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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between June 25th 2008 and March 24th 2009

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

451-690-9

EC Name:

-

Cas Number:

86273-46-3

Molecular formula:

C9H14O4

IUPAC Name:

2-[2-(ethenyloxy)ethoxy]ethyl prop-2-enoate

Test material form:

other: liquid

Details on test material:

Identity: 2-(2-Vinyloxyethoxy) ethyl acrylate
Abbreviation: VEEA
- Substance type: clear colourless liquid
- Physical state: liquid
- Lot/batch No.: 7D23-3323
- Stability: Unstable in light and heat. Stable in air. Flammable, irritating, and polymerization may occur by
radical, light and acid.
- Storage condition of test material: The test substance was shielded from light, and refrigerated (measured temperature 1 to 9ºC). Refrigeration room in the test substance storage room of Safety Research Institute for Chemical Compounds Co., Ltd.
- Other:
Safety precautions: A face mask, gloves, protection glasses (goggles) were used.

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Charles River Laboratories Japan, Inc., Atsugi Breeding Center

- Age at study initiation:
The animals were ordered at
4 weeks of age; males and females
8 weeks of age; females

Number of animals ordered and received:
4 weeks of age; 52 males and 12 females were ordered, and 54 males and 13 females were received (males in the main test groups and females in the recovery groups) 8 weeks of age; 50 females were ordered, and 52 females were received (females in the main test groups) Males in the recovery groups were selected from the main test groups on the basis of the mean body weight in each group.

Age of animals at the start of administration:
4 weeks of age; 6 weeks of age for males and females
8 weeks of age; 10 weeks of age for female

- Weight at study initiation:
4 weeks of age; 87 to 106 g for males and 65 to 75 g for females
8 weeks of age; 164 to 199 g for females

- Fasting period:
All animals were fasted from the evening on the day before necropsy,

- Housing:
Type of cages: Metal bracket-type cages (300 W × 410 D × 200 H, mm)
with wire mesh floors.

- Number of animals per cage: Three or less during the quarantine and acclimatization period, one after the group assignment, one male and one female during mating period, and one litter after parturition.

- Use of restrainers for preventing ingestion (if dermal):
Not applicable

- Diet:
γ-ray irradiated pellet diet, CRF-1 Animals, had free access to the diet using metal feeders. Each lot of the diet used (lot nos. 080305 and 080509) was analyzed for chemical and bacterial contaminants that might affect the study. The analyses were performed at Japan Food Research Laboratories (Certificate of Analysis No. 108031936-001) or Eurofins Scientific Japan K.K. (Certificate of Analysis No. AR-08-JP-000056-01) for chemical contaminants, and at the diet manufacturer for bacterial contaminants.


- Water:
Method of water supply: Animals had free access to water using an automatic watering system or water bottles.
Analysis for contaminants: Drinking water was analyzed for contaminants that might affect the study. The analysis was conducted on the water
samples collected from the end of the same piping system as that in the animal room (room no. 301) on July 1, 2008, October 1, 2008 and January 7, 2009. The analyses were performed at Nihon Eisei Co., Ltd. and the data were supplied. The contaminants assayed and the limits of acceptable values were in compliance with those prescribed in the Standard Operating Procedure of Safety Research Institute for Chemical Compounds Co., Ltd.

- Acclimation period:
For 13 days

ENVIRONMENTAL CONDITIONS

- Temperature (°C):
21 to 25ºC,
- Humidity (%):
43 to 60%)

- Air changes (per hr):
10 to 15 times/hour

- Photoperiod (hrs dark / hrs light):
Artificial lighting for 12 hours (from 8:00 to 20:00)

IN-LIFE DATES:
From day 1 of administration to the day of necropsy (the day
after day 91 of administration).

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: Not applicable

Vehicle:

other: corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulation and chemical analysis

Dose formulation method: The test substance accurately weighed was added to the vehicle, which was well mixed with a stirrer to prepare a dose formulation.

Frequency: At least every 15 days

Storage condition: At room temperature (measured temperature 21 to 25ºC)

Storage location: The test substance storage room of Safety Research Institute for Chemical Compounds Co., Ltd.

Expiry: 15 days after preparation

Safety precautions: A face mask, gloves, protection glasses (goggles) were used.

Remaining dose formulations: The remaining dose formulations were collected as industrial waste to be incinerated.

Stability of dose formulations:
Before the start of administration, dose formulations of the concentrations 10 and 80 mg/mL were analyzed to confirm stability of the test substance in the dose formulations at room temperature for 15 days (day of preparation was defined as Day 0, Appendix 3).

Confirmation of concentration of dose formulations:
Concentrations of the test substance in the dose formulations of all concentrations were confirmed in a total of 3 times; at the first dose formulation, once during the administration period, and at the final dose formulation.

DIET PREPARATION
γ-ray irradiated pellet diet, CRF-1 was used throughout the study period.

