Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 July 2012 to 4 February 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Remarks:
The body weight range at treatment start in males was 337-372 grams instead of 200- 240 grams as stated in the study plan. This deviation did not affect the study or its results.
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): 2-Phenoxyethyl Acrylate
- Physical state: Liquid
- Purity test date: No data
- Lot/batch No.: 120317147
- Expiration date of the lot/batch: 16 March 2013
- Stability under test conditions: Dose formulations stable at maximum 13 days at room temperature and protected from light
- Storage condition of test material: Under dry conditions, protected from light and at room temperature (20 ± 5 ºC)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Rat, RccHan®:WIST from Harlan Laboratories Models, Harlan Laboratories B.V.
- Age at study initiation: 13 weeks
- Weight at study initiation: Males: 337-372 g; Females: 214-246 g
- Housing: Cages with standard, granulated, softwood Lignocel S8/15 bedding. Premating period (5 animals/cage), Mating period (one male and one female/cage), Postmating (individually)
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): pelleted standard Harlan Teklad 2014C rat/mouse maintenance diet ad libitum, pelleted standard Teklad 2018C rat/mouse
maintenance diet batches ad libitum, for lactating females and pups (until day 4 postpartum).
- Water (e.g. ad libitum): Tap water in bottles ad libitum. Results of bacteriological, chemical, composition and contaminant analyses are archived at Harlan
- Acclimation period: 5 days between arrival and pretest

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

IN-LIFE DATES: From: To: 25 July 2012 to 4 February 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:
Details on mating procedure:
- M/F ratio per cage: One M/One F
- Length of cohabitation: 14 days
- Proof of pregnancy: The daily vaginal smear was sperm-positive, or/and a copulation plug was observed. referred to as day 0 postcoitum
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): housed individually
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In order to prepare the formulations, a stock solution was prepared and used to take the volume required for each day of administration into aliquots. In the case that the formulation was prepared for only one administration day, the same procedure was followed for the preparation of the stock solution.

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

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

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

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

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

The results obtained showed that mean test item concentrations found in the formulations analyzed ranged from 99.7% to 105.3% and was therefore within the acceptance range of 85-115% and that precision was below 3.28 %.
Duration of treatment / exposure:
Males: two weeks prior mating and at up to and including the day before sacrifice (minimum a total of 43 days).
Females: two weeks prior mating and at least up to and including the day before sacrifice (day 4 postpartum).
Frequency of treatment:
Once daily
Details on study schedule:
Pairing of animals within each dose group was undertaken on a one male : one female basis within each treatment group on day 15 of the study, with females subsequently being allowed to litter and rear their offspring up to day 4 of lactation.

During the lactation phase, daily clinical observations were recorded on all surviving offspring, together with litter size and offspring weights; and surface righting reflex was assessed.

Extensive functional observations were performed on five selected males from each dose group the day before sacrifice and for five selected parental females from each dose group on day 4 postpartum.

Adult males were terminated al least on Day 44, adult females on day 5 postpartum and offspring on day 4 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed in five males and five females.
Doses / concentrations
Remarks:
Doses / Concentrations:
Group 1: 0 mg/kg body weight/day (control group); Group 2: 100 mg/kg body weight/day; Group 3: 300 mg/kg body weight/day; Group 4: 800 mg/kg body weight/day
Basis:
actual ingested
The dose volume administered was 4 mL/kg body weight with an adjustment to body weight.
No. of animals per sex per dose:
80 in total (40 males and 40 females):

Group 1 (0 mg/kg): 10 M/10F
Group 2 (100 mg/kg): 10 M/10F
Group 3 (300 mg/kg): 10 M/10F
Group 4 (800 mg/kg): 10 M/10F
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on a dose-range finding toxicity study in rats (Harlan Laboratories Study S39286 14-day Oral Dose-Range-Finding Toxicity Study in the Wistar Rat), using dose levels of 0, 100, 500 and 1000 mg/kg/day.

High dose 800 mg/kg - based on the toxicity observed for animals at 1000 mg/kg in a 14-day range finder where the clinical findings suggest that more prolonged dosing during the main study together with the expected increase in exposure of pregnant females may result in more pronounced toxicity beyond the maximum tolerated dose level. This may well have significant effects upon the ability of the female to maintain any potential offspring post partum.

