Phenol, ethoxylated, esters with acrylic acid

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

NA

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See attached testing report. None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

See attached testing report. None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: • EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 2000.

Deviations:

yes

Remarks:

See attached testing report. None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

NA

Test material

Test material information

Constituent 1

Reference substance name:

Phenol, ethoxylated, esters with acrylic acid

EC Number:

500-133-9

EC Name:

Phenol, ethoxylated, esters with acrylic acid

Cas Number:

56641-05-5

Molecular formula:

C13H16O4 (PHEA-2) - C23H36O9 (PHEA-7)

IUPAC Name:

2-phenoxyethyl prop-2-enoate

Test material form:

liquid

Specific details on test material used for the study:

Identification: Phenol, ethoxylated, esters with acrylic acid
Batch (Lot) Number: 190405H27
Expiry date: 07 April 2020 (expiry date)
Physical Description: Clear colourless liquid (determined by Charles River Den Bosch)
Storage Conditions: At room temperature
Test Facility Test Item Number: 210501/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Specific gravity / density: 1.11 g/cm3 at 25°C
Stability at higher temperatures: Stable, maximum temperature: 40°C
Chemical name (IUPAC, synonym or trade name): 2-phenoxyethyl prop-2-enoate
CAS number: 56641-05-5
EC number: 500-133-9
Molecular weight: ≥236 - ≤456 g/mol
Stability in vehicle: Corn oil : Stability for at least 24 hours at room temperature under normal laboratory light conditions and 8 days in a refrigerator, is confirmed over the concentration range 1 to 200 mg/mL, Test Facility Study No. 20212880

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar rat strain (Crl:WI(Han) rats) from Charles River Deutschland, Sulzfeld, Germany.

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
On 13 Nov 2019, female Crl: WI(Han) rats were received and on 27 Nov 2019, male Crl:WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. At initiation of dosing, males were 10-11 weeks old and weighed between 296 and 337 g and females were 13-14 weeks old and weighed between 196 and 252 g. A health inspection was performed before the initiation of dosing.

A total of 40 females was selected at randomization before initiation of the pretest phase. A total of 40 females with regular estrous cycles continued in the study. The supernumerary females were removed from the study, and their estrous cycle results were kept in the raw data but not reported. Animals were randomly assigned to groups at arrival. Males and females were randomized separately.

The animals were allowed to acclimate to the Test Facility toxicology accommodation for 7 days prior to start of the pretest period (females) or 7 days before the commencement of dosing (males).

On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).

During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.

During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cageenrichment,bedding material, food and water. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The rooms in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.

Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.

Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours. Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility.

ENVIRONMENTAL CONDITIONS
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 20-22°C with an actual daily mean relative humidity of 32 to 53%. A 12-hour light/12-hour dark cycle was maintained, except when interrupted for designated procedures. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Corn oil. Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure. Trial preparation formulations were not used for dosing and were discarded after the assessment was complete. These trial preparations have a non-GLP status and were carried out in the quality assured environment of the Test Facility.Stability for at least 6 hours at room temperature under normal laboratory light conditions is confirmed over the concentration range 1 to 200 mg/mL, Test Facility Study No. 20212880.

- Concentration in vehicle: 0, 20, 60, 200 mg/mL

The oral route of administration was selected in order to optimize the potential occurrence of systemic toxicity for the purpose of regulatory hazard assessment.
The dose levels were selected based on the results of a 10-day Dose Range Finder with oral administration of Phenol, ethoxylated, esters with acrylic acid in rats (Test Facility Reference No. 2021288), and in an attempt to produce graded responses to the test item.

Details on mating procedure:

After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.

A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis. All samples to be analyzed were transferred (at room temperature under normal laboratory light conditions) to the analytical laboratory at the Test Facility.

Analyses were performed using a validated analytical procedure (Test Facility Study No. 20212880).

Duplicate sets of samples (approximately 500 mg accurately weighed) were sent to the analytical laboratory. Concentration results were considered acceptable
if mean sample concentration results were within or equal to ± 10% for suspensions of target concentration.

