2-Propenoic acid, 2-[2-(ethenyloxy)ethoxy]ethyl ester

Endpoint summary

Description of key information

Key study: 90 -day oral toxicity (OECD 408):

In males and females in the 400 mg/kg group, changes attributable to the local irritation to the first exposure site were noted. The other changes observed in this group were only minor changes that may possibly be associated with inflammation. Therefore, the no-observed-adverse-effect level of VEEA was considered 40 mg/kg based on the local effects observed in both males and females under the present study conditions. No specific target organ toxicity was observed in either sex up to the dose level of 400 mg/kg.

28-day oral toxicity (OECD 407):

50 mg/kg body weight/day  was established as the no-observed-effect-level (NOEL) and 160 mg/kg body weight/day  as the no-observed adverse- effect-level (NOAEL).

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

In life phase: February 27 to June 19 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

June 25, 2018

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Name: 2-(2-Vinyloxyethoxy) ethyl acrylate
Synonym: VEEA
Description: Colourless liquid
Lot No: 8K15
Purity: 99.6%
Supplier: Study sponsor
Storage conditions: Refrigerated

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

Species/strain commonly used in repeated dose toxicity studies and historical control data avalable at the test facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5 weeks for both males and females.
- Weight at study initiation: males weighed 161 to 184 g, females weighed 123 to 155 g.
- Fasting period before study: No
- Housing: Metal bracket ages (260 W x 380 D x 180 H, mm), with wire mesh floors. 1 to 3 animals per cage during the acclimatization period, 1 animal per cage during the study period.
- Diet (e.g. ad libitum): Pellet diet, CRF-1, supplied freely from metal feeders.
- Water (e.g. ad libitum): Sapporo-City tap water, Supplied freely from the automatic watering system or water containers.
- Acclimation period: From the day of receipt to the day of group assignment, including the quarantine period. The quarantine period was from the day of receipt (quarantine day 1 to quarantine day 6)

DETAILS OF FOOD AND WATER QUALITY:
Food quality:The diet of the lots used in this study was analyzed for contaminants that may affect the study. The analysis results of all items were within the acceptable ranges.
Drinking water: Samples of drinking water were collected at the end of the piping system of the animal room used in this study on January 7, and July 1, 2019, and analyzed for contaminants that may affect the study. The analysis results of all items were within the acceptable ranges.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3°C (actual range 19°C to 23°C)
- Humidity (%): 50% +/- 20% (actual range 37% to 56%)
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light) 12 hours artifical lighting (from 8:00 to 20:00):

IN-LIFE DATES: From: February 27 To: June 19 2019
Study Initiation: 19th February, 2019.
Start of administration: 27th and 28th February, 2019
End of administration:27th and 28th May, 2019
Necropsy at the end of administration: 28th and 29th May
Necropsy at end of recovery: 18th and 19th June
End of experiment September 17th, 2019
Study completion: 17th October, 2019

Route of administration:

oral: gavage

Details on route of administration:

A dosing solution was administered into the stomach by oral gavage using a disposable gastric tube and a disposable syringe.
Individual dose volume was calculated based on the body weight on the measurement day closet to the day of administration.
- Reason for selection of route, method. number of times and period: According to test guidelines

Vehicle:

corn oil

Details on oral exposure:

PREPARATION AND CHEMICAL ANALYSIS OF DOSING SOLUTIONS:
Preparation method: A required amount of the test article was accurately weighed out for each dose level, to which the vehicle was added to obtain each prescribed concentration, and the mixture was stirred with a stirrer to dissolve the test article.
Preparation frequency: Prepared 21 times at intervals of2 to 5 days, and used within 5 days after preparation.
Storage conditions: Dosing solutions were placed in light-resistant containers, and stored at room temperature (actual range 20.7°C to 22.2°C).

VEHICLE
- Name: Corn Oil
- Justification for use and choice of vehicle (if other than water): Test article stable in vehicle.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Confirmation of stability of dosing solutions: A preceding study confirmed stability of the 2.5 and 100 mg/mL preparations for 8 days after preparation (counted from the day of preparation designated as day 0) under room temperature (this was performed in the Prenatal Developmental Toxicity Study of VEEA in Rats, Safety Research Institute for Chemical Compounds Co., Ltd., SRI 8409). Before the start of administration, the 0.8 mg/mL preparation was confirmed to be stable for 5 days at room temperature.
Confirmation of concentrations of dosing solutions: The concentrations of the test article in the dosing solutions of all concentrations were analyzed at the first, midterm and final preparation by HPLC . The analysis results confirmed that the concentrations of the dosing solutions were appropriate (concentration within ±10 of nominal).

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Dose / conc.:

4 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

Middle dose

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

10 males and females per dose.
5 males and 5 females in 0 and 400 mg/kg recovery groups

Control animals:

yes, concurrent vehicle

Details on study design:

VEEA was orally administered to male and female Crl:CD(SD) rats at 0 ( control), 4, 40, and 400 mg/kg/day for 90 days to investigate its toxic potential and toxicological profile. Subsequently, reversibility of toxicity following withdrawal was examined in the 0 and 400 mg/kg groups by 21-day observation from the day after the end of administration.

Rational for dose selection:
In a combined repeated dose toxicity study with the reproduction/developmental toxicity study of the test article in rats, the test article was administered by oral gavage at doses of 0, 50, 140, and 400 mg/kg to 12 male and 12 female rats per group, for 91 days in males and from 14 days before mating to 5 days after parturition in females. As a result, thickening of the limiting ridge of the stomach or the forestomach mucosa was noted in males and females at 50 mg/kg and higher doses; and histopathologically squamous cell hyperplasia, ulcer, and changes associated with inflammation of the submucosal tissue were noted at these regions.
In addition, salivation was noted in males or females in the 140 mg/kg and higher dose groups, high WBC, neutrophil, and beta-Globulin fraction, low albumin and NG ratio, and low thymus weight in males or females in the 400 mg/kg group. Therefore, the high dose was set at 400 mg/kg which is expected to produce clear changes, and the middle and low doses were set at 40 and 4 mg/kg with a common ratio of 10.

Positive control:

No

Observations and examinations performed and frequency:

OBSERVATION, MEASUREMENT AND EXAMINATION:
The first day of administration was designated as Administration Day 1 (Day 1), and the day after Administration Day 90 as Recovery Day 1. The day of necropsy was the day after Administration Day 90 (Day 91) or the day after Recovery Day 21 (Recovery Day 22). Administration Days 1 to 7 were designated as Administration Week 1 (Week 1 ), and every 7 days after that were counted as subsequent Administration Weeks (to Administration Week 13 [Administration Days 85 to 90], final week of administration). Recovery Days 1 to 7 were designated as Recovery Week 1, and every 7 days after that were counted as subsequent Recovery Weeks (to Recovery Week 3 [Recovery Days 15 to 21], final week of recovery).

