Esterification products of 4,4'-isopropylidenediphenol, ethoxylated and 2-methylprop-2-enoic acid

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

A reproduction screening study (2019, OECD 422) is available on Reaction products of 4,4'-isopropylidenediphenol, ethoxylated and methacrylic acid. In this study, no adverse parental toxicity and no effects on mating or fertility were identified up to 1000 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 June 2018 - 05 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France
- Age: on the first day of treatment, the males were approximately 10 weeks and the females were approximately 11 weeks old
- Mean body weight: on the first day of treatment, the males had a mean body weight of 404 g (range: 386 to 429 g) and the females had a mean body weight of 281 g (range: 262 to 315 g)
- Fasting period before study: no
- Housing: individually
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: males were acclimated to the study conditions for 7 days before treatment and females were acclimated to the study conditions for 5 days before the beginning of estrous cycle monitoring during the pre-treatment period.
- Parental females selected according to their estrous cyclicity checked before initiation of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 03 July 2018 to 05 September 2018.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING FORMULATIONS:
- Solution in the vehicle
- Justification for use and choice of vehicle: suitable formulation in the selected vehicle
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation (mating period): until mating occurred
- Proof of pregnancy: vaginal plug or sperm in the vaginal lavage referred to as Day 0 post-coitum

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: High Performance Liquid Chromatography with UV detection (HPLC/UV)
Test item concentrations: actual concentrations of the test item in the dose formulations analyzed during the study remained within an acceptable range of variation (-2.5% to +2.2%) when compared to the nominal concentrations.
The dose formulations containing the test item and prepared at 2 mg/mL and 200 mg/mL in corn oil were found to be homogeneous. They should be magnetically stirred for 15 minutes before sampling.

Duration of treatment / exposure:

In the males:
- 2 weeks before mating,
- during the mating period,
- until euthanasia (approximately 4 weeks in total),

In the females:
- 2 weeks before mating,
- during the mating period,
- during gestation,
- during lactation until Day 13 post-partum inclusive,
- until euthanasia for females with no delivery.

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 animals

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for dose selection:
The dose levels were selected in agreement with the Sponsor, on the basis of the results of a previous study performed in the same species, in which a structural analogue of the test item was administered daily by gavage to male and female Sprague-Dawley rats at dose levels of 100, 300 or 1000 mg/kg/day in corn oil for 4 weeks. In this study, the test item was clinically well tolerated and induced ptyalism only at 1000 mg/kg/day. At laboratory investigations, the major finding was dose-related higher cholesterol levels from 100 mg/kg/day in males and from 300 mg/kg/day in females. This effect was considered to be adverse at 1000 mg/kg/day. At pathology, findings consisted of non adverse hepatocellular hypertrophy in males and females at 1000 mg/kg/day and non adverse increased vacuolation in proximal tubules of kidneys in females at 300 mg/kg/day and in males and females at 1000 mg/kg/day.
Therefore, 1000 mg/kg/day was selected as the high-dose level. The low-dose and mid-dose were selected using a ratio representing approximately a three-fold interval (i.e. 100 and 300 mg/kg/day).

- Rationale for animal assignment: stratified procedure.

Positive control:

no (not required)

Parental animals: Observations and examinations:

MORTALITY/MORBIDITY:
- Time schedule: each animal was checked for mortality or signs of morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Detailed clinical examinations were performed on all animals, once before the beginning of the treatment period and then once a week until the end of the study.

CLINICAL SIGNS:
- Time schedule: From arrival, each animal was observed once a day as part of routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

BODY WEIGHT:
- Time schedule: The body weight of each male was recorded once before the beginning of the treatment period, on the first day of treatment (Day 1), and then once a week until euthanasia.
The body weight of each female was recorded once before the beginning of the treatment period, on the first day of treatment (Day 1), then once a week until mated, on Days 0, 7, 14 and 20 post-coitum (p.c.) (and on the day of euthanasia for females which did not deliver, and Days 1, 4, 8 and 13 p.p.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by each male was measured once a week from the first day of treatment until the start of the mating period.
The quantity of food consumed by each female was measured once a week from the first day of treatment until the start of the mating period, during gestation for the interval Days 0-7, 7-14 and 14-20 p.c. and during lactation for the intervals Days 1-4, 4-8 and 8-13 p.p.
During the mating period, food consumption was not measured for males or females.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule: the last five males and the first five females euthanized on Day 14 p.p. from each group were evaluated with a Functional Observation Battery once at the end of the treatment period (for males: during the last week of treatment; for females: on Day 13 p.p. after euthanasia of the pups).
This included a detailed clinical examination, the assessment of reactivity to manipulation and to different stimuli and motor activity.
All animals were observed in the cage, in the hand and in the standard arena.