- Rate of preparation of diet (frequency):
Not applicable

- Mixing appropriate amounts with (Type of food):
Not applicable

- Storage temperature of food:
No data

VEHICLE
Identity: Corn oil
Lot number: V8A6289 and V8K7807
Manufacturer: Nacalai Tesque, Inc.
Storage condition: At room temperature (measured temperature 21 to 25ºC)
Safety precautions: Nothing specified.
Expiry: 3 years after manufacture

Details on mating procedure:

- M/F ratio per cage:
1/1 (Animals were paired on a 1 male: 1 female basis within each dose group)

- Length of cohabitation:
Up to 14 days

- Proof of pregnancy:
Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation)

- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.:
No data

- Further matings after two unsuccessful attempts:
No data

- After successful mating each pregnant female was caged:
Mated females were housed individually during the period of gestation and lactation.

- Any other deviations from standard protocol:
Not applicable

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Up to 28 consecutive days for the 400 mg/kg/day dose group and up to 54 consecutive days for the 140 and 50 mg/kg/day dose groups

Frequency of treatment:

Daily

Details on study schedule:

Composition of the test groups and animal numbers in each group are as follows:

Test group Dose level (mg/kg) Concentration(mg/mL) Number of animals (Animal number)

Control group 0 0 12 (101 to 112) 12 (151 to 162)
Low dose group 50 10 12 (201 to 212) 12 (251 to 262)
Mid dose group 140 28 12 (301 to 312) 12 (351 to 362)
High dose group 400 80 12 (401 to 412) 12 (451 to 462)

Control group 0 0 5 (101, 105, 107, 108 and 112) 5 (163 to 167)
High dose group 400 80 5 (403, 404, 405,407 and 409) 5 (463 to 467)

To the control group, only the vehicle was dosed by the same method as the other groups.

- Frequency of administration: Once daily, for consecutive days
- Administration time: Between 9:00 and 14:00 Dams during parturition at the above-mentioned administration time were dosed after the completion of parturition.
- Administration period: Males; 70 days before the start of mating, and subsequent 21 days (a total of 91 days)
Females; During 14 days before the start of mating and mating period until successful copulation. Females showing evidence of copulation were dosed during the duration of gestation and until day 5 after parturition (day 0 of lactation was defined as day 0 after parturition). Females that did not deliver were dosed until day 26 of gestation (a total of 43 to 52 days).

Females in the recovery groups; 91 days, which were the same as males

- Recovery period: 14 days after the end of administration period
- Dose volume: 5 mL/kg. Individual dose volume was calculated from the body weight on the day nearest to the administration day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

140 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose (including control).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were determined with reference to the 28-day repeated dose oral toxicity (gavage) study1) with the dose levels of 50, 160 and 400 mg/kg. The high dose level was set at 400 mg/kg, with which a tendency to anemia was observed in females, and increasing tendency of reticulocyte counts, hyperkeratosis and ulceration accompanied with chronic inflammation in the stomach, etc. were observed in males and females. The low dose level was set at 50 mg/kg, with which adaptive reactions such as increases in the liver weights, cholesterol or phospholipids were not observed. The middle dose level was set at 140 mg/kg, approximate value obtained by multiplying 50 by the square root of 8.

- Rationale for animal assignment (if not random):
Random.

- Other:
Not applicable

Positive control:

Not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Yes see atached tables.


- General clinical observation
Animals observed: All animals
Period: From day 1 of administration to the day of necropsy (the day after day 91 of administration). The day of the first administration was defined as day 1 of administration.
Frequency: Twice daily, before and after the administration. Once in the morning on the day of necropsy.
Observation method: Animals were individually observed for mortality, external appearance, behavior, etc. Any abnormal findings were recorded with their duration.

DETAILED CLINICAL OBSERVATIONS:
Yes see atached tables.

- Time schedule:
Animals observed: All animals
Time: Before the start of administration and on days 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84 and 91 of administration

BODY WEIGHT:
This method was the same for males
Measurement days: Before administration on Days 1, 3, 5, 7, 10, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84 and 91 of administration and on the day of necropsy.
Measurement method: Animals were weighed with an electronic balance and recorded as the whole number in grams.
Body weight gain and percentage body weight gain were calculated.

Off spring: All live animals were weighed on days 0, 1 and 4 after birth.

FOOD CONSUMPTION:
Animals measured: All animals
Measurement days: Food consumption was measured on the same days as body weight measurement, except on the day of necropsy and during mating period.

Measurement method: Food consumption was measured with an electronic balance and recorded as the whole number in grams. On the day before the start of administration, an adequate amount of diet was measured and set at each cage. The residual amount and the amount supplied were measured on each measurement day.
FOOD EFFICIENCY:
No

WATER CONSUMPTION:
No

HAEMATOLOGY AND CLINICAL CHEMISTRY:
Animals examined: Five animals of each group [except those used for the
functional observational battery at necropsy, 5 animals
were selected from the smallest animal number]
Time: Blood was collected at necropsy

Method of blood collection:
Rats fasted for 16 to 19 hours were anesthetized with
ether and blood samples were collected from the
abdominal aorta.