Mid–dose 300 mg/kg - based on the potential cut off point for GHS classification as “Harmful” using the criteria for a 28-day repeat dose toxicity study.

Low dose 100 mg/kg - ensures that the interval between dose levels is within the limits prescribed within the OECD test guideline number 422 and is also above the cut off point for GHS classification as “toxic” using the criteria for a 28-day repeat dose toxicity study.

- Rationale for animal assignment (if not random): Randomization method based on the similarity of the mean body weights among the groups.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily for mortality and for signs of reaction to treatment and/or symptoms of ill health before dosing and post dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed on all test and control group animals before the first exposure to the test item and once weekly thereafter, at least two hours after dosing. Observations were also performed on females with litters on Day 4 postpartum. These observations were performed outside the home cage, in a standard arena, at least two hours after dosing (where applicable) to ensure that any transient effects of treatment are identified. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: F0 males: twice weekly during the pre-pairing and pairing period and daily during postpairing period. F0 females: twice weekly during the pre-pairing and pairing period and daily until day 5 postpartum.

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

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

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

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

Sensory Reactivity Assessment: Sensory reactivity to different stimuli (e.g. auditory, visual and proprioceptive) was evaluated in the animals mentioned above.
Oestrous cyclicity (parental animals):
Stage of Estrus: All females during mating via vaginal smear. Smearing of individual females was discontinued when sperm are found.

Delivery: From day 21 postcoitum, the females were examined at least twice daily for signs of parturition. The duration of gestation (days) was recorded.

Nursing: The females that gave birth were checked to observe whether they nursed their offspring.
Sperm parameters (parental animals):
F0 male parental generations: A qualitative staging of spermatogenesis and histopathology evaluation of interstitial cells of all males from the control and high-dose groups was performed. Further, the weight of testes was recorded.
Litter observations:
Litter Size and Sexes: The number of live and dead pups, their sex and any macroscopic anomalies were recorded for each litter within 24 hours of parturition (day 0 or 1 postpartum). If parturition ends before 7 am, this day was considered as day 1 postpartum and if parturition ends after 7 am this day was considered as day 0 postpartum. The size of the litters was recorded daily.

Morbidity / Mortality: At least twice daily. Any rat sacrificed or found dead during the study was subjected to macroscopic examination. The sex of pup that was devoured by the mother was assigned at random to male or female.

Clinical Signs: Animals were observed at least once daily.

Body Weight: Days 1 and 4 postpartum

Behavior Test: Day 1 postpartum: surface righting test (righting refelex)
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: At least on day 44 of the study all surviving males were deeply anaesthetized with sodium pentobarbital administered intraperitoneally, exsanguinated and necropsied.
- Maternal animals: On day 5 postpartum, all females were deeply anaesthetized with sodium pentobarbital administered intraperitoneally, exsanguinated and necropsied. The mated females that did not give birth or show signs of pregnancy were sacrificed after 24-26 days postcoitum if no repeat
mating was done.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations. The necropsy included the examination of the external surface of the body, all orifices, cranial, thoracic and abdominal cavities and the observation of the organs both in situ and after evisceration.
Samples of tissues and organs described in the OECD 422 test guideline including reproductive organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution (10% formalin).

HISTOPATHOLOGY / ORGAN WEIGTHS
- The following organs were weighed on the day of necropsy and their organ to terminal body weight ratios and organ to brain weight ratios were determined:
Adrenals *; Kidneys*; Prostate and seminal vesicles; Thyroid and parathyroids *; Brain Liver Spleen Uterus and oviducts; Epididymides*; Ovaries* Testes*; Heart Pituitary Thymus (* Paired organs were weighed together).

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

- Slides of all organs and tissues collected at necropsy from five males and five females from groups 1 and 4 selected for clinical pathology, as well as all gross lesions, were examined. Reproductive organs (tissues shown in bold) from all control and high dose animals were examined for histopathology. Because test-item-related morphological changes were detected in stomach and liver at high dose, these organs of all remaining animals (including group 2 and 3) were examined. A description of all abnormalities was included in the report. Where possible, the microscopic findings were correlated with the gross observations.
A copy of the histopathology report is included in the test report.
Postmortem examinations (offspring):
SACRIFICE
- On day 4 postpartum, all pups were sacrificed by an intraperitoneal injection of sodium pentobarbital and subjected to macroscopic examination. F1 offspring that died during the study was examined externally. Necropsy and a macroscopic examination were carried out for all the other animals.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations. The necropsy included the examination of the external surface of the body, all orifices, cranial, thoracic and abdominal cavities and the observation of the organs both in situ and after evisceration.
Samples of tissues and organs described in the OECD 422 test guideline including reproductive organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution (10% formalin).

