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Description of key information

OECD 422 study:.

 

Three groups of ten male and ten female rats received Ethoxy ethoxy ethyl acrylate at doses of 25, 75 and 225 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation

 

Oral administration of Ethoxy ethoxy ethyl acrylatewas well toleratedwith no mortalities and no adverse effects of treatment on clinical condition, sensory reactivity, grip strength, motor activity, body weight gain, food intake or blood chemistry measurements in males and females treated with Ethoxy ethoxy ethyl acrylate.

 

Microscopic findings in the stomach of males and females treated at 75 or225 mg/kg/day are compatible with secondary (reactive) changes due to an irritant effect of the test item on the stomach when administered by oral gavage. Inflammatory lesions were present in the glandular and non-glandular region of the stomach.

 

Macroscopically enlarged local lymph nodes in males treated at 225 mg/kg/day show histological features suggestive of local immunostimulation, which could be secondary to the gastric inflammatory changes.

 

In addition, an increase in total and differential leucocyte counts were observed in males treated at 225 mg/kg/day which is likely secondary due to the minimal to moderate inflammatory changes observed at microscopic evaluation in the forestomach of 9/10 males in this group. However, no similar increase in leucocyte counts was detected in females treated at 225 mg/kg/day despite the minimal to slight microscopic stomach lesions observed in 9/10 females at this dose level.

 

A NOAELanda LOAEL were establishedto 25- and 75 mg/kgbw/day, respectively both for effects related to local stomach inflammation observed in both sexes.

Furthermore a NOAEL for systemic toxicity at 225 mg/kg kg/day can be concluded.

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
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2016 to May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
These minor discrepancies: slightly heavier male rats and one week older than written in the protocol. Considered to have no impact on the integrity of the study.
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): MIRAMER M170
- Physical state: Clear colorless liquid
- Analytical purity: 95.49% ( CAS No.: 7328-17-8; EC No.: 230-811-7)
- Impurities (identity and concentrations): Ethyl Carbitol 0.99%, 2-Ethoxy ethyl acrylate(EOEA) 0.14%, EOEOA/AA 1.28%, EOEOEA dimer 2.11%.
- Lot/batch No.: 151210177
- Expiration date of the lot/batch: 30 June 2017
- Storage condition of test material: At ambient temperature (10 to 30 degree celcius) and protected from light (although could be used for formulation in light).
Supplier: Miwon
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Rat, RccHan®:WIST from Harlan Laboratories Models, S.L.
- Age at study initiation: Males 76 to 83 days old and females 90 to 97 days old.
- Weight at study initiation: Males: 360 to 446 g Females: 239 to 306 g.
- Fasting period before study: No.
- Housing:Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.,Solid bottom cages contained softwood based bark-free fiber bedding which was changed at appropriate intervals each week..
- Diet: SDS VRF1 Certified pelleted diet - ad libitum.
- Water: Tap water ad libitum
- Acclimation period: Males six days and females 20 days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): air-conditioned with ranges for room temperature 20 -24 °C
- Humidity (%): relative humidity 40-70%
- Air changes (per hr): not specified.
- Photoperiod (hrs dark / hrs light): a 12-hour fluorescent light/12-hour dark cycle

IN-LIFE DATES: From: To: 7 December 2016 to 5 February 2017
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The stock solution was prepared by weighing the necessary amount of test item in an appropriate The required amount of test item was weighed out and approximately 50% of the final volume of vehicle was added. It was magnetically stirred until the test material was uniformly mixed. It was made up to the required volume with vehicle and mixed again with a magnetic stirrer until homogenous.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil is a generally recognized vehicle
- Concentration in vehicle: 0, 5, 15 and 45 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight.
- Lot/batch no. (if required): NA
- Purity: NA
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Fresh preparations were made weekly and stored refrigerated (2-8°C).

For each day of administration, the necessary volume was taken from the stock solution into appropriate containers. The aliquots were stored at room temperature (20 ± 5 ºC) protected from light.

Stability and homogeneity:
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. Stability of the test item was confirmed for one day at ambient temperatures (15-25°C) or for 15 days when stored refrigerated (2-8°C).

Achieved concentration:
Samples of each formulation prepared for administration in Week 1 of treatment and on Day 12 of lactation were analyzed for achieved concentration of the test item.
Duration of treatment / exposure:
Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks.
Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation.