Duplicate sets of samples (approximately 500 mg accurately weighed) were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.

Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No.20212880) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No 20212880.

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Males were treated for 33 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50 to 63 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 43 to 55 days.

Frequency of treatment:

The test item and vehicle were administered to the appropriate animals by once daily oral avage 7 days a week for a minimum of 28 days.

Details on study schedule:

After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.

A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.

Detection of mating was not confirmed in first instance for female no. 45. Evidence of mating was obtained by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continuation of di-estrus during the mating in this female. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle (corn oil)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 male/10 female per dose

Control animals:

yes, concurrent vehicle

Details on study design:

A total of 40 females was selected at randomization before initiation of the pretest phase. Any selected female classified as not having regular estrous cycles during the pretest phase was replaced before initiation of dosing by one of the 8 additional females having regular estrous cycles, if feasible. A total of 40 females with regular estrous cycles continued in the study. The supernumerary females were removed from the study, and their estrous cycle results were kept in the raw data but not reported. Animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy. During the dosing period, these observations were performed directly after dosing based on the results of the dose range finder

BODY WEIGHT: Yes
- Time schedule for examinations:
Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was expected or noted at visual inspection of the water bottles.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.

Sperm parameters (parental animals):

Parameters examined included testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology (see attached study report).

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- eight pups from each litter of equal sex distribution were selected.; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups. Particular attention was paid to the external reproductive genitals (see attachedd study report)

GROSS EXAMINATION OF DEAD PUPS:
- yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
Scheduled necropsies were conducted on the following days:
Males: Following completion of the mating period (a minimum of 28 days of administration).
Females which delivered: PND 14-16.
Females which failed to deliver: With evidence of mating: Post-coitum Day 25 (nos.65).
Without evidence of mating: 26 days after the last day of the mating period (no. 51).

All males were fasted overnight with a maximum of 24 hours before necropsy. Water was available. F0-females were not fasted.

All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were
stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

GROSS NECROPSY
- Full Gross necropsy was performed (See attached study report)

HISTOPATHOLOGY / ORGAN WEIGHTS
- A full histopathological evaluation was performed and organ weights collected in accordance to OECD 422 testing guideline requirements (see attached study report). Representative samples of tissues identified were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated. Tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. All tissues as defined under Histology – F0-Generation (section 4.12.6) were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. Target tissues identified by the study pathologist during microscopic evaluation were communicated to the Study Director; tissues were evaluated and reported. For the testes of all selected males of Groups 1 and 4 and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. A peer review on the histopathology data was performed by a second pathologist.

Organs were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in extremis. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated.

Postmortem examinations (offspring):

SACRIFICE
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 7-16 PND 14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%). The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. If possible, the pups selected for (complete) blood sampling were the same pups as selected for thyroid preservation.

GROSS NECROPSY
- Gross necropsy consisted of xternal and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
A full histopathological evaluation was perfomed and oragn weights noted in accordance to requirements in OECD 422 testing guideline (see attached study report).

Statistics:

Statistical analysis:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistivcal model.

Non-Parametric:
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).

Incidence:
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

Mating index (%); Precoital time; Fertility index (%); Gestation index (%); Duration of gestation;

Offspring viability indices:

Post-implantation survival index (%); Live birth index (%); Percentage live males/females at First Litter Check (%), Viability index (%); Lactation index (%);

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs of toxicity were noted during the observation period, and no test item-related clinical signs were noted during weekly arena observations.
Salivation seen after dosing among all animals at 1000 mg/kg/day and more incidentally across the other dose groups was considered to be a physiological response related to taste and/or irritation of the test item rather than a sign of systemic toxicity, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.