HAEMATOLOGY:
- Time schedule: the parameters were determined from the first five males and lactating females (with non-coagulated samples) from each group to be euthanized as scheduled, on the day of euthanasia.

CLINICAL CHEMISTRY:
- Time schedule: the parameters were determined from the first five males and lactating females (with non coagulated and non-hemolyzed samples) from each group to be euthanized as scheduled, on the day of euthanasia.

URINALYSIS:
- Time schedule: the parameters were determined from the first five males and the first five females euthanized on Day 14 p.p., from each group, on the day of euthanasia.

THYROID HORMONES:
- Time schedule:
* at termination on Day 14 p.p. from all F0 females (approximately 0.5 mL of blood was collected from the orbital sinus under isoflurane anesthesia),
* at termination from all F0 males (approximately 0.5 mL of blood was collected from the orbital sinus under isoflurane anesthesia).

REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded.

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning (between 7.5 and 10 a.m.):
- during the 2 weeks of the pre-treatment period,
- from the beginning of the treatment period during the pre-mating and mating periods, until the females are mated,
- on the day of euthanasia before scheduled euthanasia (Day 14 p.p.), to allow correlation with reproductive organ histopathology.

Sperm parameters (parental animals):

not analysed

Litter observations:

STANDARDISATION OF LITTERS: Yes

PARAMETERS EXAMINED:
The following parameters were examined in F1 offspring:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies,
- body weight and body weight gain,
- clinical signs,
- anogenital distance (AGD),
- presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS:
- external abnormalities

THYROID HORMONES:

- at termination on Day 4 p.p. from at least two culled pups/litter
- at termination on Day 13 p.p. from at least two pups/litter

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: after the end of the mating period (4 weeks of treatment in total),
- Female animals: on Day 14 post-partum.
Females which did not deliver were euthanized on Day 26 p.c. (after a body weight recording to check for a possible un-noticed delivery) by the same way without overnight fasting.

ORGAN WEIGHTS: see Tissue Procedure Table below
The body weight of each F0 animal euthanized as scheduled (after the end of the mating period for males or on Day 14 p.p. for females) was recorded before euthanasia. For these animals, the organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection, or after fixation for thyroids with parathyroids.
The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.

GROSS NECROPSY:
A complete macroscopic post-mortem examination was performed on all F0 animals including the prematurely sacrificed females. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.
The numbers of corpora lutea and implantation sites were recorded for females euthanized as scheduled on Day 14 p.p.
For apparently non-pregnant females, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

PRESERVATION OF TISSUES:
The tissues of F0 animals specified in the Tissue Procedure Table were preserved in 10% buffered formalin (except for the eyes with optic nerves and Harderian glands, and the testes and epididymides which were fixed in Modified Davidson's fixative).

PREPARATION OF HISTOLOGICAL SLIDES:
All tissues required for microscopic examination were trimmed based on the RITA guidelines, when applicable (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004), embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except testes and epididymides which were stained with hematoxylin/PAS).
This tissue processing was performed at Citoxlab France.

HISTOPATHOLOGY:
A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table from the first five euthanized as scheduled males and lactating females of the control and high-dose groups (groups 1 and 4),
- all macroscopic lesions of all groups,
- the following tissues of the low- and intermediate-dose groups (groups 2 and 3) based upon the results of the microscopic examination of the high-dose group: adrenals (males), kidneys (males and females), liver (males and females), mesenteric lymph nodes (males and females).