The following observations were performed:

CLINICAL OBSERVATIONS:
Animals observed: All animals
Period: From Administration Day 1 to the day of necropsy
Frequency: Animals were observed twice daily, before and after daily administration during the administration period, and twice daily in the morning and in the afternoon during the recovery period. On the day of necropsy, animals were observed once daily.
Method: Individual animals were observed for mortality, external appearance, behavior, and other changes. Any abnormalities noted were recorded with the times of onset and disappearance.

DETAILED CLINICAL OBSERVATIONS:
Animals observed: All animals
Time: Before the start of administration, and on Administration Days 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, and 90, and Recovery Days 7, 14 and 21.

Method:
Observation results scored using the predetermined scoring criteria were recorded.
Observation parameters and methods:
[1] Body position/posture, respiratory pattern, tremor/convulsion, stereotypes (rolling/repetitive circling), and bizarre behavior (self-biting) were observed from outside the cage.
[2] Ease of removal, ease of handling, muscle tone, piloerection, fur condition, appearance of skin, eyes·eyeballs, and mucous membranes, pupil size, lacrimation, salivation, and other secretions or excretions were observed when taking out of the cage.
[3] Gait, co-ordination of movement, reactivity to environmental stimuli, searching ( sniffing and standing), excretions (urination and defecation), stereotypes ( excessive grooming and unusual head movement), bizarre behavior (walking backwards and vocalization), and aggression were observed in an open field.

FUNCTIONAL OBSERVATIONS:
Animals observed: All animals
Time: Administration Week 13 and Recovery Week 3

Observation and measurement methods:
Observation results scored using the predetermined scoring criteria or values obtained with measurement equipment were recorded.

Observation and measurement parameters and methods:
[1] Sensory and motor reactivity to visual, touch, auditory, pain, and proprioceptive stimuli, and righting reflex were examined on the desk.
[2] Grip strength was measured using CPU gage (Aikoh Engineering Co., Ltd.) 3 times each for the forelimbs and hindlimbs, and recorded as whole numbers in grams.
[3] Motor activity was measured using a motor activity measuring system (Supermex and CompAct; Muromachi Kikai Co., Ltd.) for every 10 minutes during 1 hour, subsequent to the above procedures [1] and [2].

BODY WEIGHT MEASUREMENT
Animals measured: All animals
Measurement days: On Administration Days 1, 4, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, and 90, and Recovery Days 1, 7, 14, and 21, and the day of necropsy.
Method: Body weight was measured with an electronic balance

Body weight gain:
Body weight gain was calculated from the following equations:
Administration period
Body weight gain (g) = body weight (g) on Day 90 - body weight (g) on Day 1

Recovery period
Body weight gain (g) = body weight (g) on Recovery Day 21 - body weight (g) on Recovery Day 1

FOOD CONSUMPTION:
Animals measured: All animals
Measurement days: Measured on the same days as body weight measurement, except the day of necropsy.

Method:
Food consumption was measured with an electronic balance.
On Administration Day 1 or Recovery Day 1, an adequate amount of feed was measured and set at each cage. After that, the amount of feed that remained and the amount supplied in each cage were measured on each day of measurement. However, only the amount that remained was measured on Administration Day 90 and Recovery Day 21. Food consumption (g/rat/day) was calculated from the following equation.

Food consumption (g/rat/day):
amount of feed supplied (g/rat) - amount of feed that remained (g/rat) / number of days between measurements (days)

OPHTHALMOLOGY:
Animals examined: All animals before the start of administration, and all animals in the control and high dose groups in Administration Week 13. Since no changes were noted in Administration Week 13, animals in the other groups were not examined.

Timing: Before the start of administration ( during the acclimatization period) and in Administration Week 13. Since no changes were noted in Administration Week 13, examination in Recovery Week 3 was not performed.

Method:
Macroscopic and slit-lamp examinations:
Anterior chamber of both eyes was examined macroscopically and using a slit lamp (Kowa SL-2; Kowa Co., Ltd.). Then, mydriasis was induced by instillation of mydriatic (Mydrin-P; Santen Pharmaceutical Co., Ltd.), and optic media of both eyes were examined using the slit lamp.

Ocular fundus examination:
After the above examination (in mydriasis), both ocular fundi were observed with a fundus camera (Kowa RC-2, Kowa Co., Ltd.).

URINALYSIS:
Animals examined: All animals
Timing: Administration Week 13 and Recovery Week 3

Method: Urine samples were collected from non-fasted animals using metabolic cages for rats. Urine accumulated for approx. 3 hours (actual collecting time: approx. 3 to 6 hours) from immediately after dosing was used for the parameters reported, and urine accumulated for approx. 21 hours for the parameters reported. For the parameter urinary sediments obtained by centrifugation (approx. 400 x g, 4°C, for 5 minutes) were used. The urine collected was discarded after completion of the examination. In urinalysis in Week 13, as urine accumulated for approx. 3 hours could not be collected from animal No. 50153, urine was collected from this animal by forced urination. Urine volume was too small to examine the parameter.
Parameters: pH, protein, glucose, ketone body, urobilinogen, bilirubin, occult blood, color, urinanry sediments, urine volume, specific gravity.


HEMATOLOGY:
Animals examined: All animals
Timing: At necropsy
Method:
Animals fasted overnight (16 to 22 hours) from early evening were anesthetized with isoflurane (Isoflurane for animals, Mylan, Inc.), and then blood was collected from the abdominal aorta. One blood sample (approx. 1 mL) was treated with EDTA·2K and used for the reported parameters. The other blood sample was treated with 3.8% sodium citrate, and approx. 1 mL of the treated blood (0.1 mL of3.8% sodium citrate solution+ 0.9 mL of blood) was centrifuged (approx. 2000 x g, 4°C, for 10 minutes) to obtain plasma, which was used for the parameters for prothrombin. The blood and plasma obtained were discarded after completion of the examination. Since differential counts ofWBC were obtained, white blood cell smears were not prepared.
Parameters: Red blood cell count, hematocrit, hemoglobin concentration, mean corpuscular hemoglobin, mean corpusular hemoglobin concentration, platelet count, retiulocyte, white blood cell count, differential count of WBC, prothrombin time, activated partial thromboplastin time

BIOCHEMISTRY:
Animals examined: All animals
Timing: At necropsy
Method:
Animals fasted overnight (16 to 22 hours) from early evening were anesthetized with isoflurane, and then blood was collected from the abdominal aorta. One blood sample was treated with approx. 20 units per mL blood of heparin sodium and centrifuged (approx. 2000 x g, 4°C, for 10 minutes) to obtain plasma for analysis.. The other blood sample was collected into the test tube containing separator and centrifuged ( approx. 2000 x g, 4 °C, for 10 minutes) to obtain serum, which was used for the other parameters. A part of the serum was dispensed into 6 polypropylene tubes (approx. 150 μL per tube) and cryopreserved (set at -80°C), which was used for measurement of the parameters triiodothyronin, thyroxine, thyroid stimulating hormone. After the examination, the serum used for hormone measurement was cryopreserved at -70°C or below, and the other plasma and serum were stored at - 20°C or below, both of which were discarded by the day of study completion.
Parameters: Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, y-glutamyl transpeptidase, glucose, total cholestrol, triglyceride, HDL-cholestrol, LDL-cholestrol, total bilirubin, total bile acid, urea nitrogen, creatinine, sodium, potassium, chloride, calcium, inorganic phosphorous, total protein, albumin/globulin ratio, albumin, protein fraction, triiodothyronin, thyroxine, thyroid stimulating hormone.

