Registration Dossier

Administrative data

Description of key information

From an OECD 422 screening study, an oral NOAEL in relation to systemic efects was established to 300 mg/kg bw/day based on increase in liver weights in both sexes (and increased kidney weight in males) at 800 mg/kg/d. For local effects in the forestomach an oral NOAEL of 100 mg/kg bw/day was established.

In an OECD Guideline 408 study, Wistar rats (10 animals per sex/ dose level) were dosed by gavage during 90 days at levels of 0, 30, 100 and 350 mg/kg/day. No unscheduled deaths occurred, and there were no toxicological findings in relation to clinical observation or pathological findings.Salivation at the dose level of 350 mg/kg/day was considered as a test item palatability response and only slight differences in clinical laboratory investigations and some slight changes in organ weights at the high dose level were noted. Under the conditions of this study the dose of 350 mg/kg/day is to be considered as a NOAEL both in relation to systemic effects as well as local effects.

It is to be noted that 350 mg/kg bw/day was selected as the highest dose level for the study as inflammatory infiltrates in submucosa and forestomach ulcerations was observed at dose levels of 300 mg/kg bw/day and 800 mg/kg bw/day in the OECD 422 study. Thus, for longer term exposure an adaptation to the gastric irritational effects seems to occur as such effects were not observed in this 90D study at 350 mg/kg/d.


Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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:
30 July 2012 to 4 February 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed in accordance with testing guidelines with no important deviations to study plan
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. certificate)
Limit test:
no
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: 13 weeks
- Weight at study initiation: Males: 337-372 g Females: 214-246 g
- Fasting period before study: No information
- Housing: Cages with standard, granulated, softwood Lignocel S8/15 bedding (supplied by Harlan Laboratories Models, S.L.).
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2014C rat/mouse maintenance diet ad libitum, batches: 121211MA, 122911MB, 042612MA expiry dates: 7 September 2012, 18 September 2012 and 21 January 2013 (supplied by Harlan Laboratories Models, S.L.). Pelleted standard Teklad 2018C rat/mouse maintenance diet batches: 122311MA and 041112MA expiry dates: 24 September 2012 and 6 January 2013 (supplied by Harlan Laboratories Models, S.L.) ad libitum, for lactating females and pups (until day 4postpartum).
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 5 days between arrival and pretest

ENVIRONMENTAL CONDITIONS
- Temperature (°C): air-conditioned with ranges for room temperature 21 -25 °C
- Humidity (%): relative humidity 30-70%
- Air changes (per hr): 20 air changes per hour
- Photoperiod (hrs dark / hrs light): a 12-hour fluorescent light/12-hour dark cycle

IN-LIFE DATES: From: To: 25 July 2012 to 4 February 2013
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
container or into a volumetric flask and slowly adding the vehicle while mixing. Then the
formulation was transferred into a volumetric flask and the initial container rinsed with the
vehicle, when applied.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Generally recoqnized vehicle
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In order to prepare the formulations, a stock solution was prepared and used to take the volume required for each day of administration into aliquots. In the case that the formulation was prepared for only one administration day, the same procedure was followed for the preparation of the stock solution.

The stock solution was prepared by weighing the necessary amount of test item in an appropriate container or into a volumetric flask and slowly adding the vehicle while mixing. Then the formulation was transferred into a volumetric flask and the initial container rinsed with the vehicle, when applied.

Vehicle was added to top up the volumetric flask after which the formulation was transferred into an appropriate container. If there was any vehicle left, it was used to take any formulation remaining on the walls of the volumetric flask and to obtain the final volume.

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.

The concentration of dose formulation was determined in samples taken from the formulation to be administered to Groups 1 to 4 at the start and end of treatment on.

2-phenoxyethyl acrylate was used as analytical standard. Formulations were analyzed using an analytical method previously validated in-study in Study
S39286 and according to SOP BA/ML/0084.