No clinical signs were noted for control group males and for females at 100 mg/kg/day.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item.
One female at 100 mg/kg/day (No. 57) died during the blood sampling procedure on the scheduled day of necropsy. This death was considered related to the blood sampling procedure and not related to treatment with the test item. This animal showed no clinical signs and had normal body weight gain and food intake throughout treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, a slight but statistically significantly lower weight gain was recorded for males on Days 1, 8 and 15 of the mating period. Mean body weight gain for these males at the end of the treatment period was 0.77x of control. Mean absolute body weights were however not statistically significantly different to control means and did not show a dose-related trend. It was therefore considered doubtful if these weight gain changes represented an actual test item-related effect or were secondary to the marginally higher mean body weights on Day 1 of treatment.
Body weight and body weight gain of females at 1000 mg/kg/day, and of both males and females at 100 and 300 mg/kg/day was considered not affected by treatment with the test item.

Any other statistically significant changes in body weight gain were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment. These variations consisted of statistically significantly lower mean body weight gain of females at 100 and 1000 mg/kg/day on Day 8 of the premating period and higher mean body weight gain for females at 300 mg/kg/day on Day 4 of the lactation period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight was considered not affected by treatment with the test item.
For one female at 1000 mg/kg/day (No. 75) a lower food intake was recorded during the lactation period. This female also had a small litter containing only three pups. Since other animals of this dose group showed a normal food intake, this was considered not to be related to treatment with the test item.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The following (statistically significant) changes in hematological parameters were recorded. Relative differences in mean values as compared to the control group are indicated between parentheses.

• Higher mean corpuscular volume (MCV) for males at 300 and 1000 mg/kg/day (1.03 and 1.04x of control, respectively; within historical control range), and for females at 1000 mg/kg/day (not statistically significant; 1.05x of control; slightly above the historical control range).

• Higher Mean Corpuscular Haemoglobin (MCH) for females at 1000 mg/kg/day (1.05x of control; within historical control range).
Hematological parameters at 100 mg/kg/day were considered not affected by treatment.

Any other statistically significant changes in hematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, a statistically significantly lower total bilirubin was recorded for males (0.75x of control). A clear dose-related trend was absent, but the mean was slightly below the historical control range.

Other clinical biochemistry parameters were considered not affected by treatment with the test item. All other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.

Thyroid hormone analyses:
Serum levels of T4 in F0-males were considered unaffected by treatment with the test item.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional observation parameters were considered not affected by treatment with the test item.

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals.
Motor activity (total movements and ambulations) of females at 1000 mg/kg/day in the repeated/additional measurement appeared lower compared to the concurrent control mean (not statistically significant). This apparent difference was not recorded during the initial motor activity measurements. Also, one control female (No. 44) had a high motor activity (exceeding the historical control range ), resulting in a higher mean motor activity for this control group. Additionally, individual motor activity values of females at 1000 mg/kg/day were in the same range as for individual control animals (except for female No. 44).

Therefore, it was considered that there were no effects on motor activity that could be related to treatment with the test item.

All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item.

Most females had regular cycles of 4 days. An irregular cycle was recorded for two control females (Nos. 42 and 45) in the premating period (No. 42 had a normal litter and No. 45 was not pregnant). As these incidences occurred in the control group, they were not related to treatment with the test item.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were 1/10 couples (male No. 05 and female No. 45) of the control group and 1/10 couples (male No. 40 and female No. 80) of the 1000 mg/kg/day group with no offspring. A marked cyst in the cervix, corresponding with fluid in the cervix noted at necropsy, was considered to have attributed to the infertility of control female No. 45. This female also had an irregular estrous cycle (see 9.3.1). For male No. 40 and female No. 80the remaining animals, no abnormalities were seen in the reproductive organs, which could account for their lack of offspring.

Fertility index was not affected by treatment with the test item.
All mated females were pregnant, except for one control female (No. 45). Since this incidence occurred in the control group, this was not related to treatment with the test item.

At 1000 mg/kg/day, the mean number of implantation sites appeared slightly lower than the control mean (10.9 vs 12.1 in the control group; not statistically significant). This was essentially attributed to one female (No. 80, without offspring) that had 2 implantation sites only(without offspring), and to one female (No. 75) that had 3 pups6 implantation sites (with 3 pups) only. The mean number of implantation sites remained within the historical control range .
Number of implantation sites at 100 and 300 mg/kg/day was considered not to be affected by treatment with the test item.