Sacrifice and pathology:

NECROPSY:
Animals examined: All animals
Timing: Animals were necropsied on the day after Administration Day 90 or the day after Recovery Day 21.
Method: Animals were observed for external appearance and blood was collected under anesthesia with isoflurane, and then they were euthanized by exsanguination by cutting the abdominal aorta and caudal vena cava. After macroscopic observation of all organs and tissues, the organs and tissues shown below were fixed and preserved in 10% neutral buffered formalin. The lung was infused with fixative through the trachea prior to immersion in to fixative. The eyeballs and harderian glands that were fixed and preserved in Davidson's fixative, and the testes and epididymides that were fixed in Bouin's solution and preserved in 70% ethanol. For bilateral organs, both sides of them were fixed and preserved.

Observation of estrous cycle:
From all females, vaginal smears were collected before necropsy on the day of necropsy, which were stained with Giemsa solution, and observed to determine the stage of the estrous cycle under the light microscope. The data were used to assist in evaluation of organ weight and histopathology.

Organs and tissues:
Brain (including cerebrum, cerebellum, pons, and medulla oblongata), spinal cord ( cervical, mid-thoracic and lumbar), pituitary gland, thymus, thyroids, parathyroids, adrenals, spleen, heart, esophagus, stomach, duodenum, jejunum, ileum (with Peyer's patches), cecum, colon, rectum, liver, pancreas, salivary glands (submandibular glands, sublingual glands), trachea, lung and bronchus, thoracic aorta, kidneys, urinary bladder, testes, epididymides, prostate, seminal vesicles ( with coagulating glands), ovaries, uterus (horn and cervix), vagina, eyeballs, harderian glands, skin and mammary gland (right abdomen), sternum (with bone marrow), femur (with bone marrow, right) , mesenteric lymph nodes, submandibular lymph nodes, skeletal muscle (biceps femoris, right), sciatic nerve (right), and gross lesions (with a border to the normal tissue).

ORGAN WEIGHT:
Animals measured: All animals
Timing: At necropsy
Method: The organs shown below were weighed with an electronic balance. Bilateral organs were measured together. The thyroid and pituitary gland were measured after fixation.

Organs and tissues:
Brain, heart, liver, kidneys, testes, epididymides, prostate+seminal vesicles (with coagulating glands); (g) Pituitary gland, thyroids (with parathyroids), spleen, thymus, adrenals, ovaries, and uterus; (mg)

All animals
At necropsy

Relative organ weight: Relative organ weight was calculated from the absolute organ weight and the body weight on the day of necropsy.

Relative weight (g/100 g or mg/100 g) = absolute weight (g or mg) / body weight on the day of necropsy (g) x 100

HISTOPATHOLGY:
Animals examined: All organs and tissues of all animals in the control and high dose groups in the toxicity study group were microscopically examined. The stomach of males and females, in which changes considered to be effects of the test article administration were noted in the high dose group, was histopathologically examined also in all animals in the low and middle dose groups in the toxicity study group and all animals in the recovery study group. Gross lesions at necropsy (pancreaticoduodenal lymph node of all animals except animal Nos. 10401, 50455, and 50456 in the 400 mg/kg toxicity study group and animal No. 10413 of the recovery study group) were also microscopically examined.
Method: Among the organs and tissues fixed and preserved at necropsy, the organs and tissues to be examined were embedded in paraffin wax, thin-sectioned, and stained with hematoxylin and eosin and microscopically examined.
The testes were examined for spermatogenesis.

Statistics:

Please see the any other information on results section:

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Administartion period:
In males, salivation was noted after administration in 2 animals in the 400 mg/kg group between Administration Days 74 and 90. Other than this, trauma (fourth digit of the left hindlimb) was noted in 1 male in the control group and crushing of upper incisors in 1 male in the 4 mg/kg group, both of which were considered due to accidents in cages and unrelated to the test article administration.

The test article is weakly irritating to the skin and the same change was noted in the combined repeated dose toxicity study with the reproduction/developmental toxicity study of the test article; therefore, salivation was considered due to irritancy of the test article.

In females, no changes were noted in any test group.

Recovery Group: No abnormalities were noted in males or females in any group.

Detailed clinical observations:
There were no significant differences in any parameter in males or females in any test article group compared with the control group.

Recovery Group:
There were no significant differences in males or females in the 400 mg/kg group compared with the control group.

Mortality:

no mortality observed

Description (incidence):

There were no significant differences in any parameter in males or females in any test article group compared with the control group. There was no mortality in any of the treatment groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Treatment period: There were no significant differences in body weight or body weight gain in males ofemales in any test article group compared with the control group.

Recovery period:
In the 400 mg/kg group, there were no significant differences in body weight of males or females compared with that in the control group. Body weight gain was significantly high in males and tended to be high in females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Administration period: There were no significant differences in males or females in any test article group compared with the control group.

Recovery period: In the 400 mg/kg group, food consumption was significantly high in males and females on Recovery Day 14 compared with that in the control group.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No abnormalities were noted in any male or female in any group before the start of administration, or in the control or 400 mg/kg group in Administration Week 13.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

End of administartion period:
In the 400 mg/kg group, platelet was significantly higher in males and females, RBC and MCHC were significantly lower and reticulocyte, WBC, and neutrophils were significantly higher in males, compared with those in the control group. In this group, PT and APTT were significantly shortened in males; however, shortening is a change of little clinical significance, and these changes were not considered toxicologically significant. In addition, reticulocyte was significantly high in females. However, there were no changes in relevant parameters including RBC and HGB, no histopathological changes indicative of increased haematopoiesis were observed, and each individual value was within the historical control range (mean± 2 SD, 2.585% ± 1.206%). Therefore, the high reticulocyte was not considered toxicologically significant. In the 4 mg/kg group, HCT was significantly higher in females, which was considered unrelated to the test article administration because there was no dose-response relationship. In the 40 mg/kg group, there were no significant differences in any parameter in males or females.

End of recovery period:
There were no significant differences in any parameter in males or female in the 400 mg/kg group compared with the control group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

End of administartion period:
In the 40 mg/kg group, y-GTP was significantly higher in males compared with the control group. In the 400 mg/kg group, y-GTP and LDL-C in males and females and TCho and HDL-C in males were significantly high. In TP and protein fractions in this group, albumin fraction and A/G ratio were significantly low and Beta-G and y-G fractions were significantly high in males. These were considered due to a decrease in albumin and an increase in globulin because no changes were noted in TP.

In females, significantly low albumin and its related significantly low TP were noted. In addition, a1-G fraction was significantly low, which was considered a decrease in proteins constituting a1-G fraction because no changes were noted in albumin fraction or A/G ratio. The high a2- and ~Beta-G fractions were considered corresponding to an increase in proteins constituting these fractions, or opposing decreases in albumin and proteins constituting a1-G fraction.