HISTOPATHOLOGY / ORGAN WEIGTHS
- The following organs were weighed on the day of necropsy and their organ to terminal body weight ratios and organ to brain weight ratios were determined:
Adrenals *; Kidneys*; Prostate and seminal vesicles; Thyroid and parathyroids *; Brain Liver Spleen Uterus and oviducts; Epididymides*; Ovaries* Testes*; Heart Pituitary Thymus (* Paired organs were weighed together).

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

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

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

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

Fisher's exact-test was applied to the macroscopic findings.

Bonferroni-test was applied to some reproduction parameters.
Reproductive indices:
The following parameters were calculated from the individual data during the mating period of the parental generation:

i) Pre-coital time
ii) Fertility Indices
iii) Implantation Losses (%)

The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.

i) Gestation Length
ii) Gestation Index
Offspring viability indices:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using the individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

Post–natal loss = Number of offspring/Number of offspring alive on Day 4 x 100

The following viabiliyy indices were calculated for each litter as follows:

Viability Index (%) = Number of offspring alive on Day 4/Number of offspring alive on Day 1 x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Salivation was recorded in all test-item-treated groups. This clinical sign increased in frequency and number of occurrences with the dose level. Test-item-related clinical signs such as hunched back and abnormal gait were recorded at 800 mg/kg from first week of treatment. These clinical signs disappeared on week 3 (abnormal gait) and on week 6 (hunched back). In addition, ruffled fur and rales were recorded occasionally in some males at 800 mg/kg. Salivation was recorded occasionally in one male from the control group.

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

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Slight decreased in food consumption was recorded during the prepairing period at 800 mg/kg, but the difference was not statistically significant. No differences with the control group were recorded in the remaining groups.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No treatment-related differences with the control group were recorded in the mean of implantation sites per litter or corpora lutea. Higher mean postimplantation losses were recorded in all test item groups with respect to the control. Consequently, lower mean of live pups were recorded at first litter check.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No indication of toxicity in the testes. Single sperm resorption in stage VIII or IX tubules, atrophic stage II/III tubule, Sertoli’s cell vacuolation or isolated degenerated tubules were recorded in 5/10 males from control group and 1/10 males from high dose. These findings were within the range of normal background lesions which may be recorded in animals of this strain and age.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No treatment-related differences were recorded in mean or median precoital time. No treatment-related differences were recorded in the percentage of mating, gestation index, fertility index or conception rate. An Increase in postimplantation losses was observed in all test item groups, mainly at 800 mg/kg,
consequently the mean of alive pups decreased. No differences in corpora lutea, implantation sites, preimplantion losses nor sex ratio was observed with respect the control group. The length of pregnancy (days) was similar within groups.
One female from control group and one at 800 mg/kg had agalactorrhea.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Increases in liver and kidney weights were recorded in both genders at 800 mg/kg. No noticeable differences were observed in the remaining animals.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Thickened gastric wall was recorded in males and females at 800 mg/kg. All other lesions recorded were considered within the normal range of background alterations observed in rats of this strain and age and under the experimental conditions used in this study.
No treatment-related alterations were recorded in offspring.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Microscopic alterations related to test item were observed in stomach and liver. These alterations consisted in acanthosis, hyperkeratosis and/or inflammatory infiltrate in submucosa and presence of forestomach ulcerations in stomach from animals from all test item groups mainly in groups 3 and 4 and centrilobular hepatocellular hypertrophy with increased incidence of hematopoietic foci was observed in group-4 animals. All other findings recorded were considered to be within the range of normal background lesions that may be seen in rats of this strain and age and under the experimental conditions used in this study.

FUNCTIONAL PERFORMANCE TEST (PARENTAL ANIMALS)
Lower locomotor activity was recorded in males at 800 mg/kg and in females at 100 and 800 mg/kg. No relevant differences from the control group were recorded in the grip strength test in either gender. No differences from control were recorded in the sensory reactivity assessments.

