Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 422 testing:

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. 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.

Conclusions for F0 generation:

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 finding a NOAEL of 100 mg/kg bw/d in parental animals can be concluded.

Fertility/development:

The dose of 300 mg/kg can be considered the NOAEL based on the higher postimplanation losses recorded at 800 mg/kg.

Link to relevant study records
Reference
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
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
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
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.
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
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
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: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.
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'
Clinical signs:
not examined
Reproductive function: oestrous cycle:
not examined
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
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.
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)
Reproductive effects observed:
not specified

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.

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.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch score 1
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

OECD 422 testing:

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.

Conclusions for F0 generation:

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 finding a NOAEL of 100 mg/kg bw/d in parental animals can be concluded.

Fertility/development:

A dose dependent decrease in the mean number of living pups/per litter at day 0 was seen (12.4; 10.0; 9.5; 8.3, respectively), which was below the mean historical control value for the testing laboratory used (mean 11.9). This could partly be explained by the higher post implantation loss seen at 800 mg/kg bw/d but was also due to a high number of dead pups born in the high dose group (18 vs. 2 in the control) even though not statistically significant. However, the number of litters affected was statistically higher than in the control group (6 vs 2 litters).

The dose of 300 mg/kg can be considered the NOAEL based on the higher postimplanation losses recorded at 800 mg/kg.

OECD 414 testing:

The toxicity and adverse effects of 2-phenoxyethyl acrylate on prenatal development toxicity were investigated in accordance with the requirements of OECD 414 test guideline at dose levels 0, 65, 200, and 600 mg/kg bw/day. It should be noted that a higher dose level than 600 mg/kg bw/d could not be used for this study due to the observations in the dose range finding study where severe local irritation in the stomach was observed at 800 mg/kg bw/d.

The administration with 2-Phenoxyethyl Acrylate by oral gavage, did not cause mortality, changes in body-weight and food consumption and caused no adverse clinical signs at any of the doses. Salivation recorded at 600 mg/kg/ bw/day was attributable to the palatability of the test item.

A slightly increased post implantation loss of 12.2% was observed at 600 mg/kg bw/d compared to control group (8.3%). However, the available historical control data (2014-2016), indicated a considerable lower mean incidence of post implantation losses of 3.1%. No adverse effects were observed in the foetuses in relation to the skeletal and visceral examinations.

In conclusion, as the increase in postimplantation loss was not significant at the dose level of 600 mg/kg/ bw/d, a NOAEL for maternel toxicity as well as for fertility/developmental toxicity of 600 mg/kg bw/d can be concluded from the OECD 414 study when assessed on its own.

However, as postimplantation loss has also been seen in the dose-range finding study and in the OECD 422 study at a dose level of 800 mg/kg bw/d, the postimplantation loss at 600 mg/kg bw/d in the OECD 414  study may be considered related to the dosing and thus, a more precautious NOAEL of 200 mg/kg bw/d for increased postimplantation loss is concluded.

 

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 2016 - March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): MIRAMER M140
- Physical state: Colorless liquid
- Analytical purity: 84.46 % ( CAS No.: 48145-04-6; EC No.: 256-360-6)
- Impurities (identity and concentrations): cas no. 61630-25-9; 8.6%; cas no. 122-99-6; 1.6 %
- Lot/batch No.:151014142
- Expiration date of the lot/batch: 31 December 2016
- Storage condition of test material: Under dry conditions, protected from light and at room temperature (20 ± 5 ºC)

Supplier: Miwon
Species:
rat
Strain:
Wistar
Remarks:
Hannover
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Rat, Wistar Hannover from Charles River Laboratories France
- Number: 107 mated females (nulliparous and non-pregnant); 29 males
- Age at study initiation: 11 weeks (Males/Females)
- Weight at study initiation: Males: xxx g Females: 196-310 grams (day 0 post coitum)
- Fasting period before study: No information
- Housing: Cages with standard, granulated, softwood Lignocel S8/15 bedding (supplied by Harlan Laboratories Models, S.L.).
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2014C rat/mouse maintenance diet ad libitum, batches: 121211MA, 122911MB, 042612MA expiry dates: 7 September 2012, 18 September 2012 and 21 January 2013 (supplied by Harlan Laboratories Models, S.L.). Pelleted standard Teklad 2018C rat/mouse maintenance diet batches: 122311MA and 041112MA expiry dates: 24 September 2012 and 6 January 2013 (supplied by Harlan Laboratories Models, S.L.) ad libitum, for lactating females and pups (until day 4postpartum).
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 5 days between arrival and pretest

