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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld Germany
- Age at study initiation: male: 35-37 days; female: 33-35 days
- Housing: 5 animals per cage
- Diet (e.g. ad libitum): ad libitum (ground Kliba maintenance diet mouse/rat "GLP" meal)
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30-70
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12712

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Drinking water with 1% Carboxymethylcellulose, 35 µl of Cremophor EL, 25 µl hydrochloric acid
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was administered as an emulsion. To prepare the emulsion, the appropriate amount of test substance was weighed out depending on the desired concentration and filled up with the vehicle (Drinking water containing 1% Carboxymethylcellulose, 35 µl [1 drop] of Cremophor EL and 35 µl [1 drop] hydrochloric acid [1 mol/l] per 100 ml). The preparations were homogenized using a high-speed homogenizer and were kept homogenous during the administration with a magnetic stirrer. The test-substance preparations were prepared weekly.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in the vehicle (Drinking water containing 1% Carboxymethylcellulose, 35 µl [1 drop] of Cremophor EL and 35 µl [1 drop] hydrochloric acid [1 mol/l] per 100 ml) for a period of 7 days was determined before the start of the administration period (PART III, Supplement).

Homogeneity and concentration control analyses of the test-substance preparations in samples of all concentrations were performed at the start and towards the end of the administration period.
Duration of treatment / exposure:
3 months with a recovery period of 28 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 60, 120, 360 mg/kg b.w.
Basis:

No. of animals per sex per dose:
0 and 360 mg/kg b.w.: 15 animals
60 and 120 mg/kg b.w.: 10 animals
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
All animals were checked daily for any clinically abnormal signs. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE :
Individual food consumption was determined weekly over a period of 1 day and calculated as mean food consumption (g per animal and day).

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: Yes

WATER CONSUMPTION AND COMPOUND INTAKE :
Water consumption was determined weekly over a period of 4 days and calculated as mean water consumption (g per animal and day).

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: males: days 91; female: days 89
- Dose groups that were examined: Control group and high-dose group (360 mg/kg b.w.)

HAEMATOLOGY: Yes

CLINICAL CHEMISTRY: Yes

URINALYSIS: Yes

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Kruskal-Wallis test; Wilcoxon test; Dunnett`s test; WELCH test

Results and discussion

Results of examinations

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
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
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 examined
Details on results:
CLINICAL SIGNS AND MORTALITY
One female animal (no. 55) of test group 0 (control) died because of a gavage error on study day 54.
No test substance-related findings were observed. The female control animal which died because of a gavage error showed altered general state one day before its death.

BODY WEIGHT AND WEIGHT GAIN
Compared to the control animals, body weights of females of test group 3 (360 mg/kg body weight/day) were significantly lower from study day 35 onwards until the end of the administration period (max. of 11.0% less on study day 84). During the recovery period the body weights of this test group were still significantly lower (-12.5% on study day 112). As this effect correlated with the reduced food consumption, it was assessed as related to the test-substance administration.
Compared to the control animals, body weight change values of females of test group 3 (360 mg/kg body weight/day) were significantly lower from study day 28 onwards until the end of the administration period (-24.2% on study day 91) and recovery period (-28.8% on study day 119).
In male animals of test group 3 (360 mg/kg body weight/day) body weight and body weight change values were not significantly lower compared to the control animals.
For both sexes, no test substance-related deviations to the control group were seen in test group 1 (60 mg/kg body weight/day) and test group 2 (120 mg/kg body weight/day).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
A tendency to reduced food consumption could be seen in female animals of test group 3 (360 mg/kg body weight/day) during the entire administration period and recovery period. No further test substance-related findings were observed.

FOOD EFFICIENCY
No test substance-related findings were observed.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
No test substance-related overt changes in water consumption were observed.

OPHTHALMOSCOPIC EXAMINATION
The observed corneal stipplings and remainders of the pupillary membrane, which were detected towards the end of the study, were equally distributed between the test substance-treated animals and the controls. Therefore, these findings were not related to treatment and were without any toxicological relevance.

FUNCTIONAL OBSERVATIONAL BATTERY
Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between test substance-treated groups and controls and occurred only in single animals without a dose-response relationship, these observations were considered to have been incidental.
- Home cage observations: No test substance-related findings were observed.
- Open field observations: No test substance-related findings were observed.
- Sensorimotor tests/reflexes: No test substance-related findings were observed.
- Quantitative parameters: No test substance-related findings were observed.