Postmortem examinations (offspring):

SACRIFICE:
Pups were euthanized by an intraperitoneal injection of sodium pentobarbital (or by decapitation under isoflurane anesthesia on Day 4 p.p. when blood sampled), followed by exsanguination when there was a necropsy:
- pups whose mother died: as soon as possible,
- pups not selected on Day 4 p.p.: on Day 4 p.p.,
- surviving pups: on Day 13 p.p.

GROSS NECROPSY:
Pups not selected on Day 4 p.p. were discarded without further examination.
Pups euthanized on Day 13 p.p. were submitted to a detailed external examination (including orifices and buccal cavity) after euthanasia. Particular attention was paid to the external genital organs. Then, they were discarded without any further examination, or after sampling of thyroids with parathyroids for the selected pups.

PRESERVATION OF TISSUES:
Thyroids with parathyroids of the selected pups/litter euthanized on Day 13 p.p. were preserved in 10% buffered formalin.

HISTOPATHOLOGY: No.

ORGAN WEIGTHS:
The body weight of one selected pup/sex/litter (or of two pups from the same sex when there was only one sex in the litter) euthanized on Day 13 p.p. was recorded before euthanasia.

Statistics:

Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).
Hematology, blood biochemistry, urinalysis, hormone, anogenital distance, nipples/areolae and post-implantation loss data were analysed with CITOX software.
PATHDATA software was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01).

Reproductive indices:

Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live pups) on Day 1 p.p. / Number of implantation sites
Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)

Offspring viability indices:

Live birth index = 100 * (Number of live born pups on Day 1 post-partum/ Number of delivered pups)
Viability index on Day 4 post-partum = 100 * (Number of surviving pups on Day 4 post-partum before culling / Number of delivered pups)
Lactation index on Day 13 post-partum = 100 * (Number of surviving pups on Day 13 post-partum / Number of surviving pups on Day 4 post-partum (after culling))

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Ptyalism was observed with a dose-related incidence in test item-treated males and females during the premating, pregnancy and lactation periods. This sign, commonly noted when a test item is administered by gavage, was not considered to represent an adverse effect.

The other clinical signs recorded during the study, i.e. cutaneous observations on various parts of the body (area of hair loss, cutaneous lesions, scabs), chromodacryorrhea, opacity of eye, reflux at dosing, abnormal growth of teeth, half-closed eyes, pallor of extremities/eyes, round back, piloerection and/or dyspnea were considered to be unrelated to the test item treatment as they were present both in control and test item-treated animals, and/or were reported sporadically in only a few animals and/or were attributed to the gavage procedure.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No unscheduled death occurred in males at any dose level during the study.
Due to no delivery, the following females were euthanized on Day 26 p.c. without clinical signs prior to death:
- at 0 mg/kg/day: one female,
- at 300 mg/kg/day: one female.
At necropsy, these females were found to be non-pregnant.
At 1000 mg/kg/day, one female was prematurely euthanized on Day 1 p.p. due to difficulties to deliver. Pallor of extremities, round back, piloerection and ptyalism were noted prior to euthanasia. At necropsy, this female had enlarged left horn with two dead fetuses. At microscopic examination, slight subacute inflammation of the uterus was noted, together with slight necrosis of liver, suggesting probably an early systemic process.
A relationship to the administration of the test item was considered as unlikely given this isolated case.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant effects were observed on mean body weights or mean body weight changes in males or females at any dose level.
The few statistically significant differences noted between control and females given 100 mg/kg/day in mean body weight during the lactation period (i.e. on Days 4 and 13 p.p.) were not attributed to the test item treatment as they were of low magnitude (-7%) and/or not dose-related.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No relevant effects on mean food consumption were observed in males and females at any dose level.
The statistically significant differences between control and females given 100 mg/kg/day throughout the lactation period were not attributed to the test item treatment as they were not dose-related.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The hematology parameters were considered to be unaffected by the test item treatment in males and females at the end of the treatment period.
The only statistically significant differences from controls (i.e. higher eosinophil counts in males at 100 mg/kg/day, lower hemoglobin concentration and basophil counts in females at 300 mg/kg/day, variation of prothrombin time in females at 100 and 1000 mg/kg/day and shortened activated partial thromboplastin time in females at 100 mg/kg/day) were not attributed to the test item treatment as they were of low magnitude and/or isolated and/or not dose-related and/or noted with opposite trends in the same sex.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The only change attributed to the test item treatment consisted of higher mean cholesterol level in males.
Higher mean cholesterol level was noted in males given 1000 mg/kg/day (5/5 individual values were above the highest control value) with the same tendency at 100 and 300 mg/kg/day (3/5 individual values at each dose level were above the highest control value). Females were not affected.
This change was considered as non-adverse given the limited magnitude and the absence of adverse microscopic lesions.
The other statistically significant differences between control and test item-treated animals, namely in males, lower sodium and higher calcium and triglyceride levels at 300 mg/kg/day, higher total protein levels at 300 and 1000 mg/kg/day, and in females, higher sodium and chloride levels at 1000 mg/kg/day, lower albumin to globulin ratio at 1000 mg/kg/day and lower triglyceride level at 100 mg/kg/day, were reported with no dose relationship and/or were isolated and/or were of low magnitude. They were therefore not attributed to the test item treatment.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The urinary parameters were considered to be unaffected by the test item treatment in males and females at the end of the treatment period.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Motor activity
No test item-related effects were observed on motor activity (horizontal movements and rearing) in males or females at any dose level.
The higher mean numbers of horizontal movements (statistically significant) noted at 100 and 300 mg/kg/day in females were not attributed to the test item treatment as they were not dose-related and/or due to the contribution of a single female (given 100 mg/kg/day: 1024 horizontal movements).
The higher mean numbers of rearing movements (not statistically significant) noted at 100 and 300 mg/kg/day in females were not attributed to the test item treatment as they were not dose-related. The lower mean value noted at 1000 mg/kg/day (not statistically significant) was mainly due to the contribution of one female (44 rearing movements).