LABORATORY INVESTIGATIONS
Hematology: Red blood cell parameters were affected in males at 800 mg/kg as seen in lower hemoglobin, hematocrit and erythrocyte values. In addition, mature reticulocyte values were affected in both sexes at 800 mg/kg. No relevant differences were observed at 100 or 300 mg/kg in either gender.

Blood Chemistry: Higher chloride levels were recorded in males at all test item groups as well as higher sodium levels were recorded in females at 800 mg/kg.
No relevant differences were observed at 100 and 300 mg/kg.

Urine Evaluation: Urine volume collected was higher and test-item related at 800 mg/kg in both genders. Low relative density was recorded in females from high group.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No effects at the highest dose level (800 mg/kg bw/day) in the male reproductive parameters examined.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
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:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
Higher mean of dead pups was recorded at 800 mg/kg at the first litter check but this was not statistically significant. The resulting viability index was lower than in the control group (78.8% versus 82.1%, respectively). The number of litters affected was statistically higher than in the control group (6 vs 2 litters).
During the first 4 days postpartum, the percentage of postnatal losses was similar in all groups. Females no. 42 (control) and 79 (800 mg/kg) lost the entire litter. No dead pups at first litter check or on days 1-4 postpartum were recorded at 300 mg/kg.

BODY WEIGHT (OFFSPRING)
Mean body weights of pups from the groups treated with the test item at 800 mg/kg were slightly lower than those recorded in the control group. These differences affected both sexes and were not statistically significant. No differences were recorded at 100 and 300 mg/kg. At 100 mg/kg, one runt pup (no. 6 from litter no. 53) was observed. At 800 mg/kg, runt pups were observed in litter no. 75.

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

HISTOPATHOLOGY (OFFSPRING)
No treatment-related alterations were recorded in the morphological examination of the pups. Observations such as hematoma, recorded in all groups, were considered within the range of normal background findings which may be seen in rats of this strain and thus were considered incidental. One male pup from the control group had curly tail at delivery and was devoured within 24 hours of life.

Behavioral tests - Motor development (OFFSPRING)
With respect to observation of postural reflexes in the pups, a statistically lower percentage of fetuses with positive response in the surface-righting reflex at 100 mg/kg was recorded with respect to the control group. There were no treatment-related differences with respect to the control animals at 300 or 800 mg/kg.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
800 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No developmental toxicity at highest tested dose level (800 mg/kg bw/day)

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Mating Performance:

Median precoital time and mean precoital time (ca. 3 days) were similar in animals treated with the test item at 300 and 800 mg/kg with respect to the control group. However, female no. 48 from the control group did not mate with the first male (no. 8) after a pairing period of 14 days and was mated a second time with male no. 6. This female presented an anoestrus cycle during 12 days. Although no differences in median precoital time at 100 mg/kg were recorded with respect to the other groups, mean precoital time was longer (ca. 5.2 days). Female no. 56 did not mate with the first male (no. 16) after a pairing period of 14 days and was mated a second time with male no. 11. This female presented an anoestrus cycle during 11 days. Females no. 52 (first mating) and 53 took nine and thirteen days, respectively, to mate and presented a regular cycle of 4-5 days. Female no. 57 took 14 days in its second mating and presented an anoestrus cycle of 12 days.

Fertility:

No treatment-related differences were recorded in the percentage of mating, the gestation index, fertility index or conception rate in females treated at the three administered doses with respect to the control group. Females no. 49 from the control group, no. 60 at 100 mg/kg and no. 76 at 800 mg/kg, which mated with males 9, 20 and 36, respectively, were not pregnant despite the presence of sperm in the vaginal smear and vaginal plug in the first two females. Mating length of those couples was 1-2 days. To check the fertility of the males whose first pairing did not result in mating, males no. 8 from the control group and no. 16 from the 100-mg/kg group were mated a second time with one female from a reserve group and mating led to pregnancy for male no. 16.