ENVIRONMENTAL CONDITIONS
- Temperature (°C): air-conditioned with ranges for room temperature 20 -23 °C
- Humidity (%): relative humidity 35-70%
- Air changes (per hr): 15-20 air changes per hour
- Photoperiod (hrs dark / hrs light): a 12-hour fluorescent light/12-hour dark cycle

IN-LIFE DATES: From: To: 24 August 2016 to 04 October 2016
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
REPARATION OF DOSING SOLUTIONS:

Method: Oral, by gastric gavage

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Corn Oil (Sigma-Aldrich, cat.no. C8267), test item soluble in this vehicle and non-toxic in the concentration used.
- Concentration in vehicle:
Group 1: 0 mg/kg,
Group 2: 65 mg/kg
Group 3: 200 mg/kg
Group 4: 600 mg/kg

- Amount of vehicle (if gavage): 4 mL/kg /once daily
- Lot/batch no. (if required): MKBV2080V (expiry Date 05 Sep. 2018) and MKBW9504V (expiry Date 09 September 2018 / 06 October 2021)

- Purity: not specified.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The formulations were considered homogeneous when the coefficient of variation (CV) of
the results is ≤10%. The acceptance limits are 85-115% of the nominal content.
Details on mating procedure:
- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 2:1
- Length of cohabitation: After acclimatization, females were housed with sexually mature males in a cage for a period approximately of 16 hours
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- Verification of same strain and source of both sexes: [yes]
- Proof of pregnancy: [vaginal plug or sperm in vaginal smear] referred to as [day 0] of pregnancy
- Any other deviations from standard protocol:
Duration of treatment / exposure:
The females received the test item from days 6 to 19 post coitum
Frequency of treatment:
Once per day
Duration of test:
At the scheduled necropsy on day 20 post coitum, females were sacrificed by CO2 inhalation and the fetuses removed by caesarean section.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
65 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
-Group 1: 22 (0 mg/kg),
-Group 2: 29 animals, (65 mg/kg),
-Group 3: 29 animals, (200 mg/kg),
-Group 4: 27 animals, (600 mg/kg).
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 S55881Dose Range Finder for Prenatal Developmental Toxicity in Rats), using dose levels of 0, 100, 300 and 800 mg/kg/ bw/day.
Due to severe irritation of the gastric mucosa observed as thick whitish and rugous non-glandularmucosa in one female at 300 and in two females (one not pregnant) at 800 mg/kg/ bw/day, the highest dose level was considered too high for the main study. Furthermore, two mated females from the highest dose group showed abnormal gait, pilo erection,and hunched posture mainly on days 11-13 of pregnancy.
Based on these findings 600 mg/kg bw/day was chosen as the highest dose level for the main study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage signs on remaining days (together with the viability / mortality
Observation twice daily).

- Cage side observations checked in table [yes] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical signs for signs of reaction to treatment and/or
symptoms of ill health during days 6-20 of pregnancy.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, daily from 6 to 20 post coitum

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20 post coitum, females were sacrificed by CO2 inhalation
and the fetuses removed by caesarean section.
- Organs examined: all internal organs with emphasis on the uterus, uterine contents, position of fetuses in the uterus and the number of corpora lutea.