In addition, Cl was a significant low in males and females in the 400 mg/kg group. However, histopathological examination of the kidney revealed no changes related to the test article administration, there were no changes in Na, and each individual value was within the historical control range (males, mean ± 2 SD, 105.0 ± 2.6 mEq/L [n=181]; females, mean ± 2 SD, 105.5 ± 3.8 mEq/L [n=176]). Thus, the low Cl was not considered toxicologically significant. In addition, AST and ALT in males in this group were significantly lower, which were not considered toxicologically significant because these changes were of little clinical significance. Glucose of females in the 40 mg/kg group and TP and TSH of males in the 4 mg/kg group were significantly high, which were considered unrelated to the test article administration because there was no dose response relationship.

End of recovery period:
In the 400 mg/kg group, y-GTP was significantly higher in females subsequent to the end of administration. The ratio to the control group was 145% at the end of the recovery period compared to 222% at the end of administration, which showed a tendency toward recovery. The A/G ratio and albumin fraction were significantly low in females, which were not considered toxicologically significant because there were no changes in albumin, TP, or other protein fractions. Other than those above, TBA was significantly high in males and Na was significantly low in females in this group. These were considered unrelated to the test article administration because they were not observed at the end of administration; for TBA, no histopathological changes were noted in the liver; for Na, no changes were noted in Cl; and each individual value was within the historical control range (TBA in males, mean± 2 SD, 11.38 ± 10.56 μmol/L [n=5]; Na in females, mean± 2 SD, 143.1 ± 2.4 mEq/L [n=30]).

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Administartion period:
In the 400 mg/kg group, urine pH was significantly lower in males compared with the control group, which was not considered toxicologically significant because histopathological examination of the kidney revealed no changes related to the test article administration. Other than this, urine protein was significantly low in males in the 400 mg/kg group. This was a change of little clinical significance, and was not considered toxicologically significant. In the 4 and 40 mg/kg groups, there were no significant differences in any parameter in either males or females.

Recovery period:
There were no significant differences in any parameter in males or female in the 400 mg/kg group compared with the control group.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Administartion period:
In the final week of administration, motor activity measurements were significantly high for 30-40 minutes and 40-50 minutes after the start of measurement in females in the 40 mg/kg group and for 30-40 minutes and 0---60 minutes in females in the 400 mg/kg group, compared with those in the control group. These were not considered to be of toxicological significance because no changes indicating excitement were noted in clinical observation or detailed clinical observation, and individual values were within the historical control range (mean± 2 SD: 335.3 ± 502.0 for 30-40 minutes; 263.3 ± 447.2 for 40-50 minutes; 2564.6 ± 2635.8 for 0-60 minutes; n=100). In the 4 mg/kg group, there were no significant differences in either males or females. There also were no significant differences in sensory and motor reactivity to stimuli or grip strength in either males or females in any test article group.

Recovery period:
In the final week of recovery, grip strength of the forelimbs was significantly higher in females in the 400 mg/kg group compared with that in the control group. However, this change was considered unrelated to the test article administration because it was not observed in the final week of administration and each individual value was within the historical control range (mean ± 2 SD, 1254.09 ± 324.20 g; n=15). In sensory and motor reactivity to stimuli or motor activity measurements, there were no significant difference in either males or females in the 400 mg/kg group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

End of administration period:
In the 400 mg/kg group, relative liver weight of males and females, absolute liver weight of females, and absolute and relative spleen weight of males were significantly higher than those in the control group. Other than those above, relative kidney and ovary weight was significantly high in females. These were not considered toxicologically significant because there were no changes in the absolute weight and no changes related to the test article administration were noted histopathologically. In the 4 and 40 mg/kg groups, there were no significant differences in any organ in males or females.

End of recovery period:
In the 400 mg/kg group, absolute brain weight was significantly lower and relative weight of the epididymis and the prostate + seminal vesicle were significantly higher in males, and absolute liver weight was significantly higher in females compared with those in the control group. These were considered unrelated to the test article administration because these changes were observed in either absolute or relative weight, and no changes related to the test article administration were noted in histopathological examination at the end of the administration period.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

End of administration period:
In the 400 mg/kg group, thickening of the forestomach mucosa was noted in 10 males and 8 females, and large size of the pancreaticoduodenal lymph node in 9 males and 8 females. Other than those above, cyst in the pituitary gland was noted in 1 male, and histopathological examination of this organ revealed mild remnant of craniopharyngeal duct. This change was considered unrelated to the test article administration because it is a congenital legion.

Recovery period:
In the 400 mg/kg group, large size of the pancreaticoduodenal lymph node was still noted in 1 male but with a decrease in frequency compared with that at the end of administration. No abnormalities were noted in females in the 400 mg/kg group or the control group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

End of administration period:
In the stomach examined in males and females in all groups, squamous cell hyperplasia at the limiting ridge was noted in 10 male (slight 1, mild 4, moderate 5) and 10 females (slight 1, mild 5, moderate 4); in the forestomach, ulcer in 5 male (slight 2, mild 3) and 7 females (slight 6, mild 1), and squamous cell hyperplasia in 10 males (mild 1, moderate 9) and 10 females (mild 2, moderate 8); in the glandular stomach, slight pancreatic metaplasia in 4 males and 3 females, and mucosal atrophy in 6 males ( slight 5, mild 1) and 3 females (slight) in the 400 mg/kg group. In the 4 or 40 mg/kg group, no abnormalities were noted in males or females.

In the organs examined in males and females in the control and 400 mg/kg groups, various findings were occasionally noted in males. and females in the 400 mg/kg group. All of these changes are spontaneous or were also noted in the control group, and there was no enhancement in their frequency or grade; therefore, these were considered unrelated to the test article administration.

In the examination of gross lesion at the pancreaticoduodenal lymph node in the 400 mg/kg group, plasma cell infiltration was noted in 9 males (mild) and 8 females (slight 1, mild 7), hypertrophy of follicles in 9 males (slight 4, mild 5) and 8 females (slight 5, mild 3), and sinus ectasia in 8 males (moderate) and 5 females (slight 3, mild 1, moderate 1).

End of recovery period:
In the stomach examined in males and females in the control and 400 mg/kg groups, slight squamous cell hyperplasia was noted in the forestomach in 1 male and 3 females with decreased frequency and grade compared with those at the end of administration. In addition, in the pancreaticoduodenal lymph node of 1 male in the 400 mg/kg group, in which the same macroscopic findings as those at the end of administration were noted, slight follicular hyperplasia was observed.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The NOAEL was considered 40 mg/kg based on the local effects observed in both males and females under the study conditions. No specific target organ toxicity was observed in either sex up to the dose level of 400 mg/kg

Key result

Critical effects observed:

no

VEEA was orally administered to male and female Crl:CD(SD) rats at 0 (control), 4, 40, and 400 mg/kg/day for 90 days to investigate its toxic potential and toxicological profile. Subsequently, reversibility of toxicity following withdrawal was examined in the 0 and 400 mg/kg groups by 21-day observation from the day after the end of administration.

