Phenol, ethoxylated, esters with acrylic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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:

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

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

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:

Crl:WI(Han) rats were received 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 6 days prior to start of the pretest period (females) or 6 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

Details on route of administration:

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. 20212881), and in an attempt to produce graded responses to the test.

Vehicle:

corn oil

Details on oral 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 because this is a possible route of human exposure during manufacture, handling or use of the test item. 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 Study No. 20212881), and in an attempt to produce graded responses to the test item

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:

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 gavage 7 days a week for a minimum of 28 days.

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 (0, 100, 300, 1000 mg/kg bw/day)

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

Observations and examinations performed and frequency:

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.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes (Yes, see attached study report)
Blood of F0-animals (except for animals which were sacrificed in extremis or found dead) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. Due to clotting of non-serum samples of individual animals, additional blood samples were obtained in necropsy room. After collection all samples were transferred to the appropriate laboratory for analysis. F0-males were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available. F0 females were not fasted overnight. Blood of F1-animals was collected on PND 4 and PND 14-16, if possible. This was performed in the necropsy room. On PND 4 at culling, blood was collected from two surplus pups per litter (if possible) by decapitation, between 7.00 and 10.30 a.m., and samples were pooled to one sample per litter. If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much blood as possible was collected from this single pup. On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female). Any incomplete blood sample was discarded. Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure.

CLINICAL CHEMISTRY: Yes (see attached study report)
Blood of F0-animals (except for animals which were sacrificed in extremis or found dead) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. Due to clotting of non-serum samples of individual animals, additional blood samples were obtained in necropsy room. After collection all samples were transferred to the appropriate laboratory for analysis. F0-males were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available. F0 females were not fasted overnight. Blood of F1-animals was collected on PND 4 and PND 14-16, if possible. This was performed in the necropsy room. On PND 4 at culling, blood was collected from two surplus pups per litter (if possible) by decapitation, between 7.00 and 10.30 a.m., and samples were pooled to one sample per litter. If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much blood as possible was collected from this single pup. On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female). Any incomplete blood sample was discarded. Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
F0-generation: Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 12-14) These tests were performed after dosing, after completion of clinical observations (including arena observation, if applicable).The following tests were performed:
• Hearing ability (HEARING) (Score 0 = normal/present, score 1 = abnormal/absent).
• Pupillary reflex (PUPIL L/R) (Score 0 = normal/present, score 1 = abnormal/absent).
• Static righting reflex (STATIC R) (Score 0 = normal/present, score 1 =
abnormal/absent).
• Fore- and hind-limb grip strength, recorded as the mean of three measurements per
animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
• Locomotor activity (recording period: 1-hour under normal laboratory light
conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway,
USA). Total movements and ambulations were reported. Ambulations represent
movements characterized by a relocation of the entire body position like walking,
whereas total movements represent all movements made by the animals, including
ambulations but also smaller or more fine movements like grooming, weaving or
movements of the head.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table in attached study report)

HISTOPATHOLOGY: Yes (see table i attached study report)

Other examinations:

Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.

All male pups in each litter were examined for the number of areola/nipples on PND 13.

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.

Results and discussion

Results of examinations

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, 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

Description (incidence and severity):

Test item-related higher liver weights (absolute and relative to body weights) were noted in the 1000 mg/kg/day group males.

The testes weights at 1000 mg/kg/day were statistically significant higher (absolute +9 %; relative to body weight +14%) compared to the weights of the control group. This organ weight change was regarded as unrelated to treatment with the test item. This was based on the direction of the change, the absence of a microscopic correlate, the evidence of mating and since the group mean value remained within historical control data.

Any other differences, including those which reached statistical significance (thyroid gland, kidneys and adrenal gland of females) were also considered not to be related to treatment with the test item due to the direction of the change, lack of dose-related pattern, absence of microscopic correlate and/or general overlap and variability in individual values.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the stomach of both sexes at 300 and 1000 mg/kg/day, in the kidneys of males at 300 and 1000 mg/kg/day and in the urinary bladder of males at 1000 mg/kg/day.

Forestomach: Squamous cell hyperplasia (up to marked) and hyperkeratosis (up to moderate) and/or subepidermal lymphogranulocytic infiltrate (up to slight) were present at 300 and 1000 mg/kg/day in both sexes and additionally erosions (both sexes; up to slight) and/or edema (males; up to slight) were present at 1000 mg/kg/day.

Kidneys: An increased incidence and severity (up to slight) of hyaline droplet accumulation was present at 300 and 1000 mg/kg/day.

Urinary bladder: Hyperplasia of the urothelium was present in 1000 mg/kg/day males (minimal). In general, the hyperplasia was diffuse and not accompanied by underlying inflammation.

The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

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 histopathological forestomach lesions in both sexes at 1000 mg/kg/dad

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

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 of squamous cell hyperplasia (up to marked) and hyperkeratosis (up to moderate).This was accompanied by a subepidermal lymphogranulocyt icinfiltrate (up to slight) and mucosal and submucosaledema in males. In some cases, the mucosa additionally showed focal or multifocal erosions.Squamous cell hyperplasia with hyperkeratosis was 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) with hyperkeratosis (minimal) and subepidermall ymphogranulocytic 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) of hyaline droplet accumulationin males at 300 and 1000 mg/kg/day. The observed hyaline droplets resemble alpha2µ-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 of hyaline droplet accumulationat 300 and 1000 mg/kg/day was considered non-adverse. Non-adverse histopathological urinary bladder findings consisted of a minimal 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 wasconsidered 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).

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 parental toxicity was evaluated to be 300 mg/kg/day based on histopathological forestomach lesions in both sexes at 1000 mg/kg/day. 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).

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 in accordance with OECD 422. Test item was formulated in corn oil and chemical analyses of formulations conducted during the study confirmed accuracy and homogeneity as well as target concentration.

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. 

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 of squamous cell hyperplasia (up to marked) and hyperkeratosis (up to moderate).This was accompanied by a subepidermal lymphogranulocyt icinfiltrate (up to slight) and mucosal and submucosaledema in males. In some cases, the mucosa additionally showed focal or multifocal erosions.Squamous cell hyperplasia with hyperkeratosis was 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) with hyperkeratosis (minimal) and subepidermall ymphogranulocytic 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-adverse histopathological kidney findings consisted of an increased incidence and severity (up to slight) of hyaline droplet accumulationin males at 300 and 1000 mg/kg/day. The observed hyaline droplets resemble alpha2µ-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 of hyaline droplet accumulationat 300 and 1000 mg/kg/day was considered non-adverse. Non-adverse histopathological urinary bladder findings consisted of a minimal 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 wasconsidered 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).

In conclusion, 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 parental toxicity was evaluated to be 300 mg/kg/day based on histopathological forestomach lesions in both sexes at 1000 mg/kg/day. 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).