MOTOR ACTIVITY MEASUREMENT
Regarding the overall motor activity (MA), a significantly lower mean MA value was observed for the male animals of test group 2 (120 mg/kg body weight/day), but no difference occurred in male animals of test groups 1 (60 mg/kg body weight/day) and 3 (360 mg/kg body weight/day). Thus, a dose-response relationship was missing.
Furthermore, no differences occurred in female animals of test groups 1-3 when compared to the control group.
With regard to the single intervals, the mean MA was significantly decreased in male animals of test group 2 (120 mg/kg body weight/day) at interval 2, when compared to the control animals. Mean MA values of the female animals of test groups 1 (60 mg/kg body weight/day) and 2 (120 mg/kg body weight/day) were significantly increased at interval 7 and significantly decreased in test group 3 (360 mg/kg body weight/day) at interval 3.
All deviations in test groups 1-3 occurred at single time points and a dose-response relationship could not be determined.
Taken together, all detected differences occurred incidentally without a relation to dosing or gender. Therefore, they were assessed as spontaneous in nature and toxicologically irrelevant.

HAEMATOLOGY
At the end of the administration period no treatment-related, adverse changes were measured in hematology and haemostasis parameter values.
The haemoglobin values were slightly, but statistically significantly decreased in male rats of test group 3 (360 mg/kg body weight/day; 3% reduction compared to the controls). The finding was assessed as incidental and non-adverse since the other red blood cell parameters were not altered (MUELLER, A., et al., 2006).

The reticulocyte cell counts were statistically significantly decreased in females of test group 3 (360 mg/kg body weight/day). These lower reticulocyte counts were not accompanied by a change of any other red blood cell parameters. Therefore, this finding was assessed as incidental rather than adverse.

The absolute lymphocyte counts were statistically significantly increased in females of test group 3 (360 mg/kg body weight/day), and the absolute eosinophil counts were statistically significantly decreased in females of test group 1 (60 mg/kg body weight/day). Both deviations occurred without any change in the total white blood cell counts of these samples and no other changes were found in the differential blood cell counts. Therefore, these findings were regarded as incidental rather than adverse effects.

CLINICAL CHEMISTRY
After substance administration the aspartate aminotransferase (AST) activities were statistically significantly reduced in male rats of test group 2 (120 mg/kg body weight/day). This decrease was not dose-dependent, and therefore regarded as a non-adverse effect.

After the recovery period the AST activities were statistically significantly reduced and the urea levels were statistically significantly increased in the female rats of test group 3 (360 mg/kg body weight/day). The mean AST and urea values of the treated test groups were within the historical range of rats in 3-month studies (AST 1.27-1.92 U/L; urea 6.07-8.14 mmol/L; PART III, Supplement). No corresponding change was observed in the male rats. Therefore, these changes were regarded as incidental rather than treatment-related.

At the end of the administration period, the potassium levels as well as the inorganic phosphate levels were statistically significantly increased in rats of both sexes of test group 3 (360 mg/kg body weight/day). The phosphate concentration was additionally statistically significantly increased in male and female rats of test group 2 (120 mg/kg body weight/day), and in male rats of test group 1 (60 mg/kg body weight/day). In female rats of test group 3 (360 mg/kg body weight/day) the chloride levels were statistically significantly higher and the globulin levels were statistically significantly lower compared to the controls.

In contrast to test group 3 (360 mg/kg body weight/day) the increased inorganic phosphate levels were the only changed parameter values in test group 2 (120 mg/kg body weight/day; both sexes) and in the males of test group 1 (60 mg/kg body weight/day). The phosphate level increase was regarded as a treatment-related, but not adverse effect because no histopathological correlate was found (4.4. Histopathology).

URINALYSIS
With regard to the urinalysis parameters, no treatment-related adverse effects were found in the test-substance treated rats at the end of the administration period.

In the males of test groups 2 and 3 (120 and 360 mg/kg body weight/day) fewer phosphate crystals were found in the urine sediment compared to the controls. Regarding the individual pH values of the urine samples, there appeared to be slightly lower pH values in the dosed rats. Therefore, it was assumed that the phosphate crystals were dissolved in the urine of the test substance-treated males. This effect was regarded as treatment-related, but not adverse.

ORGAN WEIGHTS
Absolute organ weights
males: The tendency of a decreased terminal body weight in males of test group 3 (360 mg/kg body weight/day) was considered to be a treatment-related effect.
All other significant weight changes were assessed as being related to the decrease of terminal body weight or incidental in nature. Neither a dose-dependency nor histopathological changes could be correlated and, therefore, a biological relevance was not given.
The increase of testes weights after the 28-day recovery period was regarded to be incidental and not related to treatment.

females: The significantly decreased terminal body weight in females of test group 3 (360 mg/kg body weight/day) was considered treatment-related.
All other significant weight changes were assessed as being related to the decrease of terminal body weight or incidental in nature. Neither a dose-dependency nor histopathological changes could be correlated and, therefore, a biological relevance was not given.
The decrease of uterus weights after the 28-day recovery period was regarded to be incidental (sex-cycle dependent) and not related to treatment.

Relative organ weights
males: The increase of the relative organ weights in males of test group 3 (360 mg/kg body weight/day) was considered to be secondary to the decrease of the absolute terminal body weights and did not reflect organ specific effects.
Males (R1)
No significant weight changes were calculated.

females: The increase of the relative organ weights in females of test group 3 (360 mg/kg body weight/day) was considered to be secondary to the decrease of the absolute terminal body weights. The increase of the relative kidney weight in females of test group 2 (120 mg/kg body weight/day) was assessed as incidental and did not reflect an organ specific effect.
Females (R1)
No significant weight changes were calculated.