OTHER:
- Food consumption Periods: days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-20 post coitum.
- Body weight Days 0, 3, daily from 6 to 20 post coitum.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: When no implantation sites were evident, the uterus was placed in an aqueous solution of
ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [ half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]
Statistics:
The following comparisons are performed:
Group 1 vs Groups 2, 3 and 4 to analyze food consumption, body weights, clinical signs and reproduction data:
Means and standard deviations of various data were calculated and included in the report.
For continuous data, a parametric analysis was performed when Bartlett's test for variance homogeneity is not significant at the 1% level. Treated groups were compared to control using Williams' test, unless there is evidence against a monotonic dose-response when Dunnett's test was performed instead.
A non-parametric analysis was performed when Bartlett's test is still significant at the 1% level following both logarithmic and square-root transformations. Treated groups were compared to control using Shirley's test, unless there is evidence against a monotonic dose-response when Steel's test was performed instead.
Indices:
NA
Historical control data:
NA
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were recorded. The salivation recorded at 600 mg/kg/day (before, during and after administration) could be attributed to the palatability of the test item.

Some findings as coat hair loss (dorsal or ventral surface, head, forelimbs and hind limbs), encrustation (ear and head) and wound were observed sporadically.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test-item-related findings were recorded in the macroscopic examination at hysterectomy
in the rats treated with the test item.

Some findings were recorded among all groups: dilated uterus horns or containing reddish
fluid/hemorrhagic contents, reddish kidney medulla and alopecia in different body areas.
These findings were considered to be within the range of normal background lesions that may
be seen in rats of this strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There was no indication of a food consumption effect.
There was no effect on body weights.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The statistically significant differences observed at 600 mg/kg/day in pre-implantation losses were devoid of any toxicological relevance due to the fact that the females received the test item from days 6 to 19 post coitum, once the implantation had already occurred. They were considered a consequence of the biological variability and not test-item related. A slightly increased post implantation loss of 12.2% was observed at 600 mg/kg bw/d compared to control group (8.3%). However, the available historical control data (2014-2016), indicated a considerable lower mean incidence of post implantation losses of 3.1%.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
not specified
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Slightly reduced uterus weight and litter weight was observed at 600 mg/kg/day when compared to control group.
The reduction was related to 2 litters (No 94 and 100) with observed values outside the range of control group.
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effect found at the highest dose (600mg/kg bw/day)
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Description (incidence and severity):
No macroscopic findings which could be attributed to the treatment were observed.

0 mg/kg/day
Litter no. 12 and 13: One fetus (no. 9 in both litters) presented protrusion of intestine.

600 mg/kg/day
Litter no. 113: One fetus (no. 2) had thin skin in right side of head.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
At 600 mg/kg/day there was a slightly increased incidence of incompletely ossified cranial centers, sternebrae and sacrocaudal vertebrae compared to concurrent Control. All except cranial centers are just outside of historical control data from previous S.A.U study.

At 200 mg/kg/day there was a slightly increased incidence of incompletely ossified thoracic vertebrae compared to concurrent Control which was outside of historical control data.

At 65 mg/kg/day there was a slightly increased incidence of fused/partially fused maxilla/jugal (precocious ossification) compared to concurrent Control which was outside of historical control data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Some isolated major abnormalities were recorded in a total of four fetuses: Control group
(litters no. 12 and 13, fetuses no. 9), doses 200 mg/kg/day (fetus no. 6 and 12 from litter 68)
and 600 mg/kg/day (no. 5 from litter 103 and fetus no 1 form litter 94). As no dose-effect relationship was observed, it
cannot be considered attributable to the test item.

Some minor visceral abnormalities were observed in all the groups, including Control.
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect found at the highest dose (600mg/kg bw/day)
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Summary of Performance of Mated Females

Group
Dose (mg/kg/day)

1
0

2
65

3
200

4
600

Number of mated females

22

29

29

27

Number of pregnant females

20

21

21

23

The following females were not pregnant:

Group 1: 4 and 7

Group 2: 31, 33, 34, 39, 40, 42, 48 and 58

Group 3: 61, 62, 64, 74, 78, 79, 87 and 89

Group 4: 92, 97, 98 and 109.