Examination parameters and methods:
1. Red blood cell count (RBC)
2. Hematocrit (Ht) - Electric resistance method
3. Hemoglobin concentration
4. Mean corpuscular volume (MCV)
5. Mean corpuscular hemoglobin (MCH)
6. Mean corpuscular hemoglobin concentration (MCHC)
7. Reticulocyte count (Reticulocyte) - Brecher’s method (microscopy)
8. Platelet count (Platelet) Electric resistance method
9. White blood cell count (WBC) Electric resistance method
10. Differential count of WBC May-Grünwald-Giemsa staining (microscopy)
11. Prothrombin time (PT) Thromboplastin method
12. Activated partial thromboplastin time (APTT)

Method of blood collection for females: The same as males except for the fasting time for 17 to 19 hours, the parameters measured were the same as
Method of blood collection for the offspring: The same as the main test groups except for the fasting time for 16 to 18 hours, the parameters mwasured were the same as above.

URINALYSIS:
Animals examined: Five animals of each group [the same animals as those used for the functional observational battery]
Time: Week 13 of administration
Method of urine collection: Urine samples were collected using metabolic cages for rats (KN-646, type B-1, Natsume Seisakusyo Co., Ltd.). Urine samples collected immediately after to approximately 3 hours after administration were used for the following parameters 1 to 8, and those collected for approximately 21 hours for parameters 9 and 10. The urine samples were discarded after the completion of examination.

- Time schedule for collection of urine:
urine was collected betwenn the period immediately after dosing and 3 hours after dosing

Examination parameters and methods:
1. pH Test paper method
2. Protein Test paper method
3. Glucose Test paper method
4. Ketone body Test paper method
5. Urobilinogen
6. Bilirubin
7. Occult blood
8. Color - Macroscopic observation
9. Urine volume - Volumetric method
10.Specific gravity

Urinalysis for the offspring
Animals examined: All animals
Time: Week 2 of recovery
Method of urine collection: The same as the main test groups

NEUROBEHAVIOURAL EXAMINATION:
No

Organ weights
Animals weighed: Five animals of each group [the same animals as those used for the hematological examination]. All males for the testes and epididymides.
Time: At necropsy
Measurement method: Absolute weights of the organs described below were measured with an electronic balance. Bilateral organs were measured together. The measurement method was exactly the same for males and females.

Histopathology:
Animals examined: Preparations of all organs and tissues fixed and stored at necropsy were made, and those of 5 animals (the same animals as those used for the hematological examination) in the control and high dose groups were microscopically examined. This process was exactly the smale for male and female animals.

OTHER:
Examination of oestrous cycle
Animals examined: All females
Period: From the day of first administration to the day of successful copulation.
Method: Vaginal smears stained with Giemsa stain were prepared, and stage of oestrous cycle was examined by light microscopy.
Determination: Repetition of each stage (proestrus, estrus, metestrus, and diestrus) of oestrous cycle (one complete cycle comprises of 4 to 6 days) was determined to be normal, and the length (number of days) of oestrous cycle (interval between estrus) was calculated. Continual appearance of diestrus for 7 days or longer was determined to be persistent anestrus, and defined to be abnormal.

Oestrous cyclicity (parental animals):

Examination of oestrous cycle
Animals examined: All females
Period: From the day of first administration to the day of successful copulation.
Method: Vaginal smears stained with Giemsa stain were prepared, and stage of oestrous cycle was examined by light microscopy.
Determination: Repetition of each stage (proestrus, estrus, metestrus, and diestrus) of oestrous cycle (one complete cycle comprises of 4 to 6 days) was determined to be normal, and the length (number of days) of oestrous cycle (interval between estrus) was calculated. Continual appearance of diestrus for 7 days or longer was determined to be persistent anestrus, and defined to be abnormal.

Sperm parameters (parental animals):

Parameters examined in [all/P/F1/F2] male parental generations:
[testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology, other:]

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum:
No

PARAMETERS EXAMINED
The following parameters were examined in offspring:
General clinical observation of offspring
Body weight of offspring
Necropsy of offspring

GROSS EXAMINATION OF DEAD PUPS:

Postmortem examinations (parental animals):

GROSS NECROPSY / ORGAN WEIGHTS

Animals examined: All animals except those selected for use in the recovery groups
Time: Animals were necropsied on the day after day 91 of administration.
Examination method: Animals were observed for external appearance, and blood was collected under ether anesthesia. They were euthanized by exsanguination and macroscopically observed for all organs and tissues.

Organ weights
Animals weighed: Five animals of each group [the same animals as those used for the hematological examination]. All males for the testes and epididymides.
Time: At necropsy
Measurement method: Absolute weights of the organs described below were measured with an electronic balance. Bilateral organs were measured together. The measurement method was exactly the same for males and females.

Histopathology:
Animals examined: Preparations of all organs and tissues fixed and stored at necropsy were made, and those of 5 animals (the same animals as those used for the hematological examination) in the control and high dose groups were microscopically examined. This process was exactly the smale for male and female animals.