In the 400 mg/kg group, the following changes were noted in association with the test article administration.

During the administration period, salivation was noted in males. The test article is weakly irritating to the skin and the same change was noted in the combined repeated dose toxicity study with the reproduction/developmental toxicity study of the test article; therefore, salivation was considered due to irritancy of the test article. In this group, necropsy revealed thickening of the forestomach mucosa in males and females, and histopathological examination revealed squamous cell hyperplasia at the limiting ridge of the stomach, and ulcer/squamous cell hyperplasia of the forestomach. All of these are changes observed following oral administration of irritant substance and were considered due to the test article. Atrophy of the glandular stomach mucosa noted in males and females is a change observed as a result of previous gastric inflammation or ulceration, and pancreatic metaplasia is a change considered related to chronic gastritis. Therefore, the changes in the stomach noted in this study were considered due to direct damage on the first exposure site caused by irritancy of the test article and its resulting chronic inflammation. In the pancreaticoduodenal lymph node in which large size was noted at necropsy, plasma cell infiltration, follicle hypertrophy, and sinus ectasia were noted, which were considered secondary to inflammation at the first exposure site. The inflammations were local; i.e., no changes related to the test article administration were noted in the other organs or tissues except the stomach and its vicinity the pancreaticoduodenal lymph node. The high platelet, WBC, and neutrophil, low TP, albumin, albumin fraction and A/G ratio, and high α2-, ß- and y-G fractions noted in males or females were considered changes related to the inflanunations at the stomach. The low a1-G fraction noted in females may possibly be secondary to inflanunation because this change was slight in degree and no changes related to the test article administration were noted in histopathological examination of the liver.

In hematology, RBC and MCHC were low and reticulocyte was high in males, which were considered effects of the test article administration. These changes were slight in degree and may possibly be secondary to inflammation. The high spleen weight was also likely to be a similar change.

In addition, the liver weight and y-GTP, LDL/HDL-C or T-Cho were high in males and females. As these were slight in degree and histopathological examination of the liver including the bile ducts revealed no changes related to the test article administration; therefore, these were considered adaptive changes and not toxicologically significant. In the 40 mg/kg group, y-GTP was high in males, which was slight in degree and no changes were noted in the other hepatic function parameters or liver weight: therefore, this change was not considered toxicologically significant. In females in the 40 mg/kg group and males and females in the 4 mg/kg group, no effects of the test article administration were noted.

No effects of the test article administration were noted in detailed clinical observation, functional observations, ophthalmology, or urinalysis in any dose group.

In all changes described above noted until the end of administration, recovery or a tendency toward recovery was noted by the end of the recovery period. The high body weight gain and food consumption noted in males and females in the 400 mg/kg group during the recovery period may be associated with recovery from stomach inflammation.

As described above, in males and females in the 400 mg/kg group, changes attributable to the local irritation to the first exposure site were noted. The other changes observed in this group were only minor changes that may possibly be associated with inflammation. Therefore, the no-observed-adverse-effect level of VEEA was considered 40 mg/kg based on the local effects observed in both males and females under the present study conditions. No specific target organ toxicity was observed in either sex up to the dose level of 400 mg/kg.

Conclusions:

VEEA was orally administered to male and female Crl:CD(SD) rats at 0 (control), 4, 40, and 400 mg/kg/day for 90 days to investigate its toxic potential and toxicological profile. Subsequently, reversibility of toxicity following withdrawal was examined in the 0 and 400 mg/kg groups by 21-day observation from the day after the end of administration.

In males and females in the 400 mg/kg group, changes attributable to the local irritation to the first exposure site were noted. The other changes observed in this group were only minor changes that may possibly be associated with inflammation. Therefore, the no-observed-adverse-effect level of VEEA was considered 40 mg/kg based on the local effects observed in both males and females under the present study conditions. No specific target organ toxicity was observed in either sex up to the dose level of 400 mg/kg.

Executive summary:

VEEA was orally administered to male and female Crl:CD(SD) rats at 0 (control), 4, 40, and 400 mg/kg/day for 90 days to investigate its toxic potential and toxicological profile. Subsequently, reversibility of toxicity following withdrawal was examined in the 0 and 400 mg/kg groups by 21-day observation from the day after the end of administration. The results obtained were as follows:

In the 400 mg/kg group, the following changes were noted: In clinical observation, salivation was noted in males. In males and females, necropsy revealed thickening of the forestomach mucosa and large size of the pancreaticoduodenal lymph node; and histopathological examination revealed squamous cell hyperplasia at the limiting ridge of the stomach, ulcer/squamous cell hyperplasia of the forestomach, mucosal atrophy/pancreatic metaplasia of the glandular stomach, and plasma cell infiltration/follicle hypertrophy/sinus ectasia in the pancreaticoduodenal lymph node. In males or females, hematological examination revealed high platelet count (platelet), white blood cell count (WBC), and neutrophil; and biochemical examination revealed low total protein (TP), albumin, albumin fraction and A/G ratio and high α2-, ß- and y-Globulin fractions. All of these changes were noted in association with irritancy of the test article and its resulting inflammation at the stomach, the first exposure site. Other than those above, in males, red blood cell count (RBC) and mean corpuscular hemoglobin concentration (MCHC) were low and the reticulocyte count and spleen weight were high; and in females, a1-Globulin fraction was low. These changes may possibly be associated with inflammation.

No effects of the test article administration were noted in detailed clinical observation, functional observations, ophthalmology, or urinalysis.

In males and females in the 4 and 40 mg/kg group, no effects of the test article administration were noted.

All the changes observed by the end of administration were considered to be reversible after withdrawal.

As described above, in males and females in the 400 mg/kg group, changes attributable to the local irritation to the first exposure site were noted. The other changes observed in this group were only minor changes that may possibly be associated with inflammation. Therefore, the no-observed-adverse-effect level ofVEEA was considered 40 mg/kg based on the local effects observed in both males and females under the present study conditions. No specific target organ toxicity was observed in either sex up to the dose level of 400 mg/kg.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

between 17th March 2006 and 21st April 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd Laboratory Animal Services, Fullinsdorf, CH-4414, Switzerland.
- Age at study initiation: Approximately 7 weeks old.
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz, Switzerland).
- Diet (e.g. ad libitum):Pelleted standard Provimi Kliba 3433 (batch no. 76/05 and 001/06) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst, Switzerland). Ad libitum.
- Water (e.g. ad libitum): Mains water ad libitum.
- Acclimation period: 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3ºC.
- Humidity (%): 30 - 70%.
- Air changes (per hr): 10 - 15 changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness.

IN-LIFE DATES: Day 0 (the day of dosing) up to Day 29.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared weekly. The substance was weighed into a tared glass beaker on a
Mettler balance and the vehicle was added. It was then mixed using a magnetic stirrer and were wrapped into an aluminium foil to avoid direct sunlight and were stored at room temperature of 15-25°C. Homogeneity of the test item in the vehicle was maintained during the daily administration
period using a magnetic stirrer.