Conclusions:
The toxicity and adverse effects of 2-phenoxyethyl acrylate on prenatal development toxicity were investigated in accordance with the requirements of OECD 414 test guideline at dose levels 0, 65, 200, and 600 mg/kg bw/day. It should be noted that a higher dose level than 600 mg/kg bw/d could not be used for this study due to the observations in the dose range finding study where severe local irritation in the stomach was observed at 800 mg/kg bw/d.

The administration with 2-Phenoxyethyl Acrylate by oral gavage, did not cause mortality, changes in body-weight and food consumption and caused no adverse clinical signs at any of the doses. Salivation recorded at 600 mg/kg/ bw/day was attributable to the palatability of the test item. A slightly increased post implantation loss of 12.2% was observed at 600 mg/kg bw/d compared to control group (8.3%). However, the available historical control data (2014-2016), indicated a considerable lower mean incidence of post implantation losses of 3.1%.

In conclusion, as the increase in postimplantation loss was not significant at the dose level of 600 mg/kg/ bw/d, a formal NOAEL for maternel toxicity as well as for fertility/developmental toxicity of 600 mg/kg bw/d can be concluded in this study when assessed on its own.
However, as postimplantation loss has also been seen in the dose-range finding study and in the OECD 422 study at a dose level of 800 mg/kg bw/d the post-implantation loss at 600 mg/kg bw/d in this OECD 414 study imay be considered to be related to the dosing and thus, a more precautious NOAEL of 200 mg/kg bw/d for increased postimplantation loss may be concluded.


Executive summary:

The toxicity and adverse effects of 2-phenoxyethyl acrylate on prenatal development toxicity were investigated in accordance with the requirements of OECD 414 test guideline.

2-phenoxyethyl acrylate was administered by oral gavage to four groups (0, 65, 200, and 600 mg/kg bw/day), each of 22-29 female rats. Treatment started at Day 6 post coitum and ended at Day 19 post coitum. At day 20 post coitum caesarean section and necropsy were performed.

 

The administration with 2-Phenoxyethyl Acrylate by oral gavage, did not cause mortality, changes in body-weight and food consumption and caused no adverse clinical signs at any of the doses. Salivation recorded at 600 mg/kg/ bw/day was attributable to the palatability of the test item. A slightly increased post implantation loss of 12.2% was observed at 600 mg/kg bw/d compared to control group (8.3%). However, the available historical control data (2014-2016), indicated a considerable lower mean incidence of post implantation losses of 3.1%.

There were no test-item related changes in the observations derived from hysterectomy. The skeletal examination of the fetuses showed a slightly increased incidence in incomplete ossification considered a transient stage in fetal development. As this finding is expected to be resolved in time, this finding is not considered adverse. The slightly increased incidence of fused/partially fused maxilla/jugal (precocious ossification) is not considered to be relevant, as it was only recorded at the low dose (65 mg/kg/ bw/day). The external, skeletal and visceral examination of the fetuses showed the presence of major abnormalities in two fetuses (no. 9 from the litters no. 12 and 13) from the Control group, two fetuses (no. 6 and 12 from litter 68) at 200 mg/kg/ bw/day and two fetuses (no. 5 from litter 103 and no. 1 from litter 94) at the dose of 600 mg/kg/ bw/day. Despite the fact that these abnormalities were also observed in some of the groups treated with the test item, the incidence and distribution recorded could be considered not dose-related.

It should be noted that a higher dose level than 600 mg/kg bw/d (with a higher potential for systemic uptake and systemic toxicity) could not be used for this study due to the observations in the dose range finding study where severe local irritation in the stomach was observed at 300 and 800 mg/kg bw/d.

In conclusion, as the increase in postimplantation loss was not significant at the dose level of 600 mg/kg/ bw/d, a formal NOAEL for maternel toxicity as well as for fertility/developmental toxicity of 600 mg/kg bw/d can be concluded in this study when assessed on its own.

However, as postimplantation loss has also been seen in the dose-range finding study and in the OECD 422 study at a dose level of 800 mg/kg bw/d the postimplantation loss at 600 mg/kg bw/d in this OECD 414  study may be considered related to the dosing and thus, a more precautious NOAEL of 200 mg/kg bw/d for increased postimplantation loss may be concluded.