Precoital time was considered not to be affected by treatment. Most females showed evidence of mating within the first 4 days after cohabitation. One female (No. 52 at 100 mg/kg/day) showed evidence of mating at 14 days after cohabitation. This incidental case of a longer precoital time did not show a dose-related trend and was therefore considered not to be related to treatment with the test item.

Duration of gestation was considered not to be affected by treatment. The number of females with living pups on lactation Day 1 compared to the number of pregnant females was decreased at 1000 mg/kg/day. The gestation indices were 100% for the control, 100 and 300 mg/kg/day groups, and 80% for the 1000 mg/kg/day group. The lower gestation index in the high dose group was caused by the failed deliveries of Female Nos. 72 and 74.

Details on results (P0)

For developmental effects the following was concluded:

- At 1000 mg/kg/day, mean gestation length was considered increased compared to the control group (21.8 days vs. 21.1 days in the control group. Individual gestation length showed a trend towards an increase over the dose groups. In addition, the number of females with living pups on Day 1 compared to the number of pregnant females (gestation index) at 1000 mg/kg/day was lower than the control mean at 90% vs 100% in the control group. This was attributed to one female at 1000 mg/kg/day (No. 80) which had two implantation sites (without offspring). It could not be excluded that this incidence was related to treatment with the test item taking into consideration the other developmental effects recorded in this study. Gestation indices were 100% for the control group and for 100 and 300 mg/kg/day groups.

- No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

- At 1000 mg/kg/day, the post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was lower than the control group (78% vs 91% in the control group, below the historical control range). Post-implantation survival index at 100 and 300 mg/kg/day (91 and 90%, respectively) was considered not affected by treatment with the test item.

- At 1000 mg/kg/day, mean litter size was lower when compared to the control mean (9.0 vs 11.0 pups per litter in the control group; not statistically significant, and within the range of historical control data ). This was essentially attributed to one female (No. 75) that had 3 pups only. Mean litter size without this female was 9.8 pups per litter. Litter size at 100 and 300 mg/kg/day was considered not affected by treatment with the test item; live litter sizes were 11.7 and 12.1 living pups/litter, respectively.

- At 1000 mg/kg/day, Llive birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was slightly lower than the control mean (95% vs. 100% in the control group). Four pups at 1000 mg/kg/day were found dead at first litter check (two pups from Litter No. 72 (out of 10 pups), and one pup each from Litter Nos. 75 and 79 (out of 4 and 13 pups, respectively). It could not be excluded that these deaths were related to treatment with the test item as no deaths occurred in the other groups and taking into consideration the other developmental effects recorded in this study. Live birth indices at 100 and 300 mg/kg/day were 100%.

- Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment with the test item. Viability indices were 100% for the 100, 300 and 1000 mg/kg/day groups, and 98% for the control group. Two pups of the control group (both in Litter No. 50) were found missing on PND 2 and were most likely cannibalised. As these incidences were recorded in the control group, these were not related to treatment with the test item.

- Lactation index (the number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not affected by treatment with the test item. No pups were found dead/missing between lactation Days 5 and 13, resulting in a lactation index of 100% for all groups.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on the higher gestation length, lower gestation index and lower mean number of implantation sites at 1000 mg/kg/day

Target system / organ toxicity (P0)

Critical effects observed:

no

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:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment with the test item.
For Female No. 75 at 1000 mg/kg/day, three out of four pups that survived beyond PND 1 appeared dehydrated during most of the lactation period (for two of these pups absence of milk in the stomach was recorded at first litter check). This female also showed a reduced food intake during lactation (see 9.2.4). Since these findings were absent among other pups of the same dose group, this was considered to be incidental and not related to treatment with the test item.
The nature and incidence of other clinical signs remained within the range considered normal for pups of this age and were therefore also considered not to be related to treatment with the test item.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, Llive birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was slightly lower than the control mean (95% vs. 100% in the control group). Four pups at 1000 mg/kg/day were found dead at first litter check (two pups from Litter No. 72 (out of 10 pups), and one pup each from Litter Nos. 75 and 79 (out of 4 and 13 pups, respectively). It could not be excluded that these deaths were related to treatment with the test item as no deaths occurred in the other groups and taking into consideration the other developmental effects recorded in this study. Live birth indices at 100 and 300 mg/kg/day were 100%