Postmortem examinations (offspring):

Necropsy
Animals examined: All animals
Time: Animals were necropsied on the day after day 14 of
recovery.
Examination method: The same as the main test groups
Organs and tissues: The same as the main test groups

Organ weights
Animals weighed: All animals
Time: At necropsy
Measurement method: The same as the main test groups
Calculation of relative organ weights:
The same as the main test groups
Organs: The same as the main test groups

Statistics:

Means and standard deviations were calculated for the results of the following parameters: grip strength, locomotor activity, body weight, body weight gain and percent body weight gain, food consumption, urine volume, hematology, biochemistry, absolute and relative organ weights, length of oestrous cycle, number of corpora lutea, number of implantations and implantation index, number of pups delivered, numbers of live pups and dead pups at delivery, delivery index, live birth index, sex ratio, duration of gestation, number of live pups on day 4 after birth, and viability index, which were analyzed for homogeneity of variances by the Bartlett test. If there was an evidence of homogeneity, the One way analysis of variance (ANOVA) was used, and if there was no evidence of homogeneity, the Kruskal-Wallis test was used. When ANOVA indicated a significant difference, Dunnett’s test was used for comparison with the control group. When the Kruskal-Wallis test indicated a significant difference, the Mann-Whitney U-test was used for comparison with the control group. Body weight of offspring of each sex was analyzed on the litter basis.

As for the findings given two or more different grades in the observation parameters of the detailed clinical observation and the functional observational battery, qualitative parameters of urinalysis, urine specific gravity and histopathology, tendency of each group was analyzed by the Kruskal-Wallis test, and if there was a significant difference, the Mann-Whitney U-test was used for comparison with the control group.

Statistically significant level was set at 5% for comparison with the control group.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
(Clinical signs)
Tables 1 and 2, show the general clinical observation results.

Males: There were no abnormal findings in the 0 (control group, expressed as the control group thereafter) or 50 mg/kg group (expressed as the low dose group thereafter).

In the 140 and 400 mg/kg groups (expressed as the middle and high dose groups, respectively thereafter), salivation was sporadically observed during the administration period, which was observed in higher frequency in a few males (animal no. 310 in the middle dose group and nos. 406, 408 and 411 in the high dose group).

One male (animal no. 310) in the middle dose group showed hematuria sporadically, and another (animal no. 306) showed soft feces only once.

Females: There were no abnormal findings in the control, low or middle dose group during the administration period.
In the high dose group, salivation was sporadically observed mainly in the administration period before mating, which was observed in higher frequency in 1 female (animal no. 458).
In the recovery groups, there were no abnormal findings in the control group, and salivation was sporadically observed in 1 female (animal no. 465) in the high dose group.

Tables 3 to 8 show the detailed clinical observation results.

Administration period
Males: Salivation was sporadically observed in the middle and high dose groups, and no significant changes were observed in any treatment group (low, middle or high dose group) as compared to the control group.

Females: Salivation was observed in the high dose group, and no significant changes were observed in any treatment group as compared to the control group.

Recovery period
Males: In the high dose group, there were no significant changes as compared to the control group.

Females: In the high dose group, there were no significant changes as compared to the control group.

Functional observational battery:
Tables 9 to 12 show the functional observational battery results, grip strength and locomotor activity measurements results.

Week 13 of administration
Males: There were no significant changes in functional observational battery results or grip strength in any treatment group as compared to the control group.

A transient and significant increase was observed in the locomotor activity measurements in the low dose group, which was not dose-dependent.

Females: There were no significant changes in the high dose group as compared to the control group.

Day 4 of lactation:

Females: A significant decrease was observed in the locomotor activity measurements in the low dose group as compared to the control group, which was not dose-dependent. There were no significant changes in the middle or high dose group.

Week 2 of recovery:

Males: There were no significant changes in the high dose group as compared to the control group.

Females: Significant increases were observed in grip strength of the hindlimbs in the high dose group as compared to the control group, which was not observed during week 13 of administration.
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

Figures 1 to 3, Tables 13 to 17 show the body weight changes.

Administration period
Males: In the low dose group, there were no significant changes in body weight changes, body weight gain, or percent body weight gain as compared to the control group.

In the middle dose group, there were no significant changes in body weight changes or body weight gain. Percent body weight gain significantly increased, which was not dose-dependent.

In the high does group, there were no significant changes in body weight changes or body weight gain. Percent body weight gain significantly decreased.

Females: There were no significant changes in body weight changes, body weight gain or percent body weight gain during the administration period before mating, gestation or lactation period, in any treatment group as compared to the control mgroup. In the high dose group in the recovery group, there were no significant changes in body weight changes or body weight gain. Percent body weight gain significantly increased.

Recovery period
Males: In the high dose group, there were no significant differences in body weight changes as compared to the control group, and body weight gain and percent body weight gain significantly increased.
Females: In the high dose group, there were no significant changes in body weight changes, body weight gain or percent body weight gain as compared to the

Food consumption:
Figures 4 to 6, Tables 18 to 22 show the food consumption.

Administration period
Males: There were no significant changes in any treatment group as compared to the control group.
Females: During the administration period before mating, there were no significant changes in any treatment group as compared to the control group. During the gestation period, there were no significant changes in the low or middle dose group. In the high dose group, food consumption significantly decreased on days 1 and 3 of gestation. During the lactation period, there were no significant changes in any treatment group.
In the high dose group in the recovery group, there were no significant changes.