 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
600 mg/kg bw/day
Species:
rat
Quality of whole database:
high quality
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Toxicity to reproduction: other studies

Additional information

Conclusions from the OECD 422 study:

In the OECD 422 study, the most significant finding was a higher percentage of post implantation loss. This was observed in all dose groups (100; 300; 800 mg/kg bw/d), and most severely (24%) in the highest dose group (800 mg/kg bw/d). This finding was also higher than the mean historical control value for the testing laboratory used (mean 8.2%). The number of affected dams were 5, 4, 8, 9 out of 9 total animals (10 in the mid dose) respectively for the control and dosed groups.

A dose dependent decrease in the mean number of living pups/per litter at day 0 was seen (12.4; 10.0; 9.5; 8.3, respectively), which was below the mean historical control value for the testing laboratory used (mean 11.9). This could partly be explained by the higher post implantation loss seen at 800 mg/kg bw/d but was also due to a high number of dead pups born in the high dose group (18 vs. 2 in the control) even though not statistically significant. However, the number of litters affected was statistically higher than in the control group (6 vs 2 litters).

The resulting live birth index was lower at 800 mg/kg bw/d (79%) and below the historical control data (84-99%). The postnatal loss (day 4 p.p.) was not significantly increased compared to control.

The resulting viability index at day 4 postpartum was lower (but not significantly) at 800 mg/kg bw/d than in the control group (78.8% versus 82.1%, respectively). However, the viability index in the control group was at the lowest range observed in historical control data (82.1-99.1%).

Regarding maternal toxicity, 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. This finding was deemed to be of metabolic nature. Further, 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. The remaining macroscopic findings recorded at necropsy could be considered of no toxicological relevance and within the range of normal background lesions that may be seen in animals of this strain.

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

Conclusions from the OECD 414 study:

In the OECD 414 study in rats, a slightly increased post implantation loss of 12.2% was observed at 600 mg/kg bw/d (high dose level group) compared to control group (8.3%). However, the available historical control data (in the period 2014-2016), indicated a considerable lower mean incidence of post implantation losses of 3.1%.

No adverse effects were observed in the foetuses in relation to the skeletal and visceral examinations.

With respect to maternal toxicity the administration with 2-Phenoxyethyl Acrylate by oral gavage, did not cause mortality, changes in body-weight and food consumption and caused no clinical signs at any of the doses other than salivation recorded at 600 mg/kg/ bw/day. 

It should be noted that a higher dose level than 600 mg/kg bw/d could not be used for this study due to the observations in the dose range finding study where severe local irritation in the stomach was observed at 800 mg/kg bw/d.

Justification for classification or non-classification

From the OECD 422 study, a significant increase in post implantation loss was observed at the highest dose of 800 mg/kg/day.

No adverse effects on fertility was observed in the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422) in rats: No treatment-related differences were recorded in the percentage of mating, fertility index or conception in rat. Moreover, no adverse effects on reproductive organs were showed in the 90-day repeated toxicity study (OECD 408) in rats. Based on these results, 2-phenoxyethyl acrylate is considered to not alter the fertility in rats. However, adverse developmental toxicity (increased post implantation loss) related to 2-phenylethyl acrylate was observed in this study (OECD 422) at the highest dose level of 800 mg/kg bw/d.

Due to severe gastric effects observed in the dose range finder study for the OECD 414 study on pregnant females at 800 mg/kg bw/day, a highest dose level of 600 mg/kg bw/d was chosen for the main study. In this OECD 414 study, the highest dose level of 600 mg/kg bw/d induced an increase in post-implantation loss and although not statistically significant, i.e confirming the effects observed in the OECD 422 study.

Based on the reproducible effects observed in two studies in rats, a classification as Repr.2 (H361d): Suspected of damaging the unborn child, is concluded.

An Extended One-Generation Reproductive Toxicity Study (OECD 443) is not needed in the Annex IX of REACH due to the absence of effects on fertility and on reproductive organs in the available data (OECD 422, 408), and due the classification already proposed for the developmental toxicity.