The results obtained showed that mean test item concentrations found in the formulations analyzed ranged from 99.7% to 105.3% and was therefore within the acceptance range of 85-115% and that precision was below 3.28 %.
Duration of treatment / exposure:
Males: two weeks prior mating and at up to and including the day before sacrifice (minimum a total of 43 days).
Females: two weeks prior mating and at least up to and including the day before sacrifice (day 4 postpartum).
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
Group 1: 0 mg/kg body weight/day (control group); Group 2: 100 mg/kg body weight/day; Group 3: 300 mg/kg body weight/day; Group 4: 800 mg/kg body weight/day
Basis:
actual ingested
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 (100 mg/kg): 10 M/10F
Group 3 (300 mg/kg): 10 M/10F
Group 4 (800 mg/kg): 10 M/10F
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
The dose levels were selected based on a dose-range finding toxicity study in rats (Harlan Laboratories Study S39286 14-day Oral Dose-Range-Finding Toxicity Study in the Wistar Rat), using dose levels of 0, 100, 500 and 1000 mg/kg/day.
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: Detailed clinical observations were performed on all test and control group animals before the first exposure to the test item and once weekly thereafter, at least two hours after dosing. Observations were also performed on females with litters on Day 4 postpartum. 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: twice weekly during the pre-pairing and pairing period and daily during postpairing period. F0 females: twice weekly during the pre-pairing and pairing period and daily until day 5 postpartum.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- F0 males: once weekly during the pre-pairing period and during the two weeks of the postpairing period. F0 females: once weekly during the pre-pairing period and on days 0-7, 7-14, 14-21 postcoitum and days 1-4 postpartum.
No food consumption was examined during the mating period.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: F0 males: during the pre-pairing period and during the two weeks of the postpairing period. F0 females: during the pre-pairing period, pregnancy and days 1-4 postpartum.
No water consumption was examined during the mating period.

Clinical Laboratory Investigations:
Blood samples were drawn from the retro-orbital plexus of five animals/sex/group under light isoflurane anesthesia. The animals were fasted before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood Sampling was perfomed for males ( at least on day 44 (day of sacrifice)) and females (on day 5 postpartum). Haematology, coagulation and clinical biochemistry investigations was perfomed. Further, urinanalysis was performed.

Functional Performance Tests: Grip strength (fore- and hind limbs) and motor activity were measured in five randomly selected males per group once during the final week of treatment and at least two hours after dosing. Five randomly selected females per group were similarly evaluated on day 4 postpartum.

Sensory Reactivity Assessment: Sensory reactivity to different stimuli (e.g. auditory, visual and proprioceptive) was evaluated in the animals mentioned above.
Sacrifice and pathology:
Sacrifice of F0 Females
On day 5 postpartum, all females were deeply anaesthetized with sodium pentobarbital
administered intraperitoneally, exsanguinated and necropsied. The mated females that did not
give birth or show signs of pregnancy were sacrificed after 24-26 days postcoitum if no repeat
mating was done.

Sacrifice of F0 Males
At least on day 44 of the study all males were deeply anaesthetized with sodium pentobarbital
administered intraperitoneally, exsanguinated and necropsied.

Sacrifice of F1 Generation
On day 4 postpartum, all pups were sacrificed by an intraperitoneal injection of sodium
pentobarbital and subjected to macroscopic examination.
F1 offspring that died during the study was examined externally. Necropsy and a macroscopic
examination were carried out for all the other animals.
Other examinations:
Organ Weights
The following organs were weighed on the day of necropsy and their organ to terminal body weight ratios and organ to brain weight ratios were determined:
Adrenals, Kidneys, Prostate and seminal vesicles; Thyroid and parathyroids; Brain, Liver, Spleen, Uterus and oviducts; Epididymides, Ovaries; Testes,Heart;Pituitary, Thymus.

Paired organs were weighed together.

Macroscopic examination
The necropsy included the examination of the external surface of the body, all orifices, cranial,
thoracic and abdominal cavities and the observation of the organs both in situ and after
evisceration.
Samples of the following tissues and organs were collected from all animals at necropsy and
fixed in neutral phosphate buffered 4% formaldehyde solution (10% formalin) unless otherwise
indicated.
Adrenal glands
Aorta (thoracic)
Bone with bone marrow- (Sternum)
Bone marrow smear (femur) (air-dried smear)
Brain (cerebrum, cerebellum, medulla/pons)
Epididymides (Bouin's solution)
Peyer’s patches
Pharynx
Pituitary
Prostate, coagulating gland and seminal
vesicles
Salivary glands (mandibular, sublingual and
Esophagus
Eyes with optic nerve (Davidson’s fixative)
Femur (with articular surface)
Heart (with papillary muscle)
Intestine, large (cecum, colon and rectum)
Intestine, small (duodenum, jejunum and
ileum)
Kidneys
Larynx
Liver
Lungs, with main bronchi and bronchioles
Lymph nodes (mandibular and mesenteric)
Nose (the entire head will be collected)
Mammary gland area
Ovaries
Pancreas
parotid)
Sciatic nerve
Skeletal muscle
Skin (abdominal)
Spinal cord (cervical, thoracic and lumbar)
Spleen
Stomach (glandular and nonglandular)
Testes (Bouin's solution)
Thymus
Thyroid (incl. parathyroid gland, if possible)
Tongue
Trachea
Urinary bladder
Uterus (cervix, corpus and oviducts)
Vagina
All gross lesions