DIET PREPARATION
Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The stability of the test material formulations were determined at the test laboratory. They are considered to be stable for at least 7 days when kept under storage conditions.
- Amount of vehicle (if gavage): 5 ml/kg bw.
- Lot/batch no: 120471944705164.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The content, homogeneity and stability of application formulations of the test substance has been tested. The test item concentrations were determined by HPLC coupled to an UV detector and quantified with the area under the peak.
The identity of the test substance was confirmed by its retention time which was similar to that measured in the working standards. The test item content in all
samples was found to be within the accepted range of ±20% of the nominal content. In addition, the homogenous distribution of the test substance in PEG 300 was demonstrated. The application formulations were considered to be stable for at least 7 days when kept under storage conditions.

Duration of treatment / exposure:

28 days.

Frequency of treatment:

Daily.

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

160 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 animals per sex per dose and the control group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: According to the study report, the dose levels were chosen based on the results of a preliminary range-finder performed (see the details in 'any other information on materials and methods incl. tables'). However, the recommended dose levels in the range-finding does not match to the doses used in this study. The reason for this not in the report.
- Rationale for animal assignment (if not random): Recognised by the international guidelines as the recommended test system.

Positive control:

Not applicable.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for mortality/viability were recorded twice daily. The animals were observed for clinical signs once before commencement of administration; twice daily on days 1-3; as well as once daily on days 4-28.
- Cage side observations checked: Mortality and clinical signs .

DETAILED CLINICAL OBSERVATIONS: Yes
The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1 - 3) thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly during the acclimatisation and the treatment periods and before necropsy.

FOOD CONSUMPTION: Yes
- The food consumption was recorded once during the acclimatization period and weekly thereafter/
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable.

FOOD EFFICIENCY: Yes
- Food efficiency (the ratio of mean bodyweight change and mean food consumption) was calculated.

WATER CONSUMPTION: No.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the study (Day 28).
- Anaesthetic used for blood collection: Yes, light isoflurane.
- Animals fasted: Yes. For approximately 18 hours before blood sampling.
- How many animals: All animals from each test and control group.
- Parameters checked:
(Haematology) RBC, HB, HCT, MCH, RDW, MCV, MCHC, HDW, RETI, L RETI, M RETI, H RETI, WBC, Differential in NEUT, EOS, BASO, LYMPH, MONO, LUC and PLATELETS.
(Clinical chemistry) Urea, glucose, CREAT, BILI-T, CHOLEST, TRIGLY, PHOS-LIP, ASAT, ALAT, LDH, GLDH, ALP, GGT and CK.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:Daily.
- Dose groups that were examined: All animals.
- Parameters examined: Forelimb/hind limb grip strength measurements and locomotor activity.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals surviving to scheduled necropsy were anesthetised by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution; Adrenals, aorta, bone & bone marrow (femur), bone & bone marrow (sternum), brain, caecum, colon, duodenum, epididymides, esophagus, eyes with optic nerve, harderian gland, heart, ileum, jejunum, kidneys, larynx, lacrimal gland, liver, lungs, lymph nodes, mammary gland, nasal cavity, ovaries, pancreas, pituitary, prostate, rectum, salivary glands, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder, uterus, vagina and gross lesions.
Organ weights:
The organs (see below) were recorded on the scheduled dates of necropsy. The organ to terminal body weight ratios as well as organ to brain weight ratios were determined; brain, heart, liver, thymus, kidney, adrenals, spleen, testes and epididymes.

HISTOPATHOLOGY: Yes
Slides of all organs and tissues below that were collected at scheduled sacrifice from the animals of control and high-dose groups were examined:
Adrenals, bone & bone marrow (femur), brain, caecum, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes, ovaries, prostate, rectum, sciatic nerve, seminal vesicles, spinal cord, spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder, uterus, vagina and gross lesions.

Other examinations:

Not reported.

Statistics:

The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, macroscopic findings, organ weights and ratios as well as clinical laboratory data:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could 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 can not be assumed to follow a normal distribution.
• Fisher's exact-test was applied to the macroscopic findings.
• Student's T-test was applied to locomotor activity and grip strength.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: No deaths and no signs of toxicity were observed.

BODY WEIGHT AND WEIGHT GAIN: As no clear dose response relationship could be established .

FOOD CONSUMPTION & FOOD EFFICIENCY: There was no apparent adverse effect on food consumption during the study.

HAEMATOLOGY: Reduced red blood cell count, hemoglobin level, mean corpuscular hemoglobin concentration in female rats at 400 mg/kg/day.

CLINICAL CHEMISTRY: There were no blood chemical changes which could be considered toxicologically significant.

ORGAN WEIGHTS: Dose-related increases in mean absolute (in females: p<0.05 at 160 mg/kg/day and p<0.01 at 400 mg/kg/day) and relative (Iiver-to-body weight ratios in both sexes: p<0.01 at 400 mg/kg/day and liver-to-brain weight ratios in females at all doses: p<0.05 and p<0.01) liver
weights were recorded in rats of both sexes.

GROSS PATHOLOGY: Crateriform stomach retractions were recorded in both sexes at 400 mg/kg/day and deemed to be treatment-related. No further gross lesion could be attributed to treatment. The latter lesions were unremarkable and consisted of renal pelvis dilation and discoloration/discolored foci in the thymus of several animals.

HISTOPATHOLOGY: In the forestomach of animals at 400 mg/kg/day, there were indicators of irritation consisting of basal cell and epithelial hyperplasia in combination with or without hyperkeratosis, chronic inflammation in the submucosa of the forestomach and occasionally with ulceration. Males
were more affected than females. At 160 mg/kg/day, there was only a male that exhibit minimal hyperkeratosis at the limiting ridge.

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the effects observed in haematology at 400 mg/kg/day and the effects observed in the liver weight (absolute and relative) and liver metabolism at and above 160 mg/kg/day.

Dose descriptor:

NOAEL

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the effects observed in haematology at 400 mg/kg/day.

Critical effects observed:

no

Conclusions:

Based on the results of this study, 50 mg/kg bw/day of the test substance was established as NOEL and 160 mg/kg bw/day as NOAEL.

Executive summary:

Introduction, materials & methods

In this subacute toxicity study, the test substance was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 50, 160 and 400 mg/kg body weight/day for a period of 28 days.Acontrol group was treated

similarly with the vehicle, PEG 300, only. The groups comprised 5 animals per sex, which were sacrificed after 28 days of treatment.Clinical signs, outside cage observation, food consumption and body weights were recordedperiodically during the acclimatisation and the treatment periods. Functional observationalbattery, locomotor activity and grip strength were performed during week4.At the end of the dosing period, blood samples were withdrawn for hematology and plasmachemistry analyses. All animals were killed, necropsied and examined post mortem.

Histological examinations were performed on organs and tissues from all control and highdose animals, and stomach and all gross lesions from all animals.

 

Results

MORTALITY /VIABILlTY:All animals survived until scheduled necropsy.

CLINICAL SIGNS:No findings of toxicological relevance were noted during the daily or weekly observations(weeks 1-3).

FUNCTIONAL OBSERVATIONAL:No findings of toxicological relevance were noted during the functional observation battery(week4).