Functional Observation Battery
There were no test item-related neurologic abnormalities.
The higher mean landing foot splay values recorded in females given 1000 mg/kg/day (107 mm vs. 91 mm in controls) and lower mean rectal temperature noted in females given 100 or 1000 mg/kg/day (37.8°C and 38.1 °C, respectively, vs. 38.9°C in controls) were not attributed to the test item treatment as they did not correlate with any other findings and/or were within physiological values and/or were not dose-related.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Unscheduled deaths
One female given 1000 mg/kg/day had slight subacute inflammation of the uterus, together with slight necrosis of liver, suggesting probably an early systemic process.

At the end of the treatment period
Test item-related non-adverse changes were noted in the liver, kidneys, mesenteric lymph node, and adrenal glands from males and/or females treated at 100, 300 or 1000 mg/kg/day.

. Liver
Minimal diffuse hepatocellular hypertrophy was noted in the liver from one male at 100 mg/kg/day, two females treated at 300 mg/kg/day, and from 4 males and 5 females treated at 1000 mg/kg/day.
This correlated with minimally increased liver weights in females at 1000 mg/kg/day. It was of low magnitude and with no associated degenerative or necrotic lesions correlates, and thus it was considered to be non adverse.

The relationship of the minimal hepatocellular hypertrophy in 1/5 males treated at 100 mg/kg/day to test item-administration was considered to be equivocal in view of the low incidence and low severity of this change and the absence of this finding at 300 mg/kg/day in males.

. Kidneys
Minimal tubular vacuolation was noted in the kidneys from one male and one female treated at 1000 mg/kg/day. This finding was not associated with any degenerative or necrotic lesions and was of low magnitude. Thus, it was considered to be non-adverse.
In males treated at = 100 mg/kg/day, non-adverse minimal tubular basophilia was noted. This finding was not observed in females.

. Mesenteric lymph node
Minimal sinusoidal erythrocytes (i.e. hemorrhage) were noted in the mesenteric lymph node from one male treated at 300 mg/kg/day, one male treated at 1000 mg/kg/day and from females treated at = 100 mg/kg/day. This finding was not associated with any degenerative or necrotic lesions and was of low magnitude. Thus, it was considered to be non-adverse.

In one female treated at 1000 mg/kg/day, increased mast cells cellularity and minimal hyperplasia of lymphoid cells were noted. The relationship of these findings to test item-administration was equivocal.