Histotechnique
All organ and tissue samples, except for the nose, to be examined by the study pathologist (see
Histopathology, below) were processed, embedded and cut at an approximate thickness of
2-4 micrometers and stained with hematoxylin and eosin.
The bone marrow smears were stained using the May Grünwald-Giemsa method.

Histopathology
Slides of all organs and tissues collected at necropsy from five males
and five females from groups 1 and 4 selected for clinical pathology, as well as all gross lesions,
were examined. Reproductive organs (tissues shown in bold) from all control and high dose
animals were examined for histopathology.
Because test-item-related morphological changes were detected in stomach and liver at high dose,
these organs of all remaining animals (including group 2 and 3) were examined. A description of
all abnormalities was included in the report. Where possible, the microscopic findings were
correlated with the gross observations.
Statistics:
The following statistical methods were used to analyze food consumption, body weight, clinical laboratory data, organ weights and ratios as well as macroscopic findings:

- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied
if the variables can be assumed to follow a normal distribution for the comparison of the
treated groups and the control groups for each sex.

- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the
data cannot be assumed to follow a normal distribution.

- Fisher's exact-test was applied to the macroscopic findings.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No mortality was observed
Mortality:
mortality observed, treatment-related
Description (incidence):
No mortality was observed
Body weight and weight changes:
no effects observed
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):
effects observed, treatment-related
Ophthalmological findings:
not examined
Description (incidence and severity):
not officially examined, see details on results; sensory activity assessments
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Description (incidence and severity):
The observed effects are believed to be related to the physical administration, leading to local irritation and ulceration of mucosa
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
observed effects are believed to be related to the physical administration, leading to local irritation and ulceration of mucosa
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality was observed in either males or females.

F0 Males; substance-related clinical signs such as hunched
back and abnormal gait were recorded at 800 mg/kg from first week of treatment. These clinical
signs disappeared on week 3 (abnormal gait) and on week 6 (hunched back). In addition, ruffled
fur and rales were recorded occasionally in some males at 800 mg/kg.
Functional performance tests
No differences in fore- or hind limb grip strength were recorded in males after at least 43
administrations.
Slightly lower locomotor activity was recorded in males at 800 mg/kg, but the differences were
not statistically significant. No differences were recorded in the remaining groups.
Sensory reactivity assessments
All animals responded positively to the different reflexes such as blink, pinna, righting and iridic
reflexes and pain, auditory startle and handling response (push-off).

Females;
Salivation was recorded in all test item groups. This clinical sign increased in frequency and
number of occurrences with the dose level. Test item-related clinical signs such as hunched back
and abnormal gait were recorded at 800 mg/kg in all animals during pretreatment period and in
some animals during pregnancy. In addition, ruffled fur and rales at breathing were recorded in
some females at 800 mg/kg. During lactation, only salivation was recorded.
No clinical signs were recorded in the control group.

BODY WEIGHT AND WEIGHT GAIN
Males;
Statistically lower body-weight gain was recorded at 800 mg/kg during prepairing after which
there was a recovery in body weight.
Slight lower body-weight gain was recorded at 300 mg/kg during postpairing. It was statistically
significant on day 5.
No differences with the control group were recorded at 100 mg/kg.

Females;
Slightly lower body-weight gain was recorded at 100 mg/kg at pregnancy and statistically
significant differences were recorded on days 3 and 5 of pregnancy. No differences were
recorded in the remaining group.
Lower body-weight-gain was recorded at lactation in all test item groups. Statistically significant
differences were recorded at 300 and 800 mg/kg.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Males;
Slight decreased in food consumption was recorded during the prepairing period at 800 mg/kg,
but the difference was not statistically significant.
No differences with the control group were recorded in the remaining groups.