Animals of the F1 generation were not dosed.
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1 (vehicle control)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
225 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
80 in total (40 males and 40 females):

Group 1 (0 mg/kg): 10 M/10F
Group 2 (25 mg/kg): 10 M/10F
Group 3 (75 mg/kg): 10 M/10F
Group 4 (225 mg/kg): 10 M/10F
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:

The dose levels were selected in conjunction with the Sponsor, based on the results of the 2-week preliminary toxicity study in the Sprague Dawley rat (Envigo Study No. SJ71TP). using doses up to 1000 mg/kg/day. Death was observed in the group dosed 1000 mg/kg/day (due to serosal inflammation/peritonitis) and doses at 325 mg/kg/day caused stomach ulceration.

Based on findings in the 2-week study the highest dose in the present study was set to 225 mg/kg/day a dose that induce some toxicity, but not severe toxicity or death. Intermediate and low doses were set at 75 and 25 mg/kg/day to allow evaluation of any dose related trends. The low dose of 25 mg/kg/day was expected to be a no effect level for stomach lesions
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily for mortality and for signs of reaction to treatment and/or symptoms of ill health before dosing and post dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and on Days 0, 7, 14 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena
observations were performed on each animal. These observations were performed outside the home cage, in a standard arena, at least two hours after dosing (where applicable) to ensure that any transient effects of treatment are identified. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males: Before dosing on the day that treatment commenced (Week 0) and weekly thereafter. On the day of necropsy.

F0 females: Before dosing on the day that treatment commenced (Week 0) and weekly before pairing. Days 0, 6, 13 and 20 after mating. Day 1, 4, 7 and 13 of lactation. On the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Weekly, from the day that treatment commenced.
For females after mating food consumption was performed to match the body weight recording: Days 0-5, 6-12 and 13-19 after mating Days 1-3, 4-6 and 7-12 of lactation. From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.
No food consumption was examined during the mating period, with exception for males in week 4.

WATER CONSUMPTION AND COMPOUND INTAKE: not specified.

Clinical Laboratory Investigations:
Blood samples were collected after overnight withdrawal of food at the following occasion: Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 - 0.7 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant or lithium heparin as anticoagulant and examined for hematology or blood chemistry. Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate.

In addition, blood samples were collected from animals for Thyroid Hormone Analysis at the following occasions:
-At termination: All surviving F0 adult males and females
-Day 4 of age: F1 offspring, two females per litter (where possible)* one for T4 and one for TSH.
* No pups were eliminated where the resultant litter size would have dropped below ten per litter.



Sensory reactivity and grip strength assessments (Approach response, Pinna reflex, Auditory startle reflex, Tail pinch response, and Grip strength) were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the five lowest numbered surviving lactating females in each group at Day 7-9 of lactation.
For females, animals were moved into individual cages prior to transport to the testing room.

During Week 5 of treatment for males and at Day 7-9 of lactation for females, the motor activity of the five lowest numbered surviving males and the five lowest numbered surviving lactating females in each group was measured (before dosing) using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.
Sacrifice and pathology:
Sacrifice of F0 Females
At Day 14 of lactation (following terminal blood sampling) exsanguinated and necropsied.
F0 females failing to produce a viable litter: killed at Day 25 after mating using carbon dioxide asphyxiation with subsequent exsanguination and then necropsied

Sacrifice of F0 Males
At week 5 of the study all males were killed using carbon dioxide asphyxiation with subsequent exsanguination and then necropsied.

Sacrifice of F1 Generation:
Selected offspring for Day 4 thyroid hormone analysis
- Day 4 of age.
Scheduled kill - Day 13 of age were killed by decapitation.
Offspring - not selected for thyroid hormone sampling killed by Intraperitoneal injection of sodium pentobarbitone.

Necropsy and a macroscopic examination were carried out for all the other animals.
No animal was exposed to carbon dioxide until after completion of blood sampling hematology, blood chemistry and thyroid hormone assays.
Other examinations:
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

See any other information on materials and methods for a full pathology table.
Statistics:
All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.

The following statistical methods were used to analyze food consumption, body weight, clinical laboratory data, organ weights and ratios as well as macroscopic findings and comparisons were performed between Group 1 vs 2, 3 and 4.