Grip Strength:No toxicologically relevant test item-related changes were noted in mean fore- and hind limbgrip strength of males and females when compared to controls.

Locomotor Activity:Minor differences noted in the total and mean locomotor activity of males treated either with160 mg/kg/day or with 400 mg/kg/day were not seen in females at these doses and thereforewere considered to be incidental.

FOOD CONSUMPTION:The mean food consumption and the relative food consumption of the test item-treated malesand females compared favorably with their respective controls during the treatment period.

BODYWEIGHT: Minor and transient differences in mean body weights and body weight gain were noted infemale rats treated either with 50 mg/kg/day or with 160 mg/kg/day. As no clear doseresponse relationship could be established and as males were completely unaffected,therefore these findings were considered toxicologically irrelevant.

Hematology: Female rats treated with 400 mg/kg/day showed reduced red blood cell count, hemoglobinlevel, and mean corpuscular hemoglobin concentration. All differences remained within theranges of the historical control data. The mean absolute and relative reticulocyte counts wereelevated when compared to the control females and were accompanied by a marginallyshifted reticulocyte maturity index toward high fluorescent reticulocytes. These findings wereconsidered to be test item related. Although no statistical significance was noted whencompared to control males, a similar trend was observed in male rats treated with 400mg/kg/day.

Clinical Biochemistry:No test item-related changes of toxicological relevance were noted in test item-treatedfemales at any dose level.Dose-related increases in cholesterol and phospholipids concentrations were noted in malerats treated with 160 mg/kg/day or 400 mg/kg/day, which values exceeded the upper rangeof the historical control data. These differences correlated with increased absolute and

relative liver weights and were considered to be related to changes in liver metabolism as aresponse to the treatment with the test item.

ORGAN WEIGHTS:Dose-related increases in mean absolute and relative (liver-to-body weight ratios in bothsexes and liver-to-brain weight ratios in females at all doses) liver weights were recorded inrats of both sexes. These changes can be correlated with increased cholesterol andphospholipids concentrations and were therefore considered to be related to changes in livermetabolism as a response to the treatment with the test item.

MACROSCOPICIMICROSCOPIC FINDINGS:Crateriform stomach retractions were recorded at necropsy in both sexes at 400 mg/kg/day.The histological correlate revealed an irritative potential of the test substance consisting of basal cell and epithelial hyperplasia with or without hyperkeratosis,chronic inflammation in the submucosa and occasionally with ulceration. Males were moreaffected than females.

 

Conclusions:

Based on the results of this study, 50 mg/kg bw/day of the test substance was established as NOEL and 160 mg/kg bw/day as NOAEL.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

40 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

OECD guideline study and GLP study.

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

The 90-day oral toxicity study (OECD 408) has been reported as the key study as it showed adverse effects and provided a NOAEL result, where the OECD 422 study does not provide a definite NOAEL/NOEL and only shows local irritancy effects.

90 -day oral toxicity study (OECD 408):

VEEA was orally administered to male and female Crl:CD(SD) rats at 0 (control), 4, 40, and 400 mg/kg/day for 90 days to investigate its toxic potential and toxicological profile. Subsequently, reversibility of toxicity following withdrawal was examined in the 0 and 400 mg/kg groups by 21-day observation from the day after the end of administration. The results obtained were as follows:

In the 400 mg/kg group, the following changes were noted: In clinical observation, salivation was noted in males. In males and females, necropsy revealed thickening of the forestomach mucosa and large size of the pancreaticoduodenal lymph node; and histopathological examination revealed squamous cell hyperplasia at the limiting ridge of the stomach, ulcer/squamous cell hyperplasia of the forestomach, mucosal atrophy/pancreatic metaplasia of the glandular stomach, and plasma cell infiltration/follicle hypertrophy/sinus ectasia in the pancreaticoduodenal lymph node. In males or females, hematological examination revealed high platelet count (platelet), white blood cell count (WBC), and neutrophil; and biochemical examination revealed low total protein (TP), albumin, albumin fraction and A/G ratio and highα2-, ß- and y-Globulin fractions. All of these changes were noted in association with irritancy of the test article and its resulting inflammation at the stomach, the first exposure site. Other than those above, in males, red blood cell count (RBC) and mean corpuscular hemoglobin concentration (MCHC) were low and the reticulocyte count and spleen weight were high; and in females, a1-Globulin fraction was low. These changes may possibly be associated with inflammation.

No effects of the test article administration were noted in detailed clinical observation, functional observations, ophthalmology, or urinalysis.

In males and females in the 4 and 40 mg/kg group, no effects of the test article administration were noted.

All the changes observed by the end of administration were considered to be reversible after withdrawal.

As described above, in males and females in the 400 mg/kg group, changes attributable to the local irritation to the first exposure site were noted. The other changes observed in this group were only minor changes that may possibly be associated with inflammation. Therefore, the no-observed-adverse-effect level ofVEEA was considered 40 mg/kg based on the local effects observed in both males and females under the present study conditions. No specific target organ toxicity was observed in either sex up to the dose level of 400 mg/kg.

28 -day oral toxicity study (OECD 407):

In this subacute toxicity study, the test item was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 50, 160 and 400 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, PEG 300, only.

The groups comprised 5 animals per sex, which were sacrificed after 28 days of treatment.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during the acclimatization and the treatment periods. Functional observational battery, locomotor activity and grip strength were performed during week 4.

At the end of the dosing period, blood samples were withdrawn for hematology and plasma chemistry analyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high

dose animals, and stomach and all gross lesions from all animals.

MORTALITY /VIABILlTY: All animals survived until scheduled necropsy.

CLINICAL SIGNS: No findings of toxicological relevance were noted during the daily or weekly observations (weeks 1-3).

FUNCTIONAL OBSERVATIONAL BATTERY: No findings of toxicological relevance were noted during the functional observation battery (week 4).

Grip Strength: No toxicologically relevant test item-related changes were noted in mean fore- and hind limb grip strength of males and females when compared to controls.

Locomotor Activity: Minor differences noted in the total and mean locomotor activity of males treated either with 160 mg/kg/day or with 400 mg/kg/day were not seen in females at these doses and therefore were considered to be incidental.

FOOD CONSUMPTION: The mean food consumption and the relative food consumption of the test item-treated males

and females compared favorably with their respective controls during the treatment period.

BODY WEIGHT: Minor and transient differences in mean body weights and body weight gain were noted in female rats treated either with 50 mg/kg/day or with 160 mg/kg/day. As no clear dose response relationship could be established and as males were completely unaffected, therefore these findings were considered toxicologically irrelevant.

CLINICAL LABORATORY INVESTIGATIONS

Hematology:

Female rats treated with 400 mg/kg/day showed reduced red blood cell count, hemoglobin level, and mean corpuscular hemoglobin concentration. All differences remained within the ranges of the historical control data. The mean absolute and relative reticulocyte counts were elevated when compared to the control females and were accompanied by a marginally shifted reticulocyte maturity index toward high fluorescent reticulocytes. These findings were considered to be test item related. Although no statistical significance was noted when compared to control males, a similar trend was observed in male rats treated with 400 mg/kg/day.