Females;
Slightly significant lower food consumption was recorded at 100 and 800 mg/kg during days 0-7
of pregnancy. In addition, slightly lower food consumption was recorded at 300 and 800 mg/kg
during lactation, but the difference was not statistically significant.
No differences with the control group were recorded in the remaining groups.

FOOD EFFICIENCY

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Males;
Higher water consumption was recorded at 800 mg/kg throughout the treatment period. The
differences were statistically significant on days 3-8 of the postpairing period.
No differences with the control group were recorded in the remaining group.

Females;
Higher water consumption was recorded at 800 mg/kg during prepairing period and pregnancy.
The differences were statistically significant during days 2-7 and 16-18 of pregnancy.
Slightly higher water consumption was recorded at 300 mg/kg on days 4-14 of pregnancy, but
the difference was not statistically significant.
No differences with the control group were recorded at 100 mg/kg.

OPHTHALMOSCOPIC EXAMINATION

HAEMATOLOGY
Males;
At 800 mg/kg, statistically lower hemoglobin, hematocrit and erythrocyte values were observed
with respect to the control group.
In addition, high fluorescence reticulocyte ratio (HFR) value increased with respect to the
control group. These differences were not statistically significant.
At 300 mg/kg, slightly lower hemoglobin, hematocrit and erythrocyte values were observed in
males compared to the control group.
No differences with the control group were recorded at 100 mg/kg.
Females;
At 800 mg/kg, high fluorescence reticulocyte ratio (HFR) and reticulocyte values increased with
respect to the control group. These differences were not statistically significant.
No differences with the control group were recorded at 100 or 300 mg/kg.

Blood Chemistry
Males;
Statistically significant increases in chloride values were recorded in all test-item-treated groups
in a dose dependent manner. Additionally, lower calcium values and higher potassium, urea and
creatinine values were observed at 800 mg/kg. The differences recorded were not statistically
significant with respect to the control group.
Females;
At 800 mg/kg, decreases in creatinine values and increases in glucose and sodium values were
observed. The differences in the sodium values were statistically significant. Moreover, higher
cholesterol was recorded at 100 mg/kg and higher triglycerides at 800 mg/kg.

CLINICAL CHEMISTRY

URINALYSIS
Higher urine volume was recorded in both genders at 800 mg/kg and in females the differences
were statistically significant. Consequently, the urine density decreased and the pH decreased
slightly.
No differences were recorded in remaining groups.

NEUROBEHAVIOUR

Sensory reactivity assessments
All animals responded positively to the different reflexes such as blink, pinna, righting and iridic
reflexes and pain, auditory startle and handling response (push-off).

ORGAN WEIGHTS
At the dose of 800 mg/kg, higher liver weights were recorded in both genders. These differences
were statistically significant in relative to body and brain weight in males. In males, statistically
higher kidney weights relative to body weight were recorded.
In addition, lower thymus weights were recorded in females and higher prostate/seminal gland
weights were observed in males. These differences were not statistically significant.
No relevant differences from the control group were recorded at 100 or 300 mg/kg.

GROSS PATHOLOGY
Offspring;
No findings were recorded in the control, 100 or 300 mg/kg pups.
One male (no. 3) from litter no. 74 (800 mg/kg) had dilated left renal pelvis (variation) on day 4
post partum. This was considered to be within the range of normal background findings which
may be seen in rats.

Parental generation
At 800 mg/kg, thickened gastric mucosa was observed in 9/10 male and 9/10 females. Two of
these males also presented reddish foci in gastric mucosa. Likewise, reddish mesenteric and
mandibular lymph nodes were observed in one male and three females. One female had reddish
foci in thymus and two had small thymus.
At 300 mg/kg, reddish/ dark foci in gastric mucosa were recorded in three males and in one
female. Moreover two females had thick stomach. Likewise, reddish foci in the thymus of one
male and reddish mandibular lymph nodes in one female were observed. One female had small
thymus.
At 100 mg/kg, reddish focus in gastric mucosa was observed in two males. Likewise, reddish
mandibular lymph nodes were observed in two males and reddish thymus in one male. Three
females had thymus reduced in size, one of them with pale kidneys.
At control group, one male had reddish mandibular lymph node and another one had reddish foci
on thymus. Three females had small thymus.
Yellowish gastric mucosa was recorded in few animals from all groups.
The observed effects are believed to be of no toxicological relevance as it is expected to be related to the physical administration of the test substance, leading to local irritation and ulceration.