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment with the test item. Viability indices were 100% for the 100, 300 and 1000 mg/kg/day groups, and 98% for the control group. Two pups of the control group (both in Litter No. 50) were found missing on PND 2 and were most likely cannibalised. As these incidences were recorded in the control group, these were not related to treatment with the test item.

Lactation index (the number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not affected by treatment with the test item. No pups were found dead/missing between lactation Days 5 and 13, resulting in a lactation index of 100% for all groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, mean pup body weights were marginally lower on PND 13 (0.95x and 0.94x of control for male and female pups, respectively; not statistically significant, means remained within the range of historical control data. Body weights of pups at 100 and 300 mg/kg/day were considered not affected by treatment with the test item.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Total T4 levels in male and female PND 14-16 pups were considered not affected by treatment with the test item. The statistically significantly lower total T4 in males at 100 mg/kg/day occurred in the absence of a dose-related trend and was therefore considered not to be related to treatment with the test item.

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not affected by treatment with the test item.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Areola/nipple retention was not affected by treatment with the test item. No nipples were observed at PND 13 for any of the examined male pups.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment with the test item.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age and were therefore considered not to be related to treatment.

Histopathological findings:

no effects observed

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

For developmental effects the following was concluded:

- At 1000 mg/kg/day, mean gestation length was considered increased compared to the control group (21.8 days vs. 21.1 days in the control group. Individual gestation length showed a trend towards an increase over the dose groups. In addition, the number of females with living pups on Day 1 compared to the number of pregnant females (gestation index) at 1000 mg/kg/day was lower than the control mean at 90% vs 100% in the control group. This was attributed to one female at 1000 mg/kg/day (No. 80) which had two implantation sites (without offspring). It could not be excluded that this incidence was related to treatment with the test item taking into consideration the other developmental effects recorded in this study. Gestation indices were 100% for the control group and for 100 and 300 mg/kg/day groups.

- No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

- At 1000 mg/kg/day, the post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was lower than the control group (78% vs 91% in the control group, below the historical control range). Post-implantation survival index at 100 and 300 mg/kg/day (91 and 90%, respectively) was considered not affected by treatment with the test item.

- At 1000 mg/kg/day, mean litter size was lower when compared to the control mean (9.0 vs 11.0 pups per litter in the control group; not statistically significant, and within the range of historical control data ). This was essentially attributed to one female (No. 75) that had 3 pups only. Mean litter size without this female was 9.8 pups per litter. Litter size at 100 and 300 mg/kg/day was considered not affected by treatment with the test item; live litter sizes were 11.7 and 12.1 living pups/litter, respectively.

- At 1000 mg/kg/day, Llive birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was slightly lower than the control mean (95% vs. 100% in the control group). Four pups at 1000 mg/kg/day were found dead at first litter check (two pups from Litter No. 72 (out of 10 pups), and one pup each from Litter Nos. 75 and 79 (out of 4 and 13 pups, respectively). It could not be excluded that these deaths were related to treatment with the test item as no deaths occurred in the other groups and taking into consideration the other developmental effects recorded in this study. Live birth indices at 100 and 300 mg/kg/day were 100%.

- Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment with the test item. Viability indices were 100% for the 100, 300 and 1000 mg/kg/day groups, and 98% for the control group. Two pups of the control group (both in Litter No. 50) were found missing on PND 2 and were most likely cannibalised. As these incidences were recorded in the control group, these were not related to treatment with the test item.