Recovery period
Males: There were no significant changes in the high dose group as compared to the control group.
Females: Food consumption significantly increased in the high dose group on day 7 of recovery as compared to the control group.
Urinary findings:
Tables 23 to 26 show the urinary findings.

Week 13 of administration
Males: There were no significant changes in any treatment group as compared to the control group.
Females: There were no significant changes in the high dose group as compared to the control group.

Week 2 of recovery
Males: There were no significant changes in the high dose group as compared to the control group.
Females: There were no significant changes in the high dose group as compared to the control group.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Not examined.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Reproductive and developmental toxicity:
Examination of oestrous cycle
Tables 48 and 49 show the results of the examination of oestrous cycle.

Main test group
In the incidence of females showing normal oestrous cycle and the length of oestrous cycle, there were no significant differences in any treatment group as compared to the control group. In the 3 females in the low dose group and 1 female in each of the middle and high dose groups, which were determined to be persistent anestrus and their length of oestrous cycle was not calculated, copulation was observed in week 2 or 3 (re-mating period) of mating period. These females, except one in the low dose group and 2 in the middle dose group, were confirmed to be pregnant.

Recovery group:
In the high dose group set for investigation of recovery, there were no significant differences in the incidence of females showing normal oestrous cycle and the length of oestrous cycle, as compared to the control group. There were no females determined to be persistent anestrus.

Examination of reproduction performance:
Table 48 and INDIVIDUAL DATA 18-1 to 18-4 show the results of the examination of reproduction performance. Copulation was not observed in the 2-week mating period in 2 males in the low dose group and 1 male in each of the middle and high dose groups, but there were no significant differences in the copulation index as compared to the control group. As for the females paired with these males in the mating period, copulation with proved males was observed in the succeeding re-mating period. There were no significant differences in the fertility index, gestation index, duration of gestation, or nursing index on day 4 of lactation in any treatment group.

Pregnancy, parturition, nursery condition and viability index of pups Table 50 and INDIVIDUAL DATA 19-1 to 19-4 show the results of pregnancy, parturition, nursery condition and viability index of pups. There were no significant differences in the number of corpora lutea, number of implantations, implantation index, delivery index, number of pups delivered, sex ratio of pups, number of live pups and live birth index on day 0 of lactation, number of live pups and viability index on day 4 of lactation in any treatment group as compared to the control group.


ORGAN WEIGHTS (PARENTAL ANIMALS)
Organ weights
Tables 38 to 41 show the absolute and relative organ weights.

The end of administration period
Males: In the low and middle dose groups, there were no significant changes as compared to the control group. In the high dose group, absolute and relative thymus weights significantly decreased.
Females: In the low dose group, there were no significant changes as compared to the control group. In the middle dose group, relative liver weights significantly increased, which was not dose-dependent. In the high dose group, there were no significant changes.

The end of recovery period Males: In the high dose group, there were no significant changes as compared to the control group.
Females: In the high dose group, absolute weights of the kidney, adrenal and ovary significantly increased and the relative liver weights significantly increased as compared to the control group. These changes were not observed at the end of administration.

GROSS NECROPSY:
Necropsy findings
Tables 35 to 37 show the necropsy findings.

The end of administration period
Males: In the control group, atrophy of the right testis and epididymis was observed in 1 male.

In the low dose group, thickening of the limiting ridge of the stomach was observed in all of the 12 males. In the middle dose group, thickening of the limiting ridge of the stomach was observed in all of the 12 males, and diffuse (3 males) and multifocal (6 males) papillary thickening of the forestomach mucosa was observed.

In the high dose group, thickening of the limiting ridge of the stomach, diffuse papillary thickening of the forestomach mucosa, and hypertrophy of the pancreaticosplenic lymph node were observed in all of the 7 males. Multifocal black patches (1 male) and fine black patches (2 males) in the glandular stomach mucosa were observed.

Females: In the control group, there were no abnormal findings in any of the 12 females on day 6 of lactation.

In the low dose group, out of the 11 females on day 6 of lactation, focal thickening (1 female), multifocal thickening (1 female) and multifocal papillary thickening (1 female) of the forestomach mucosa were observed. There were no abnormal findings in the one non-pregnant female.

In the middle dose group, out of the 10 females on day 6 of lactation, thickening of the limiting ridge of the stomach was observed in 8 females, multifocal (4 females) and focal (1 female) papillary thickening of the forestomach mucosa, and focal (2 females), diffuse (1 female) and multifocal (1 female) thickening of the forestomach mucosa were observed. Among the non-pregnant 2 females, focal thickening of the forestomach mucosa was observed in 1 female, and thickening of the limiting ridge of the stomach and focal papillary thickening of the forestomach mucosa were observed in 1 female.

In the high dose group, thickening of the limiting ridge of the stomach and hypertrophy of the pancreaticosplenic lymph node were observed in all of the 12 females on day 6 of lactation. Diffuse (10 females) and multifocal (2 females) papillary thickening of the forestomach mucosa was observed, and adhesion to the lateral left lobe of the liver was observed in 1 female.


The end of recovery period
Males: In the control group, atrophy of the right testis and epididymis was observed in 1 male.