. Adrenal glands
Minimal atrophy of cortex (1/5 males) together with vacuolation (2/5 males) were noted at 1000 mg/kg/day at low incidence. The atrophy correlated with the decreased organ weights in both animals. These findings were not associated with any degenerative or necrotic lesions and were of low magnitude. Thus, they were considered to be non-adverse.
No finding in this organ was observed in females.

. Thyroid glands
Minimal follicular hypertrophy was noted in the thyroid glands from 1/5 males treated at 1000 mg/kg/day. In view of the low incidence and magnitude of this change, the relationship of this finding to test item administration was unlikely.

The other changes were considered to be part of the spontaneous background of the rats kept under laboratory conditions, including no test item change on mammary glands.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormones:
The thyroid hormone levels were considered to be unaffected by the test item treatment in F0 males.
The statistically significant higher mean T4 level found at 300 mg/kg/day was considered to be fortuitous as it was not dose-related and remained within the range of the historical control data.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related effects were observed on the estrous cycle at any dose level during the treatment period.
The statistically significant higher mean number of estrous days at 300 mg/kg/day during the pre-mating period (4.9 vs. 4.1 in controls) was not attributed to the test item treatment as this difference was of low magnitude and not dose-related.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Pairing, mating and fertility:
No test item-related effects were observed on the mating or fertility data.
All females mated between 2 and 4 days on average (two control females and one female at 300 mg/kg/day took 5, 8 and 14 days to mate, respectively).
All females were pregnant, except one control female and one female given 300 mg/kg/day, which accounts for the slightly lower mean fertility index noted at these dose levels. These isolated cases were considered as incidental.

Delivery data :
All pregnant females delivered, but one female given 1000 mg/kg/day was sacrificed on Day 1 due to difficulty to deliver. This isolated occurrence was considered to be unrelated to the test item treatment.

No test item-related effects were observed on the number of corpora lutea, pre- and post-implantation losses or pups delivered, or on the duration of gestation at any dose level.
The higher mean pre- and post-implantation losses noted in females given 100 mg/kg/day (10.2 and 22.4 vs. 3.5 and 16.1 in controls) were not attributed to the test item treatment as these differences were due to the contribution of single female and/or not dose-related. Additionally, the values remained within the range of our Historical Control Data.

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEL

Remarks:

(reproduction performance)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

See table 2.
No test item-related clinical signs and no abnormal behaviour were observed at any dose level.
The few findings recorded during the lactation period were not attributed to the test item treatment as they were reported in isolated animals and/or were not dose-related and/or are routinely observed in rat pups of this strain and age (e.g. thinning of hair).

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No test item-related effects on the incidence of pups found dead/sacrificed moribund or cannibalized were noted at any dose level.
The higher number of pups found dead/cannibalized between Days 1 and 4 p.p. in females given 100 mg/kg/day (21 vs. 8 in controls) was not attributed to the test item as the difference was mainly due to the contribution of one female (9 pups found dead on Day 1 p.p.).
No test item-related effects were noted on the live birth, viability or lactation index at any dose level.
The lower live birth and viability indexes at 100 mg/kg/day, correlating with the higher number of pups found dead/cannibalized between Days 1 and 4 p.p., was not attributed to the test item treatment as these differences were not dose-related.
At 1000 mg/kg/day, the lower mean live birth index, mainly due to the contribution of one female euthanized on Day 1 p.p. due to difficulty to deliver, was considered to be incidental.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, when compared to controls, mean body weight gain was lower on Days 4-13 p.p. (-12 and -13% in males and females, respectively) leading to a lower mean terminal body weight (-9% in both sexes) without statistically significance. Although the values remained within the range of our Historical Control Data, these effects were attributed to the test item treatment, but considered as non-adverse in view of the low magnitude.
At 100 and 300 mg/kg/day, the same tendency was noted but of lower magnitude (Day 13 p.p. body weight was 4 to 5% lower than that of controls) and with poor dose relationship.