HISTOPATHOLOGY: NON-NEOPLASTIC
At 800 mg/kg, thickened gastric mucosa was observed in 9/10 male and 9/10 females. Two of
these males also presented reddish foci in gastric mucosa. Likewise, reddish mesenteric and
mandibular lymph nodes were observed in one male and three females. One female had reddish
foci in thymus and two had small thymus.
At 300 mg/kg, reddish/ dark foci in gastric mucosa were recorded in three males and in one
female. Moreover two females had thick stomach. Likewise, reddish foci in the thymus of one
male and reddish mandibular lymph nodes in one female were observed. One female had small
thymus.
At 100 mg/kg, reddish focus in gastric mucosa was observed in two males. Likewise, reddish
mandibular lymph nodes were observed in two males and reddish thymus in one male. Three
females had thymus reduced in size, one of them with pale kidneys.
At control group, one male had reddish mandibular lymph node and another one had reddish foci
on thymus. Three females had small thymus.
Yellowish gastric mucosa was recorded in few animals from all groups.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)

HISTORICAL CONTROL DATA (if applicable)

OTHER FINDINGS
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: F0 generation, liver weight
Critical effects observed:
not specified
Conclusions:
The repeated dose toxicity was studied in an OECD Guideline 422 study. With respect to repeated dose toxicity of the parental generation a systemic NOAEL of 300 mg/kg bw/day could be established with respect to increase in liver weights for both sexes (LOAEL at 800 mg/kg/d) , while a NOAEL of 100 mg/kg/d can be concluded for local toxic effects in the stomach (LOAEL at 300 mg/kg/d).
Executive summary:

The repeated dose toxicity of 2-pehnoxyethyl acrylate was investigated in a combined repeated toxicity and reproductive/developmental toxicity study (OECD 422) at the dose levels of 0, 100, 300 and 800 mg/kg bw/d.

No mortality was recorded in either male or female animals. Hunched back, ruffled fur, abnormal gait and decrease in motor activity were recorded in both genders at 800 mg/kg. These clinical signs decreased in frequency and incidence with the number of administrations received. Body weight decreased in males during the pre-pairing period and slightly in females during pregnancy at 800 mg/kg. Test-item-related higher liver weight was observed in males and females at 800 mg/kg associated to minimal centrilobular hepatocellular hypertrophy with increased incidence of hematopoietic foci.  Test-item-related thick gastric wall was recorded at 300 and 800 mg/kg in both genders, related in some animals with reddish foci/focus associated with acanthosis, hyperkeratosis and/or inflammatory infiltrate in submucosa and presence of forestomach ulcerations. Based on these finding a NOAEL of 300 mg/kg bw/d can be concluded in relation to systemic effects for both sexes while a NOAEL of 100 mg/kg/d applies to local effects in the stomach.

Endpoint:
sub-chronic toxicity: oral
Remarks:
90D study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 2016 - November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
The order of sacrifice was not decided at random. No statistical analysis was done for food or water consumption due to the small sample size. These deviations were considered not to have affected the integrity or validity of the study.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Name of test material (as cited in study report): MIRAMER M140
- Physical state: Colorless liquid
- Analytical purity: 84.46 % ( CAS No.: 48145-04-6; EC No.: 256-360-6)
- Impurities (identity and concentrations): cas no. 61630-25-9; 8.67%; cas no. 122-99-6; 1.48 %
- Lot/batch No.: 151014142
- Expiration date of the lot/batch: 31 December 2016
- Storage condition of test material: Under dry conditions, protected from light and at room temperature (20 ± 5 ºC)
Supplier: Miwon
Species:
rat
Strain:
Wistar
Remarks:
Wistar Hannover
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Rat, Wistar Hannover
- Females (if applicable) nulliparous and non-pregnant: yes.
- Age at study initiation: 6-9 weeks.
- Weight at study initiation: Males: 231-285 g, Females: 152-199 g.
- Fasting period before study: NO.
- Housing: Same-sex groups of no more than five; housed in Makrolon type IV cages with Lignocel S8-15 sawdust bedding (supplied by J. Rettenmaier & Söhne).
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: 7 days.