In the high dose group, thickening of the limiting ridge of the stomach and focal thickening of the forestomach mucosa were observed in 2 males, diffuse thickening of the forestomach mucosa was observed in 1 male, and diffuse (1 male) or multifocal papillary (1 male) thickening of the forestomach mucosa was observed. Hypertrophy of the pancreaticosplenic lymph node was observed in 3 males.

Females: There were no abnormal findings in any of the 5 females in the control group. In the high dose group, thickening of the limiting ridge of the stomach was observed in 2 females, and diffuse (1 female) and focal (4 females) thickening of the forestomach mucosa, and hypertrophy of the pancreaticosplenic lymph node in 4 females.


HISTOPATHOLOGY (PARENTAL ANIMALS)
Tables 42, 44, 45 and 47, show the histopathological findings.

Tables 43 and 46 show the results of stages of spermatogenesis in the testis.

The end of administration period
Males:
[Forestomach]
Squamous cell hyperplasia was observed in 1 (slight; +) of the 12 males in the low dose group, 9 (slight to severe; + to +++) of the 12 males in the middle dose group, and all (moderate to severe; ++ to +++) of the 7 males in the high dose group, and were significant differences in the middle and high dose groups as compared to the control group. Ulcer was observed in 1 male (+) in the middle dose group and 6 males (+ to ++) in the high dose group, and there was a significant difference in the high dose group. Submucosal cellular infiltration of inflammatory cells was observed in 8 males (+) in the middle dose group and all males (+ to ++) in the high dose group, and there were significant differences in the middle and high dose groups. Submucosal fibrosis was observed in 1 male (+) in the middle dose group and all (+ to ++) in the high dose group, and there was a significant difference in the high dose group. Submucosal edema was observed in 3 males (+) in the middle dose group.
[Limiting ridge of the stomach] Squamous cell hyperplasia was observed in all males, and there were significant differences in the low (+), middle and high (+ to ++) dose groups as compared to the control group. Ulcer was observed in 1 male (++) in the high dose group.
[Glandular stomach]
Eosinophilia of secretory granule of the chief cell was observed in 1 male (+) in each of the middle and high dose groups, and erosion in 3 males (+) in the high dose group.

[Pancreaticosplenic lymph node, at which gross findings were made at necropsy in all males in the high dose group]
Medullary proliferation of plasmacytes and reactive hyperplasia of the follicles were observed in all of the 7 males (+ to ++) and sinus dilatation was observed in 6 males (+ to ++).
[Other organs and tissues]
There were no findings related to the test substance administration or those with increasing incidence.
[Spermatogenesis in the testis]
In the high dose group, significant increases were observed in the number and the number per sertoli cell of pachytene spermatocytes and round spermatids in the stages VII to VIII as compared to the control group. There were no significant changes in the other Stages, and all of these significant differences were considered incidental and were not related to the test substance administration.

Females:
[Forestomach]
Squamous cell hyperplasia was observed in 3 (slight to moderate; + to ++) of the 12 females in the low dose group, 10 (slight to moderate; + to ++) of the 12 females in the middle dose group, and all (moderate to severe; ++ to +++) of the 12 females in the high dose group, and there were significant differences in the middle and high dose groups as compared to the control group. Ulcer was observed in 3 females (+ to ++) in the low dose group, 3 females (+) in the middle dose group, and 9 females (+ to ++) in the high dose group, and there was a significant difference in the high dose group. Submucosal cellular infiltration of inflammatory cells was observed in 3 females (+ to ++) in the low dose group, 10 females (+ to ++) in the middle dose group and 11 females (+ to ++) in the high dose group, and there were significant differences in the middle and high dose groups. Submucosal fibrosis was observed in 3 females (+ to ++) in the low dose group, 7 females (+ to ++) in the middle dose group, and 11 females (+ to ++) in the high dose group, and there were significant differences in the middle and high dose groups. Submucosal edema was observed in 2 females (+) in the low dose group, 7 females (+) in the middle dose group, and 3 females (+) in the high dose group, and there was a significant difference only in the middle dose group.

[Limiting ridge of the stomach]
Squamous cell hyperplasia was observed in 1 female (+) in the low dose group, and all in the middle (+) and high (+ to ++) dose groups, and there were significant differences in the middle and high dose groups as compared to the control group. [Pancreaticosplenic lymph node, at which gross findings were made at necropsy in all females in the high dose group] Medullary proliferation of plasmacytes was observed in all of the 12 females (++), reactive hyperplasia of the follicles in 9 females (+ to ++), and sinus dilatation in 2 females (+).
[Other organs and tissues]
There were no findings related to the test substance administration or those with increasing incidence.

The end of recovery period
Males:
[Forestomach]
Squamous cell hyperplasia was observed in all of the 5 males (+ to ++) in the high dose group, and there was a significant difference as compared to the control group. Submucosal cellular infiltration of inflammatory cells was observed in 4 males (+) in the high dose group, and there was a significant difference. Submucosal fibrosis was observed in 4 males (+ to ++).
[Limiting ridge of the stomach]
Squamous cell hyperplasia was observed in 4 males (+) in the high dose group, and there was a significant difference as compared to the control group. [Pancreaticosplenic lymph node, at which gross findings were made at necropsy in 3 males in the high dose group] Reactive hyperplasia of the follicles was observed in 3 males (+) and fibrosis in 2 males (+).