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:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related effects on the anogenital distance were observed in males and females at any dose level.
The statistically significant higher mean anogenital distance corrected for body weight measured in male and/or female pups at 100 mg/kg/day group were not attributed to the test item treatment as the differences from controls were not dose-related and were of low magnitude (between +8% and +16%).

Nipple retention in male pups:

effects observed, treatment-related

Description (incidence and severity):

No nipples were noted in any male pups.
Areolae were observed in the following male pups on Day 12 p.p.:
- three pups at 300 mg/kg/day from three litters (two, three or four areolae per pup),
- five pups at 1000 mg/kg/day, from two litters (two, three or four areolae per pup).
As the incidences of this finding were dose-related and higher to the reference mean values, a relationship to the test item treatment could not be excluded.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Decreased final body weights were noted in male pups at 1000 mg/kg/day on Day 13 p.p.

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Sex ratio: The higher % of males at 300 mg/kg/day was not attributed to the test item treatment as this difference was not dose-related.

Thyroid effects: The thyroid hormone levels were considered to be unaffected by the test item treatment in pups.
The slightly lower mean T4 and TSH levels in pups at 1000 mg/kg/day was considered to be of no toxicological importance as these differences were of low magnitude, included in the historical control range and not statistically significant.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: retention of areolae in male pups from 300 mg/kg/day

Critical effects observed:

no

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Based on the experimental conditions of this study:
- the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day in males and females based on the absence of adverse findings at this dose level,
- the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day based on the absence of effects on mating or fertility at this dose level,
- the NOEL for toxic effects on progeny was considered to be 100 mg/kg/day given the dose-related retention of areolae in male pups from 300 mg/kg/day on Day 12 p.p.

Executive summary:

The objective of this OECD 422 study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, during mating and, for the females, through gestation until Day 13 post-partum (p.p.).

This study provides information:

. on the possible health hazards (including neurological effects) likely to arise from repeated exposure over a relative limited period of time,

. on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

 

Methods

Three groups of ten male and ten female Sprague-Dawley rats received the test item daily by the oral route (gavage) at dose levels of 100, 300 or 1000 mg/kg/day, under a constant dosage volume of 5 mL/kg/day. Another group of ten male and ten female rats received the vehicle only (corn oil) under the same experimental conditions and acted as a control group.

Males were treated for an overall period of approximately 4 weeks: 2 weeks before mating, during the mating period (up to 2 weeks) until the day before euthanasia. Females were treated for an overall period of 7 to 9 weeks: 2 weeks before mating, through mating (up to 2 weeks) and gestation (3 weeks) until Day 13 post-partum (p.p.) inclusive.         

The actual test item concentrations in the dose formulations were determined in Weeks 1, 3 and 6 using a validated HPLC-UV analytical method.

Animals were checked daily for clinical signs and mortality. Functional Observation Battery and motor activity were performed on five males and five females during the last days of treatment.

Body weights and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. Estrous cycle stages were determined daily from two weeks before mating until the females had mated and on Day 14 p.p. before euthanasia.

The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until Day 13 p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. The size of each litter was adjusted on Day 4 p.p. by culling extra pups to obtain as nearly as possible four males and four females per litter. Pups not selected on Day 4 p.p. were sampled, euthanized and discarded without further examination.

The pups were observed daily for clinical signs or abnormal behaviour and were weighed on Days 1, 4, 8 and 13 p.p. The physical development of pups was assessed by measuring the anogenital distance on Day 1 p.p. and by counting the number of nipples and areolae in male pups on Day 12 p.p.

Hematology and blood biochemistry investigations were performed on the first five males and females in each group at scheduled euthanasia. Thyroid hormones (TSH and T4) were determined in males at sacrifice and in at least 2 pups/litter sacrificed on Day 13 p.p.

Males were euthanized after completion of the mating period. Dams were euthanized on Day 14 p.p.

A full macroscopic post-mortem examination was performed, with a particular attention to the reproductive organs. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from the first five euthanized males and lactating females in the control- and high-dose groups, on all macroscopic lesions and on kidneys, liver, mesenteric lymph nodes, thyroids and adrenals (males only) of the low-and intermediate-dose groups.

Pups were euthanized on Day 13 p.p. and submitted to a detailed external examination with a particular attention to the external genital organs.