- Lactation index (the number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not affected by treatment with the test item. No pups were found dead/missing between lactation Days 5 and 13, resulting in a lactation index of 100% for all groups

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on lower post-implantation survival index, lower mean litter size and lower live birth index at 1000 mg/kg/day

Target system / organ toxicity (F1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

System:

female reproductive system

Organ:

other: based on lower post-implantation survival index, lower mean litter size and lower live birth index at 1000 mg/kg/day

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

Treatment related:

yes

Applicant's summary and conclusion

Conclusions:

Wistar Han rats were treated with Phenol, ethoxylated, esters with acrylic acid by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg/day in accordance with OECD 422. Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the no-observed-adverse-effect levels (NOAEL) for reproduction was evaluated to be 300 mg/kg/day (based on lower mean number of implantation sites at 1000 mg/kg/day). For developmental toxicity, A NOAEL of 300 mg/kg/day was established based on the higher gestation length, lower gestation index, lower post-implantation survival index, lower mean litter size and lower live birth index at 1000 mg/kg/day.

Executive summary:

Wistar Han rats were treated with Phenol, ethoxylated, esters with acrylic acid by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg/day. The rats of the control group received the vehicle, corn oil, alone. 

Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 33 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, duringpost-coitum, and at least 13-15 days of lactation (for 50 to 63 days). Females that failed to deliver pups were treated for 43 to 55 days.

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

Parental results

Adverse parental changes were confined to histopathological lesions in the forestomach (i.e.non-glandular part of the stomach)of most animals at 1000 mg/kg/day and consisted ofsquamous cell hyperplasia(up to marked)andhyperkeratosis(up to moderate).This was accompanied by a subepidermal lymphogranulocyticinfiltrate(up to slight) and mucosal and submucosaledema in males. In some cases, the mucosa additionally showed focal or multifocal erosions.Squamous cell hyperplasia with hyperkeratosiswas supported macroscopically by irregular surface of the forestomach.Based on the high severities of the hyperplasia and hyperkeratosis of the forestomach epithelium including erosions and involvement of the submucosa (edema), the combination of these findings was regarded to be adverse in nature. These forestomach lesions recorded at a 20% test item concentration in corn oil were regarded to be a local response to the gavage administration of the test item. The non-glandular stomach is a rat specific entity, which is not present in humans and most non-rodent species.

 

At 300 mg/kg/day (a 6% test item concentration in corn oil),non-adverse forestomach lesions consisted ofsquamous cell hyperplasia(minimal) withhyperkeratosis(minimal) and subepidermallymphogranulocytic infiltrate(up to slight), severities of which were much lower than recorded at 1000 mg/kg/day. Furthermore, there were no erosions and edema. Therefore, this combination of forestomach findings (squamous cellhyperplasia with hyperkeratosis andlymphogranulocyticinfiltrate) at 300 mg/kg/day was regarded non-adverse.

 

Other non-adverse histopathological lesions were recorded in the kidneys and urinary bladder. Non-adversehistopathologicalkidney findings consisted of an increased incidence and severity (up to slight) ofhyaline droplet accumulationin males at 300 and 1000 mg/kg/day. The observedhyalinedroplets resemblealpha2µ-globulin, a normal protein present in the kidneys of male rats. This finding is absent in female rats, as well as in humans. There were no other test-item-related findings in the kidneys. Therefore, this increased incidence and severity ofhyaline droplet accumulationat 300 and 1000 mg/kg/day was considered non-adverse(Ref. 3). Non-adverse histopathologicalurinary bladder findings consisted of aminimal degree of hyperplasia of the urothelium(the lining epithelium of the urinary bladder) in two males at 1000 mg/kg/day.Since the hyperplasia was diffuse and present at a low degree (minimal) and not accompanied by any other degenerative or proliferative lesion, this finding was regarded non-adverse.

A non-adverse higher liver weight was recorded for males at 1000 mg/kg/day (19% higher than the control mean for liver to body weight ratio). In the absence of correlating microscopic lesions or adverse clinical pathology changes, this higher liver weight was considered non-adverse.