[Other organs and tissues]
There were no findings related to the test substance administration or those with increasing incidence.

[Spermatogenesis in the testis]
In the high dose group, significant increases were observed in the number of round spermatids in the Stages I to IV; the number per sertoli cell of pachytene spermatocytes, and the number and the number per sertoli cell of round spermatids in the Stages VII to VIII; the number per sertoli cell of pachytene/diplotene spermatocytes in the stages XII to XIV, as compared to the control group. These significant differences, as those at the end of administration, were considered incidental because there were no significant changes in the Stages IX to XI.

Females:
[Forestomach]
Squamous cell hyperplasia was observed in all (+) of the 5 females in the high dose group, and there was a significant difference as compared to the control group. Submucosal cellular infiltration of inflammatory cells was observed in 2 females (+) in the high dose group. Submucosal fibrosis was observed in all females (+ to ++), and there was a significant difference in the high dose group.

[Limiting ridge of the stomach]
Squamous cell hyperplasia was observed in 4 females (+) in the high dose group, and there was a significant difference as compared to the control group. [Pancreaticosplenic lymph node, at which gross findings were made at necropsy in 4 females in the high dose group] Reactive hyperplasia of the follicles was observed in 4 females (+), fibrosis in 3 females (+) and sinus dilatation in 1 female (+).
[Other organs and tissues]
There were no findings related to the test substance administration or those with increasing incidence.

HAEMATOLOGY
Hematological findings
Tables 27 to 30 show the hematological findings.
The end of administration period

Males: There were no significant changes in the low dose group as compared to the control group.
In the middle dose group, platelet count significantly increased, which was not dose-dependent.
In the high dose group, white blood cell count significantly increased. In the differential counts of white blood cells, stab form and segmented neutrophils significantly increased and lymphocytes significantly decreased.

Females: There were no significant changes in the low dose group as compared to the control group.
In the middle dose group, red blood cell count and hemoglobin concentration significantly decreased, which were not dose-dependent. In the high does group, there were no significant changes.

The end of recovery period

Males: In the high dose group, red blood cell count significantly decreased as compared to the control group, which was not observed at the end of administration.

Females: There were no significant changes in the high dose group as compared to the control group.

BIOCHEMICAL FINDINGS:
Tables 31 to 34 show the biochemical findings.

The end of administration period
Males: In the low dose group, there were no significant changes as compared to the control group. In the middle dose group, total cholesterol significantly increased, which was not dose-dependent.

In the high dose group, γ-GTP and β-globulin fraction significantly increased. Females: In the low dose group, there were no significant changes as compared to the control group.

In the middle dose group, calcium significantly increased, which was not dose-dependent. In the high dose group, albumin and A/G ratio significantly decreased, and potassium and β-globulin fraction significantly increased.

The end of recovery period

Males: In the high dose group, there were no significant changes as compared to the control group.

Females: In the high dose group, γ-GTP significantly increased as compared to the control group, and total bilirubin significantly decreased. These were not observed at the end of administration.

Effect levels (P0)

Dose descriptor:

NOEL

Remarks:

Reproductive and development toxicity

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

not examined

Details on results (F1)

CLINICAL SIGNS (OFFSPRING)
Pups found dead at the observation at the end of parturition were as follows; 2 males and 1 female in the control group, 2 females in the low dose group, 2 males and 2 females in the middle dose group, and 1 male and 1 female in the high dose group.

Pups found dead and missing up to day 4 of lactation were as follows: 1 female in the control group, 5 males and 2 females in the low dose group, 3 females in the middle dose group, and 2 females in the high dose group.

Milk-band negative live and dead pups were sporadically observed in all treatment groups and there were no other abnormalities.

BODY WEIGHT (OFFSPRING)
There were no significant differences in males or females in any treatment group as compared to the control group.

SEXUAL MATURATION (OFFSPRING)
Not applicable

ORGAN WEIGHTS (OFFSPRING)
Not applicable

GROSS PATHOLOGY (OFFSPRING)

HISTOPATHOLOGY (OFFSPRING)
Not exammined for offspring

OTHER FINDINGS (OFFSPRING)
NECROPSY:
In the dead pups, dilatation of the renal pelvis in the kidney was observed in 2 males in the low dose group, and there were no other noticeable abnormal findings in males or females in any treatment group.

In the surviving pups on day 4 of lactation, yellowish brown discoloration of the liver was observed in 1 female in the control group, dilatation of the renal pelvis of the kidney in 2 females in the low dose group, and black mass in the liver of 1 female in the high dose group. There were no other abnormal findings in males or females in any treatment group.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No change related to the test substance administration in the examination of viability index of pups, general clinical observation, body weight changes or necropsy findings in pups

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

To investigate health effects of 2-(2-Vinyloxyethoxy) ethyl acrylate, combined repeated dose toxicity with reproduction/developmental toxicity study was performed in compliance with the OECD Guideline for Testing of Chemicals, No. 422. 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 groups were not used for mating.

