most findings
were equally distributed between treated groups and controls, were without a dose-response
relationship or occurred in single animals only, these observations were considered to have
been incidental.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In 3 of 10 males and 2 of 10 females of the high concentration group (350 ppm) and in 1 of
10 males and 2 of 10 females in the mid-high concentration group (100 ppm), a focal,
minimal (grade 1) infiltration of lymphoid cells was present in the pituitary gland. This lesion is
regarded a treatment-related finding. The slight (grade 2) goblet cell hypertrophy/ hyperplasia
in the respiratory epithelium of the nasal cavity (level I) in 2 of 10 female high concentration
animals may express a slight irritation of the respiratory epithelium due to treatment.
The focal, tubular degeneration of testes parenchyma in 2 of 10 high concentration males
versus 1 of 10 control males, and the single, diffuse tubular degeneration in one high
concentration male (1 of 10 high concentration males) are regarded to be most probably
incidental or spontaneous in nature and not related to treatment.
All other findings noted are considered to be incidental or spontaneous in nature and not
related to treatment.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Sperm analysis:
Concerning the sperm motility, the spermatid counts in the testes and the cauda epididymidis
as well as the percentages of abnormal sperms no treatment-related findings could be found.

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

Effect levels

1

Key result

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/m³ or 102.1 mg/kg bw/day

2

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: = 352 mg/m³ or 102.1 mg/kg bw/day

3

Dose descriptor:

LOAEC

Remarks:

systemic effects

Effect level:

350 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: = 1232 mg/m³ or 375.4 mg/kg bw/day

4

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 or 375.4 mg/kg bw/day

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a valid guideline study, the no-observed adverse effect level (NOAEL) for both, local and systemic effects is 100 ppm for male and female rats exposed by whole body inhalation for 90 days.

Executive summary:

In a valid guideline study acc. to OECD 413 ( Subchronic inhalation toxicity: 90 day exposure of rats), methacrylic acid 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 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) for both, local and systemic effects, in this study is 100 ppm (352 mg/m³) for male and female rats.