A non-adverse slightly lower weight gain was recorded for males at 1000 mg/kg/day during the last two weeks of treatment (0.77x of control for mean body weight gain at the end of the treatment period). Mean body weights did not show a dose-related trend. It was therefore considered doubtful if these weight gain changes represented an actual test item-related effect or were secondary to the marginally higher mean body weights on Day 1 of treatment.

Non-adverse minor changes in clinical pathology parameters consisted of higher mean corpuscular volume and/or higher mean corpuscular haemoglobin for males and/or females at 300 and/or 1000 mg/kg/day, and lowertotal bilirubin for males at 1000 mg/kg/day. As these changes were of a minor degree and occurred in the absence of supportive histopathological or clinical pathology changes, these were considered not to represent adverse effects of treatment with the test item. Also, for total bilirubin the opposite effect (i.e. an increase) would be expected to occur in case of liver toxicity.

No treatment-related changes were noted in any of the other parameters investigated in this study (i.e. clinical appearance, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), food consumption, clotting parameters and male T4 thyroid hormone levels).

Reproductive results

At 1000 mg/kg/day, the mean number of implantation sites appeared slightly lower than the control mean at 1000 mg/kg/day (10.9 vs 12.1 in the control group). This was essentially attributed to one female (No. 80, without offspring) that had 2 implantation sites only, and to one female (No. 75) that had 3 pups only.Based on overall reproductive/developmental data, it was considered that these changes may be considered to represent an adverse effect of treatment with the test item (see also Developmental results).

No test item-related changes were noted in any of the other reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Developmental results

At 1000 mg/kg/day, mean gestation length was slightly increased at 21.8 daysvs21.1 days in the control group with a trend towards increased individual gestation lengths recorded across the dose groups. Gestation index (the number of females with living pups on Day 1 compared to the number of pregnant females) at 1000 mg/kg/day was lower than the control mean at 90% vs 100% in the control group. This was attributed to one female at 1000 mg/kg/day which had two implantation sites (without offspring). Also, live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was slightly lower than the control mean (95% vs. 100% in the control group). This was due to four pups at 1000 mg/kg/day that were found dead at first litter check (two pups in one litter and one pup each in two litters. A lower post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was recorded at 1000 mg/kg/day (78% vs 91% in the control group, below the historical control range of 83-98%. In addition, the mean litter size at 1000 mg/kg/day appeared slightly lower (9.0 vs 11.0 in the control group). This was essentially attributed to one female (No. 75) that had 3 pups only. All other animals of this dose group had a litter size that was essentially within the individual control litter size range.

Overall, these developmental findings which were all recorded at the highest dose tested represented generally small changes and/or occurred at low frequencies (with exception of the post-implantation survival index). However, given that these data were obtained in a (limited) screening study setting, a relationship to treatment with the test item could not be excluded. Therefore, within the context of this study, these combined changes may be considered to represent an adverse effect of treatment with the test item.

At 1000 mg/kg/day, mean pup body weights were marginally lower on PND 13 (0.95x and 0.94x of control for male and female pups, respectively. This represented only a minor (statistically non-significant) change and means of which remained well within the range of historical control data. Moreover, there were no adverse changes in other developmental parameters. As such, this minor variation in pup body weights was considered not to represent an adverse effect on pup development.

No test item-related changes were noted in any of the developmental parameters investigated in this study (i.e. viability and lactation indices, parturition, sex ratio, maternal care, and early postnatal pup development consisting of clinical signs, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

The developmental toxicity observed in this study occurred at dose levels which also resulted in parental toxicity (histopathological lesions in the forestomach). No reproduction or developmental toxicity was observed at dose levels which were non-toxic to the parents.

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) of Phenol, ethoxylated, esters with acrylic acid were established:

Reproduction NOAEL: 300 mg/kg/day (based on lower mean number of implantation sites at 1000 mg/kg/day).

Developmental NOAEL: 300 mg/kg/day (based on the higher gestation length, lower gestation index, lower post-implantation survival index, lower mean litter size and lower live birth index at 1000 mg/kg/day).