GROSS PATHOLOGY
All gross lesions noted were single occurrences and considered to be incidental or spontaneous in nature and not related to treatment.

HISTOPATHOLOGY:
All histopathological findings noted occurred either incidentally as single cases or were equally distributed over the test groups as well as the control and were interpreted as being spontaneous in nature. Most of the gross lesions could be correlated with a sound histomorphological finding.

Effect levels

Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Effects on body weight gain and some blood chemistry parameters at 360 mg/kg bw/d

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Absolute organ weights Males (F1)

Organs

Test groups

 

1

2

3

Terminal body weights

 -3%

 -2%

-10%

Brain

 -4%*

+1%

 -3%

Heart

 -6%

 -4%

-12%**

Spleen

-15%*

 -3%

-15%*

Thymus

-21%**

-16%*

-26%**

* for p 0.05

** for p 0.01

Males (R1)

Organs

Test groups

 

 

 

3

Testes

 

 

+12%**

* for p 0.05

** for p 0.01

Females (F1)

Organs

Test groups

 

1

2

3

Terminal body weights

 -2%

    0%

 -9%**

Adrenal glands

-15%*

 -1%

-21%**

Thyroid glands

-13%*

-10%

 +11%

* for p 0.05

** for p 0.01

Females (R1)

Organs

Test groups

 

1

2

3

Uterus

 

 

-31%*

* for p 0.05

** for p 0.01 Relative organ weights Males (F1)

Organs

Test groups

 

1

2

3

Kidneys

-1%

+17%

+15%**

Liver

-3%

+7%

+11%**

* for p 0.05

** for p 0.01

Females (F1)

Organs

Test groups

 

1

2

3

Brain 

 -1%

 0%

+7%*

Kidneys

+4%

 +6%*

+14%**

Liver

  0%

+2%

+12%**

* for p 0.05

** for p 0.01

 

Applicant's summary and conclusion

Conclusions:
In conclusion, the oral administration of 2-Ethylhexyl Methacrylate by gavage over a period of 3 months with a recovery period of 28 days revealed signs of general systemic toxicity in male as well as female rats at a dose level of 360 mg/kg body weight/day. The NOAEL was found at 120 mg/kg body weight/day in female and male Wistar rats.

Therefore, under the conditions of the present study, the no observed adverse effect level (NOAEL) was 120 mg/kg body weight/day in male and female Wistar rats.

Executive summary:

In an OECD 408 study performed according GLP, 2-Ethylhexyl Methacrylate was administered daily by gavage to male and female Wistar rats at dose levels of 0 (test group 0), 60 (test group 1), 120 (test group 2) and 360 (test group 3) mg/kg body weight/day over a period of 3 consecutive months. Control and high-dose groups consisted of 15 animals per sex and group, whereas low- and mid-dose groups consisted of 10 animals per sex and group. After 3 months of treatment, 10 animals per sexof all dose groups were sacrificed (main groups). The remaining 5 animals per sex of controland high-dose groups were maintained for another 28 days without administration of the testsubstance (recovery groups). Food consumption and body weight were determined weekly. The animals were examinedfor signs of toxicity or mortality at least once a day. Detailed clinical examinations in an openfield were conducted prior to the start of the administration period and weekly thereafter. A functional observational battery (FOB) and measurement of motor activity were carried out during the last week of exposure. Clinico-chemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. All animals were assessed by gross pathology, followed by histopathological examinations.

The following test substance-related, relevant findings were noted in test group 3 (360 mg/kg body weight/day): a statistically significantly lower body weights in female animals from study day 35 onwards until the end of the administration period (max. of 11.0% lower on study day 84) and during the recovery period (12.5% lower on study day 112), a statistically significantly lower body weight changes in female animals from study day 28 onwards until the end of the administration period (max. of 25.5% lower) and during the recovery period (-28.8% on study day 119), a statistically significantly lower terminal body weight in female animals (9% less compared to the controls), a statistically significantly increased potassium levels in both sexes, and a statistically significantly increased chloride levels and decreased globulin levels in females. During the recovery period the body weights of the female animals remained below the control animals (13.2% less on study day 119). However, the clinical pathology parameters reverted to a normal physiological range.

In test groups 2 and 1 (120 and 60 mg/kg body weight/day), no treatment-related, adverse findings were observed concerning clinical examinations, clinical pathology and pathology parameters.

In conclusion, the oral administration of 2-Ethylhexyl Methacrylate by gavage over a period of 3 months with a recovery period of 28 days revealed signs of general systemic toxicity in male as well as female rats at a dose level of 360 mg/kg body weight/day. Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 120 mg/kg body weight/day in male and female Wistar rats.

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