Clinical Biochemistry:

No test item-related changes of toxicological relevance were noted in test item-treated females at any dose level.

Dose-related increases in cholesterol and phospholipids concentrations were noted in male rats treated with 160 mg/kg/day or 400 mg/kg/day, which values exceeded the upper range of the historical control data. These differences correlated with increased absolute and relative liver weights and were considered to be related to changes in liver metabolism as a response to the treatment with the test item.

ORGAN WEIGHTS:

Dose-related increases in mean absolute and relative (liver-to-body weight ratios in both sexes and liver-to-brain weight ratios in females at all doses) liver weights were recorded in rats of both sexes. These changes can be correlated with increased cholesterol and phospholipids concentrations and were therefore considered to be related to changes in liver metabolism as a response to the treatment with the test item.

MACROSCOPIC I MICROSCOPIC FINDINGS

Crateriform stomach retractions were recorded at necropsy in both sexes at 400 mg/kg/day.

The histological correlate revealed an irritative potential of the test item consisting of basal cell and epithelial hyperplasia with or without hyperkeratosis, chronic inflammation in the submucosa and occasionally with ulceration. Males were more affected than females.

Assessment:

Oral administration of the test item to Wistar rats at doses of 50, 160 and 400 mg/kg/day, for 28 days resulted in no mortality, no clinical symptoms of toxicological relevance during daily or weekly observations (weeks 1-3) or during functional observational battery (week 4), no effects on grip strength or locomotor activity and no effects on food consumption or body weight.

Test item-induced adverse changes at 400 mg/kg/day included:

• Reduced red blood cell count, hemoglobin level, mean corpuscular hemoglobin concentration in female rats,

• Elevated mean absolute and relative reticulocyte counts accompanied by a shifted reticulocyte maturity index toward high fluorescent reticulocytes in females at 400 mg/kg/day with a similar trend in male rats treated with 400 mg/kg/day,

• Irritative potential in the stomach, evidenced by crateriform retractions correlated with basal cell and epithelial hyperplasia in combination with or without hyperkeratosis, chronic inflammation in the submucosa and occasionally with ulceration.

Test item-related changes of adaptive nature were found in the liver. They consisted of changes in liver metabolism and included:

• Dose-related increases in cholesterol and phospholipids concentrations in male rats treated either with 160 mg/kg/day or with 400 mg/kg/day,

• Dose-related increases in mean absolute and relative (liver-to-body weight ratios in both sexes and liver-to-brain weight ratios in females at all doses) liver weights in rats of both sexes.

Based on the results of this study, 50 mg/kg body weight/day was established as the no-observed-effect-level (NOEL) and 160 mg/kg body weight/day as the no-observed adverse-effect-level (NOAEL).

OECD 422 (Combined Repeated Dose Toxicity with Reproduction/ Developmental Toxicity Study of 2-(2-Vinyloxyethoxy) Ethyl Acrylate in Rats):

The test substance was repeatedly administered by oral gavage to male and female Crl:CD(SD) rats, 12 males and 12 females per group, at the doses of 0 (control, corn oil), 50, 140 and 400 mg/kg/day. The test substance was administered orally to male rats, before mating, during mating period and after mating, for a total of 91 days, and to female rats before mating, during mating and gestation periods, and up to day 5 after parturition, to investigate effects of its repeated administration on male and female rats, and effects on reproduction performance of males and females and development of pups. In the control and 400 mg/kg groups, 5 of the 12 males and 5 females were added to constitute recovery groups to investigate recovery in 14-day recovery period following administration. Females in the recovery group were not used for mating. The results of this study are as follows:

Repeat dose toxicity:

In males and females in the low dose (50 mg/kg) and above groups, local irritation at the application site of the test substance was observed at the limiting ridge of the stomach and in the forestomach. These changes consisted of thickening in necropsy findings and

squamous cell hyperplasia in histopathological findings, accompanied by ulcer and cellular infiltration of inflammatory cells.

In relation to the above changes, salivation was observed in the middle dose (140 mg/kg) and high dose (400 mg/kg) groups in the general clinical observation and detailed clinical observation; increases in white blood cell count, stab form and segmented

neutrophils in the differential counts of white blood cells, and β-globulin fraction in males in the high dose group and decreases in albumin and A/G ratio, and increases in β-globulin fraction in females in the high dose group in hematology and biochemistry; decreases in absolute and relative thymus weights in males in the high dose group.

On the basis of the above-mentioned results, the no-observed-adverse-effect level (NOAEL) and the no-observed-effect level (NOEL) of 2-(2-Vinyloxyethoxy) ethyl acrylate were estimated to be less than 50 mg/kg/day in both of males and females under the

conditions of this study because thickening in the forestomach and at the limiting ridge of the stomach, which was related to local irritation at the application site of the test substance, was observed at the doses of 50 mg/kg/day and above.

Justification for classification or non-classification

The substance is not currently classified for specific target organ toxicity-repeated exposure (STOT-RE) based on review of the available data.

90-day oral toxicity study:

No specific target organ toxicity was observed in either sex up to the dose level of 400 mg/kg.

The changes observed at 400 mg/kg were associated with the irritancy of the substance and resulting inflammation at the stomach (first exposure site). The other changes observed in this group were only minor changes that may possibly be associated with inflammation and were considered to have minimal toxicological importance, be adaptive responses and were not associated with histopathological findings.

These changes were observed at the 400 mg/kg bw/day dose. The CLP guidance value (for a 90 -day study) for classification as STOT-RE Category 2 is 10 - 100 mg/kg bw/day when significant toxic effects are seen to occur within this guidance range. In animals in the 4 and 40 mg/kg bw/day dose groups, no effects of administration were noted.

Based on these results, classification as STOT-RE based on the 90-day oral toxicity study is not considered appropriate.

28 -day oral toxicity study:

In this study adverse effects (in haemetology) were observed in addition to effects associated with the irritancy of the substance. However, the toxicologicial relevance in these changes in heametology are unclear. These effects occurred at the 400 mg/kg bw/day dose. The CLP guidance value (for a 28 -day study) for classification as STOT-RE Category 2 is 30 - 300 mg/kg bw/day when significant toxic effects are seen to occur within this guidance range.

The effects observed at 160 mgkg bw/day do not support classification based on the CLP criteria as they were considered to be changes of adaptive nature and the changes in organ weights (liver) were not associated with organ dysfunction.

OECD 422 study:

Based on the results of the study the NOAEL and NOEL were estimated to be less than 50 mg/kg bw/day. However, classification for STOT-RE is not considered appropriate as these results were due to thickening in the forestomach and at the limiting ridge of the stomach, which was related to local irritation at the application site of the test substance.

All changes observed in the study (hematology, biochemical findings, necrospy, organ weights, histopathology) were considered accompanying inflammotory changes.

The changes related to test substance administration were limited to irritation and associated changes and no other toxicity was observed.

Therefore, classification as STOT-RE based on the OECD 422 study is not considered appropriate.