Parametric analysis (t-test and ANOVA, Willliams test, or Dunnetts test) was performed if Bartlett's test for variance homogeneity was not significant at the 1% level and i significant at 1% level after both logarithmic and square-root transformations a non-parametric analysis (Kruskal-Wallis, Wilcoxon rank sum test, shirley's or steel's test) was performed

For grip strength, motor activity, clinical pathology, litter size and survival indices, if 75% of the data (across all groups) were the same value, for example c, Fisher’s exact tests (Fisher 1973) were performed.

Significant differences between the groups compared were expressed at the 5% (p<0.05) or
1% (p<0.01) level.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs observed at the detailed physical examinations that were considered to be attributable to treatment with Ethoxy ethoxy ethyl acrylate.
Mortality:
no mortality observed
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
Description (incidence and severity):
Hematological examination revealed high mean total and differential leucocyte counts and large unstained cell counts in males treated at 225 mg/kg/day when compared with controls.

In females treated at 25, 75 or 225 mg/kg/day red cell distribution width was high when compared with controls attaining statistical significance at all levels, however the magnitude of change from controls showed no correlation to a dose-related trend. Platelet counts were observed to be low in females treated at 225 mg/kg/day when compared with controls attaining statistical significance however there was no relationship to dose level with values at 25 mg/kg/day and 75 mg/kg/day
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Biochemical evaluation revealed no clear effect of treatment.

In males and females treated at 225 mg/kg/day, marked increases in mean alanine phosphatase and aspartate transferase concentrations were observed, however a review of the individual data attributed this finding to markedly increased levels for both parameters in one male and one female (Nos. 2 and 46 respectively). No relationship to treatment is inferred.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Motor activity was considered to be unaffected by treatment.

Among males, the only statistically significant intergroup difference was that mean high beam scores in all groups of treated males were lower than Controls at 18 minutes; there was no dose response. This isolated difference was considered to have arisen by chance and to be unrelated to treatment.

Motor activity scores for females treated with Ethoxy ethoxy ethyl acrylate were comparable to Controls.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Group mean adjusted adrenal weights were slightly increased in females treated with Ethoxy ethoxy ethyl acrylate at all dose levels (25, 75 or 225 mg/kg/day); these changes were minor and did not attain statistical significance.

Mean adjusted liver and spleen weights were slightly high in males treated at 225 mg/kg/day.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic examination following 5 weeks of treatment revealed forestomach depressions and forestomach thickening in males treated at 75 mg/kg/day or 225 mg/kg/day (75 mg/kg/day: 2/10 and 1/10 males respectively; 225 mg/kg/day: 7/10 and 10/10 males respectively). In addition, 7/10 males treated at 225 mg/kg/day were found to have enlarged pancreatic lymph nodes.

In females on Day 14 of lactation, macroscopic examination similarly revealed forestomach depressions and forestomach thickening in females treated at 225 mg/kg/day (6/10 and 10/10 respectively); furthermore corpus stomach depressions were observed in females treated at 75 mg/kg/day or 225 mg/kg/day (2/10 and 4/10 respectively).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with Ethoxy ethoxy ethyl acrylate were seen in the stomach of both males and females and pancreatic lymph nodes of males.

These consisted of diffuse hyperplasia of the squamous epithelium, with a focal to multifocal exophytic growth pattern, along with hyperkeratosis, inflammation of the mucosa and submucosa (occasionally extending down the muscle and serosal layers at the high dose), as well as focal to multifocal erosions/ulcerations in some animals.

In the glandular region of the stomach of a few females treated at 75 or 225 mg/kg/day (1/10 and 3/10 respectively), focal to multifocal areas of slight to moderate mucosal necrosis or erosion were observed.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: F0 generation, stomach irritation/inflammation.
Key result
Dose descriptor:
NOAEL
Effect level:
225 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no systemic effects observed
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
75 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Summary table of treatment related findings in the stomach for animals killed at the end of treatment:

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

25

75

225

0

25

75

225

Squamous Cell Hyperplasia -Nonglandular Region

 

 

 

 

 

 

 

 

Minimal

0

0

1

1

0

0

2

1

Slight

0

0

0

4

0

0

0

4

Moderate

0

0

1

5

0

0

0

5

Total

0

0

2

10

0

0

2

10

Hyperkeratosis –

Nonglandular Region

 