DETAILS OF FOOD AND WATER QUALITY:
Each batch of food is analyzed by the manufacturer for contaminants and to check its composition. Results of analysis for composition and contaminants will be archived at Envigo CRS, S.A.U.
Results of drinking water bacteriological, chemical and contaminant analyses are included in the raw data.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC.
- Humidity (%):30-75%.
- Air changes (per hr): 15-20 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light/12 hours dark.

IN-LIFE DATES: From: To: 04 May 2016 to 10 August 2016.
Route of administration:
oral: gavage
Details on route of administration:
Oral, by gastric gavage,
The amount of test item to be administered was calculated according to the most recent body weight recorded.
Control animals were administered with the vehicle following the same dosing regimen as that used for animals treated with the test item.
Vehicle:
corn oil
Remarks:
from Sigma-Aldrich
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
The test item is soluble in corn oil that is non-toxic in the volume used.
- Concentration in vehicle: NA
- Amount of vehicle (if gavage): 4 mL/kg
- Lot/batch no. (if required): MKBV2080V & MKBQ9948V
- Purity: NA
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Results obtained in the analysis done during the study showed:
I. An accuracy interval for all formulations of 86-100% and 0.2-2.6 for precision.
II. The analysis of Group 1 formulations showed that no contamination was present.
The results obtained demonstrate that the means of the concentrations deviated by less than 15% from their nominal value (accuracy range: 85.65-99.79%). Precision was below 4%.
Duration of treatment / exposure:
Single gastric gavage
Frequency of treatment:
Once daily seven days per week.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle only
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
2-Phenoxyethyl Acrylate
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
2-Phenoxyethyl Acrylate
Dose / conc.:
350 mg/kg bw/day (nominal)
Remarks:
2-Phenoxyethyl Acrylate
No. of animals per sex per dose:
Groups 1 to 4: 10 males and 10 females each (Main study).
Groups 1 and 4: 5 males and 5 females each (Recovery).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random): NA
- Rationale for selecting satellite groups: to test the reversibility or progression of any test-item-related changes was assessed in recovery animals (recovery group) after a 4-week treatment-free recovery period.
- Post-exposure recovery period in satellite groups: 4 weeks
- Section schedule rationale (if not random): NA
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, clinical signs daily and
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical signs were recorded once weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: Once during acclimatization, twice weekly during treatment and recovery periods and before sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined before treatment start and once a week during treatment period

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: Not specified


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Observations were performed once at acclimatization or pretest and in treatment week 13.
- Dose groups that were examined: Groups 1 and 4

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13 of treatment (Main Study animals) and
Week 4 of recovery (Recovery animals).
- Anaesthetic used for blood collection: Yes (identity) light isoflurane anesthesia.
- Animals fasted: Yes
- How many animals: all animals in the respective groups.
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13 of treatment (Main Study animals) and
Week 4 of recovery (Recovery animals).
- Animals fasted: Yes
- How many animals: all animals in the respective groups.
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Week 13 of treatment (Main Study animals) and
Week 4 of recovery (Recovery animals).
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters checked in table [No.?] were examined.

FOB (functional observational battery): yes
-sensory reactivity to stimuli of different types was measured for all rats in week 13 and after a 4 week recovery period
-grip strength in week 13
-locomotor acitivity in week 13

IMMUNOLOGY: No
- Time schedule for examinations: NA
- How many animals: NA
- Dose groups that were examined: NA
- Parameters checked in table [No.?] were examined. NA

OTHER:
FOB (functional
observational battery)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Sacrifice:
After 13 weeks of treatment: 80 animals (main study)
After 4 weeks of recovery: 20 animals (recovery)
As the total number of animals exceeds the number that can be sacrificed in one day, the necropsies were carried out on three consecutive days. As far as possible, the same number of animals of each group and sex was sacrificed each day. The animals continued receiving the test item or vehicle until the day before sacrifice.
The animals surviving to the end of the observation period were deprived of food and deeply anesthetized with sodium pentobarbital administered intraperitoneally and killed by exsanguination.
A full necropsy was performed on all main study and recovery animals. The necropsy included the examination of the external surface of the body, all orifices, cranial, thoracic and abdominal cavities and the observation of the organs both in situ and after evisceration. Descriptions of all macroscopic abnormalities were recorded.
Samples of tissues and organs were collected and weighed (see list in Annex 5) from all animals at necropsy and fixed, unless otherwise indicated, in neutral phosphate-buffered 4% formaldehyde solution (10% formalin) or the correspond fixative. Animal identification (such as ear tattoo) was retained for identification purposes.