  Dose-levels mg/kg bw/d  0  100  300  800
 Post-implantation loss, no.  7  12  15  27
 % of impl. sites  5.8  11.8  13.6  24.3 *
 mean (impl. sites lost per litter)  0.8  1.3  1.5  3*
 Number of dams affected  5  4  8  9

* Fisher´s exact test, singnificant at 5% level

Reproduction Data:

No treatment-related differences with the control group were recorded in the mean of implantation sites per litter or corpora lutea. Higher mean postimplantation losses were recorded in all test item groups with respect to the control. Consequently, lower mean of live pups were recorded at first litter check. Female no. 73 at 800 mg/kg presented 100% implantation site loss. This animal had blood on vagina on day 22 of pregnancy. No differences in sex ratio were recorded.

Breeding Data:

The length of pregnancy was similar in all groups with a mean of 22 days. Pregnancy lasted 23 days for females no. 45 and 79 from the control- and 800-mg/kg groups, respectively. Female no. 42 from the control group and female no. 79 at 800 mg/kg had agalactorrhea and did not nurse their pups; consequently, the pups died or were devoured between days 0 and 2 of lactation. Dead pups did not have milk in the stomach. Female no. 79 showed enlarged mammary glands, whereas female no. 42 did not. One of the above females, no. 42 from control, also showed maternal neglect and did not properly clean the pups after giving birth. In addition, some pups in the litter females no. 53 at 100 mg/kg and no. 75 and 80 at 800 mg/kg showed no milk in stomach on day 1 postpartum and consequently they died.

Pathology:

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

Histopathology:

Acanthosis in stomach in animals from all groups, with greater incidence and severity in groups 3 and 4, with hyperkeratosis, increased incidence and/or severity of inflammatory infiltrate in submucosa and presence of forestomach ulcerations in animals from groups 3 and 4 were recorded. Minimal centrilobular hepatocellular hypertrophy with increased incidence of hematopoietic foci was observed in group-4 animals. All other findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

Applicant's summary and conclusion

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

No mortality was recorded in either gender. Hunched back, ruffled fur, abnormal gait and decrease in motor activity were recorded in both genders at 800 mg/kg. These clinical signs decreased in frequency and incidence with the number of administrations received. Body weight decreased in males during the prepairing period and slightly in females during pregnancy at 800 mg/kg. Test-item-related higher liver weight was observed in males and females at 800 mg/kg associated to minimal centrilobular hepatocellular hypertrophy with increased incidence of hematopoietic foci. Test-item-related thick gastric wall was recorded at 300 and 800 mg/kg in both genders, related in some animals with reddish foci/focus associated with acanthosis, hyperkeratosis and/or inflammatory infiltrate in submucosa and presence of forestomach ulcerations.

Based on these findings, NOAEL of 100 mg/kg bw/d was found for maternal toxicity in relation to local gastric irritation and ulceration, while a NOAEL of 300 mg/kg bw/d for toxicity to reproduction was found based on an increase in post-implantation loss at 800 mg/kg bw/d.


Executive summary:

The toxicity and adverse effects of 2-phenoxyethyl acrylate on reproduction (including offspring development) were investigated in accordance with the requirements of OECD 422 test guideline. 2-phenoxyethyl acrylate was administered orally by gavage to three groups, each consisting of ten male and ten female RccHan®:WIST rats, for up to eight weeks (including two weeks prior to mating through mating, pregnancy and early lactation for females), at dose levels of 0 mg/kg body weight/day (control group); 100 mg/kg body weight/day; 300 mg/kg body weight/day and 800 mg/kg body weight/day.

In terms of mating and fertility, no treatment-related differences in mean or median precoital time were observed, nor in the percentage of mating, gestation index, fertility index or conception rate.

Further, an increase in postimplantation losses was observed in all test item groups, mainly at 800 mg/kg. No differences in corpora lutea, implantation sites, preimplantion losses nor sex ratio was observed with respect the control group.

In terms of breeding data, the length of pregnancy (days) was similar within groups. One female from control group and one at 800 mg/kg had agalactorrhea.

Treatment with 2-phenoxyethyl acrylate caused no indication of toxicity in the testes including testes weight. For the sperm stage evaluation, the sperm stages were complete in all animals. There was no indicator for induced maturation arrest, increased resorption, necrosis or any lesion in interstitial cells.

Based on these findings, NOAEL of 100 mg/kg bw/d was found for maternal toxicity in relation to local gastric irritation and ulceration, while a NOAEL of 300 mg/kg bw/d for toxicity to reproduction was found based on an increase in post-implantation loss at 800 mg/kg bw/d.