 

 

 

 

 

 

 

Minimal

0

0

1

1

0

0

2

0

Slight

0

0

1

4

0

0

0

2

Moderate

0

0

0

5

0

0

0

6

Marked

0

0

0

0

0

0

0

1

Total

0

0

2

10

0

0

2

9

Erosion/Ulceration –

Nonglandular Region

 

 

 

 

 

 

 

 

Minimal

0

0

0

5

0

0

0

4

Slight

0

0

1

1

0

0

0

1

Total

0

0

1

6

0

0

0

5

Submucosal/Mucosal Inflammation – Nonglandular Region

 

 

 

 

 

 

 

 

Minimal

0

0

1

2

0

0

1

6

Slight

0

0

0

4

0

0

0

3

Moderate

0

0

0

3

0

0

0

0

Total

0

0

1

9

0

0

1

9

Mucosal Necrosis/Erosion -Glandular Region

 

 

 

 

 

 

 

 

Slight

0

0

0

0

0

0

0

2

Moderate

0

0

0

0

0

0

1

1

Total

0

0

0

0

0

0

1

3

Number of tissues examined

5

5

6

10

9

10

10

10

Pancreatic lymph nodes:

Macroscopically enlarged pancreatic lymph nodes in some males treated at 225 mg/kg/day were assessed histologically. In all of them, slightly increased germinal centre development and slight to moderate plasmacytosis were detected.

Summary table of treatment related findings in the pancreatic lymph nodes for animals killed at the end of treatment:

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

25

75

225

0

25

75

225

Increased Germinal Centre Development

 

 

 

 

 

 

 

 

Slight

0

0

0

7

0

0

0

0

Total

0

0

0

7

0

0

0

0

Plasmacytosis

 

 

 

 

 

 

 

 

Slight

0

0

0

4

0

0

0

0

Moderate

0

0

0

3

0

0

0

0

Total

0

0

0

7

0

0

0

0

Number of tissues examined

0

0

0

7

0

0

0

0

Conclusions:
The study was performed according to OECD Guideline with minor deviations, which did not compromise the integrity of the study. A NOAEL and a LOAEL were established to 25 and 75 mg/kg bw/day, respectively both for stomach inflammation observed in both sexes. For systemic effects a NOAEL of 225 mg/kg bw/day can be concluded as no systemic toxic effects were noted in the study.
Executive summary:

The study was performed according to OECD Guideline 422 with minor deviations, which did not compromise the integrity of the study.

 

Three groups of ten male and ten female rats received Ethoxy ethoxy ethyl acrylate at doses of 25, 75 and 225 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation

 

Oral administration of Ethoxy ethoxy ethyl acrylatewas well toleratedwith no mortalities and no adverse effects of treatment on clinical condition, sensory reactivity, grip strength, motor activity, body weight gain, food intake or blood chemistry measurements in males and females treated with Ethoxy ethoxy ethyl acrylate.

 

Microscopic findings in the stomach of males and females treated at 75 or 225 mg/kg/day are compatible with secondary (reactive) changes due to an irritant effect of the test item on the stomach when administered by oral gavage. Inflammatory lesions were present in the glandular and non-glandular region of the stomach.

 

Macroscopically enlarged local lymph nodes in males treated at 225 mg/kg/day show histological features suggestive of local immunostimulation, which could be secondary to the gastric inflammatory changes.

 

In addition, an increase in total and differential leucocyte counts were observed in males treated at 225 mg/kg/day which is likely secondary due to the minimal to moderate inflammatory changes observed at microscopic evaluation in the forestomach of 9/10 males in this group. However, no similar increase in leucocyte counts was detected in females treated at 225 mg/kg/day despite the minimal to slight microscopic stomach lesions observed in 9/10 females at this dose level.

 

A NOAEL and a LOAEL were established to 25- and 75 mg/kg bw/day, respectively both for effects related to local stomach inflammation observed in both sexes.

Furthermore a NOAEL for systemic toxicity at 225 mg/kg kg/day can be concluded.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
225 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
OECD guideline study

Additional information

Justification for classification or non-classification

The data does not fulfill the criteria a STOT RE classification.