HISTOPATHOLOGY: Yes
All organ and tissue samples were examined by the study pathologist (see Annex 5) processed, embedded and cut at a nominal thickness of 2-4 micrometers and stained with hematoxylin and eosin.
The bone marrow smears were stained using the May Grünwald-Giemsa Method and kept at the test facility for possible further investigation.
Other examinations:
Specialist observations:

-Ophthalmoscopic examination: Observations were performed once at acclimatization or pretest and in treatment week 13 (Groups 1 and 4).
Pupils were dilated by instillation of ®Colircusí Tropicamida eye drops (batch numbers: 5KFP1B, 5LCK1D and 6FSF1B expiry dates: September 2018, November 2018 and January 2019, respectively).
Observation areas included: cornea, lens, conjunctiva, sclera, iris and fundus


-FOB (functional observational battery): In week 13 of treatment: sensory reactivity to stimuli of different types was measured for all rats (main study and recovery). Relevant parameters form a modified Irwin screen test (blink, pinna, iridic light reflex, push-off (hind legs), pain response, startle/hearing and righting reflex).
Observations were based on procedures used for the detailed weekly observations. Any abnormal findings were recorded and rated in severity.
In week 4 of recovery: sensory reactivity was not recorded in all recovery animals, as findings recorded during week 13 were considered not to be treatment related.

-Grip strength: In week 13 of treatment: hind- and forelimb grip strength were measured with a push-pull strain gauge in the main study.

-Locomotor activity: In week 13 of treatment: was measured quantitatively (beam counts, number of light/shadow (changes)) in main and recovery animals. Decreased or increased activity was recorded. Activity was measured with an AMS from Medical Instruments GmbH (FMI) and DeMeTec-Ams version 2.0 GmbH. Activity of the animals was recorded in 10-minute intervals over a 60-minute period with fluorescent light. These data and the total activity over 60 minutes were reported.
In week 4 of recovery: all recovery animals were measured in order to discard any possible treatment-related effect observed during week 13.
Statistics:
All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.
The following data types were analyzed at each time point separately:
Food and water consumptions
Body weight
FOB: grip strength and locomotor activity
Hematology and Coagulation
Blood chemistry
Urinalysis
Macropathology
Organ weights, absolute and adjusted to body weight values

The following comparisons were performed:
Group 1 vs 2, 3 and 4

A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations
For clinical pathology data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s exact tests (Fisher, 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control
For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlström, 1963), unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights.
Significant differences between the groups compared were expressed at the 5% (p ≤ 0.05) or 1% (p ≤ 0.01) level.
No statistical analysis was done on data obtained in the food / water consumption or during recovery period given the small sample size.

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation was observed in Group 4 (350 mg/kg/day), in both sexes, during the course of the study.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no differences compared to controls in the overall body-weight gain that could be related to treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The ophthalmic examination in week 13 did not indicate any test-item related finding.
Haematological findings:
no effects observed
Description (incidence and severity):
No alterations in hematology or coagulation were observed.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No changes of toxicological relevance were observed in blood biochemistry parameters.
At the end of treatment period, the following statistically significant differences with respect to the Control group (Group 1) were recorded:
I. In males from Group 4, higher triglyceride and potassium and lower phosphorus values.
II. In females from Groups 3 and 4, higher glucose and urea values.
III. In females from Group 2, higher protein and albumin values.

However, these differences were devoid of any toxicological significance and were attributed to the normal biological variability.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No noteworthy changes were recorded in the urinalysis.

Some minor variations among groups, such as a statistically significant higher pH in the females from Groups 3 and 4 with respect to Group 1 were recorded after 13 weeks of treatment. However, these differences were devoid of any toxicological significance
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no noteworthy differences from Group 1 for organ weights that were considered of toxicological relevance.