In general clinical observation, salivation was sporadically observed in the middle dose (140 mg/kg) and high dose (400 mg/kg) groups. These were observed also in detailed clinical observation, and there were no statistically significant differences. It was considered that the salivation was related to local irritation at the application site of the test substance because this disappeared after withdrawal and was not observed in the recovery period, and was observed also in the 28-day repeated dose toxicity study1). In one male in the middle dose group, hematuria was observed sporadically, which was considered incidental because this was not dose-dependent, and no changes were observed in the kidney at necropsy. In the functional observational battery, no changes related to the test substance administration were observed. In body weight changes, there were no significant changes in males or females during the administration period. In the high dose group, percent body weight gain significantly decreased in males during the administration period, and body weight gain and percent body weight gain significantly increased during the recovery period. These changes were considered to be indicative of recovery following withdrawal. In females in the recovery groups, percent body weight gain significantly increased during the administration period. Considering that there were no changes in body weight changes in males or females, and the significant differences observed in males and females were in the opposite direction, these changes were determined not toxicologically important.

Food consumption decreased significantly in females in the high dose group on days 1 and 3 of gestation, and increased in females in the high dose group on day 7 of recovery. These changes were not considered toxicologically important because significant differences were observed transiently.

In urinary findings, there were no changes related to the test substance administration.

In hematological findings, significant increases were observed in white blood cell count, stab form and segmented neutrophils in the differential counts of white blood cells in males in the high dose group. These changes were considered accompanying inflammatory changes in the forestomach, etc. in the necropsy and histopathological examination.

In biochemical findings, significant increases were observed in γ-GTP and β-globulin fraction in males, significant decreases in albumin and A/G ratio, and significant increases in potassium and β-globulin fraction in females in the high dose group. The changes in the protein fractions were considered accompanying inflammatory changes in the forestomach, etc. The significant increases in γ-GTP and potassium were not considered toxicologically important because the former did not accompany any hepatobiliary changes, and as for the latter, there were no changes in the parameters related to the renal function (urea nitrogen, creatinine), and because there were no histopathological changes in the liver or kidneys.

In necropsy findings, thickening of the limiting ridge of the stomach and focal thickening of the forestomach mucosa were observed in males and females in the low, middle and high dose groups, which were considered related to the test substance administration. Changes including the thickening of the limiting ridge of the stomach were observed at the doses of 160 mg/kg and above in the 28-day repeated dose study1), and it was considered that these occurred at the low dose group in this study because of, longer administration period. These changes were histopathologically squamous cell hyperplasia accompanied by ulcer and cellular infiltration of inflammatory cells. The areas involved were limited to the stomach and surrounding tissues (pancreaticosplenic lymph node), and the changes related to the test substance administration were limited to the application site (local irritation), and no other toxicity was observed.

In organ weights, significant decreases were observed in the absolute and relative thymus weights in males in the high dose group. These changes were considered stress-related or wasting changes accompanying inflammatory changes in the forestomach, etc.

Histopathological findings included squamous cell hyperplasia, etc. at the limiting ridge of the stomach and forestomach, which were irritating changes caused by the test substance, and there were no other changes related to the test substance administration. In spermatogenesis in the testis, there were no changes related to the test substance administration.

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 of 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.

Reproductive and developmental toxicity

There were no changes related to the test substance administration in the examination of oestrous cycle in any treatment group in the main test and recovery test. As for the 3 females in the low dose group and 1 female in each of the middle and high dose groups, which were determined to be persistent anestrus and their length of oestrous cycle was not calculated, dose-dependency and relation to the test substance administration were not observed.

There were no changes related to the test substance administration in the examination of reproduction performance, pregnancy, parturition, nursery condition, viability index of pups, general clinical observation, body weight changes or necropsy findings in pups.

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 on the reproduction performance of the parental animals and on the development of pups were estimated to be 400 mg/kg/day under the conditions of this study because no changes related to the test substance administration were observed in the 400 mg/kg/day group.

Applicant's summary and conclusion

Conclusions:

There were no changes related to the test substance administration in the examination of reproduction performance, pregnancy, parturition, nursery condition, viability index of pups, general clinical observation, body weight changes or necropsy findings in pups.

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 on the reproduction performance of the parental animals and on the development of pups were estimated to be 400 mg/kg/day under the conditions of this study because no changes related to the test substance administration were observed in the 400 mg/kg/day group.

Executive summary:

To investigate health effects of 2-(2-Vinyloxyethoxy) ethyl acrylate, combined repeated dose toxicity with reproduction/developmental toxicity study was performed in compliance with the OECD Guideline for Testing of Chemicals, No. 422. 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:

1. Repeated 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.

2. Reproductive and developmental toxicity

There were no changes related to the test substance administration in the examination of oestrous cycle, examination of reproduction performance, gestation, parturition and nursery condition, viability index of pups, general clinical observation, body weight changes or necropsy findings in pups. 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 on the reproduction performance of the parental animals and on the development of pups were estimated to be 400 mg/kg/day under the conditions of this study.