A slightly higher statistically significant increase in absolute (about 10%) and adjusted liver weight was recorded in males from Group 4 treated at 350 mg/kg/day with respect to the Control group (Group 1).
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No histopathological changes related with treatment with 2-Phenoxyethyl Acrylate were seen in this study.
At the end of the treatment period, centrilobular hepatocellular hypertrophy was observed in a single female in Group 4 at a minimal level. However, given its low incidence and distribution, it was considered to be incidental and unrelated to treatment.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
350 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic and local effects
Key result
Critical effects observed:
no
Conclusions:
The study was performed according to OECD Guideline 408 with minor deviations that did not compromising the integrity of the study. A NOAEL was established to 350 mg/kg bw/day for systemic and local effects for both sexes.
Executive summary:

In this OECD Guideline 408 study Wistar rats (10 animals per sex/ dose level) were dosed by gavage during 90 days at levels of 0, 30, 100 and 350 mg/kg/day. No unscheduled deaths occurred, and there were no toxicological findings in relation to clinical observation or pathological findings. Salivation at the dose level of 350 mg/kg/day was considered as a test item palatability response and only slight differences in clinical laboratory investigations and some slight changes in organ weights at the high dose level were noted. Under the conditions of this study the dose of 350 mg/kg/day is to be considered as a NOAEL both in relation to systemic effects as well as local effects.

It is to be noted that 350 mg/kg bw/day was selected as the highest dose level for the study as inflammatory infiltrates in submucosa and forestomach ulcerations was observed at dose levels of 300 mg/kg bw/day and 800 mg/kg bw/day in a dose range finding study for an oral OECD 422 study. Thus, for longer term exposure an adaptation to the gastric irritational effects seems to occur as such effects were not observed in this 90D study.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
350 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Klimisch score 1

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In the OECD 422 study, 2-phenoxyethyl acrylate was administered orally by gavage to three groups, each consisting of ten male and ten female RccHan®:WIST rats, for at least four weeks (including two weeks prior to mating through mating, pregnancy and early lactation for females), at dose levels of Group 1: 0 mg/kg bw/day (control group given corn oil); Group 2: 100 mg/kg bw/day; Group 3: 300 mg/kg body weight/day, Group 4: 800 mg/kg bw/day.

No mortality was recorded in either gender. Hunched back, ruffled fur, abnormal gait and decrease in motor activity were recorded in both genders (F0) at 800 mg/kg. These clinical signs decreased in frequency and incidence with the number of administrations received. Higher liver weight was observed in males and females (F0) at 800 mg/kg associated to minimal centrilobular hepatocellular hypertrophy with increased incidence of hematopoietic foci. Higher kidney weight was observed in males at 800 mg/kg.

Macroscopic findings was restricted to thick gastric wall recorded at 300 and 800 mg/kg in both genders (F0), related in some animals with reddish foci/focus associated with acanthosis, hyperkeratosis and/or inflammatory infiltrate in submucosa and presence of forestomach ulcerations in stomach. The remaining macroscopic findings recorded at necropsy could be considered of no toxicological relevance and within the range of normal background lesions.

For males and females, a NOEL of 100 mg/kg bw/day was established for toxic effects ( local effects in the forestomach) and a NOAEL of 300 mg/kg bw/day for systemic effects. The NOAEL of 300 mg/kg bw/day was based on clinical signs recorded at 800 mg/kg, higher liver weights and an increase in hematopoietic foci in liver at 800 mg/kg bw/day, higher kidney weights for males at 800 mg/kg bw/day and forestomach ulceration at 300 and 800 mg/kgbw/day.

In an OECD Guideline 408 study Wistar rats (10 animals per sex/ dose level) were dosed by gavage during 90 days at levels of 0, 30, 100 and 350 mg/kg/day. No unscheduled deaths occurred, and there were no toxicological findings in relation to clinical observation or pathological findings.Salivation at the dose level of 350 mg/kg/day was considered as a test item palatability response and only slight differences in clinical laboratory investigations and some slight changes in organ weights at the high dose level were noted. Under the conditions of this study the dose of 350 mg/kg/day is to be considered as a NOAEL both in relation to systemic effects as well as local effects.

It is to be noted that 350 mg/kg bw/day was selected as the highest dose level for the study as inflammatory infiltrates in submucosa and forestomach ulcerations was observed at dose levels of 300 mg/kg bw/day and 800 mg/kg bw/day in a dose range finding study for an oral OECD 422 study. Thus, for longer term exposure an adaptation to the gastric irritational effects seems to occur as such effects were not observed in this 90D study.

Thus, an overall oral NOAEL of 350 mg/kg bw/d for systemic effects in rats can be concluded for repeated dose toxicity.

Justification for classification or non-classification

Based on the available findings, no severe adverse systemic toxicity was observed and no classification is proposed for repeated dose toxicity.