 

Results

Actual concentrations of the test item in the dose formulations analyzed during the study remained within an acceptable range of variation (-2.5% to +2.2%) when compared to the nominal concentrations.

 

F0 animals:

No test item-related deaths occurred during the study.

Ptyalism was observed with dose-related increased incidences. This sign, commonly noted when a test item is administered by gavage, was not considered to represent an adverse effect.

The functional observation battery and motor activity were unaffected by the test item treatment.

Body weight and food consumption were not impaired by the test item treatment.

The estrous cycle was not impacted by the test item treatment.

The mating, fertility and delivery data were unaffected by the test item treatment.

At laboratory investigations, higher cholesterol level was noted in males given 1000 mg/kg/day (1.9-fold vs. controls) with the same tendency at 100 and 300 mg/kg/day (+26 to +28% vs. controls). This isolated finding was considered to be non-adverse.

Hematology and urinary parameters were not impacted by the test item treatment.

Thyroid hormone analysis did not reveal any disturbances in F0 males at sacrifice.

At pathology, no gross test item-related changes were noted. Decreased adrenal weights were noted in males treated at 300 or 1000 mg/kg/day and correlated with microscopic non-adverse cortical atrophy at 1000 mg/kg/day. There was a trend towards increase in liver weights in males treated at 300 mg/kg/day and in females treated at 1000 mg/kg/day that correlated with non-adverse microscopic hepatocellular hypertrophy. Non-adverse microscopic hepatocellular hypertrophy was also observed in males at 1000 mg/kg/day.

 Indeed, at microscopic examination, non-adverse tubular vacuolation was noted in the kidneys from one male and one female treated at 1000 mg/kg/day, together with non-adverse tubular basophilia in males treated at = 100 mg/kg/day.

Non-adverse sinusoidal erythrocytes (i.e.hemorrhage) were noted in the mesenteric lymph node from males treated at = 300 mg/kg/day and from females treated at = 100 mg/kg/day.

 

Pups:

Observations of the pups from birth to Day 13 p.p. did not show any effects on mortality, viability, clinical signs, sex ratio or anogenital distance.

Lower body weight gain (-12 and -13%, respectively, on Days 4-13 p.p.) and non-statistically lower body weight (-9% on Day 13 p.p. in both sexes) were observed in male and female pups at 1000 mg/kg/day. The same tendency was noted at 100 and 300 mg/kg/day, but with lower magnitude (Day 13 p.p. body weight was 4 to 5% lower than that of controls) and with poor dose relationship. 

Presence of areolae was observed in three and five male pups at 300 and 1000 mg/kg/day, respectively.

Thyroid hormone analysis did not reveal any disturbance in pups on Day 13 p.p.

 

Conclusion

The test item was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and (for females) throughout gestation and until Day 13 post-partum, at dose levels of 100, 300 or 1000 mg/kg/day.

 

Based on the experimental conditions of this study:

. the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day in males and females based on the absence of adverse findings at this dose level,

. the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day based on the absence of effects on mating or fertility at this dose level,

. the NOEL for toxic effects on progeny was considered to be 100 mg/kg/day given the dose-related retention of areolae in male pups from 300 mg/kg/day on Day 12 p.p.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Screening study is considered to be reliable (performed according to study guidelines and had a klimisch score of 1).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Screening test on reproduction (2019, OECD 422)

The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, during mating and, for the females, through gestation until Day 13 post-partum (p.p.). This study provides information: on the possible health hazards (including neurological effects) likely to arise from repeated exposure over a relative limited period of time, and, on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

 

Three groups of ten male and ten female Sprague-Dawley rats received the test item daily by the oral route (gavage) at dose levels of 100, 300 or 1000 mg/kg/day, under a constant dosage volume of 5 mL/kg/day. Another group of ten male and ten female rats received the vehicle only (corn oil) under the same experimental conditions and acted as a control group.

Males were treated for an overall period of approximately 4 weeks: 2 weeks before mating, during the mating period (up to 2 weeks) until the day before euthanasia. Females were treated for an overall period of 7 to 9 weeks: 2 weeks before mating, through mating (up to 2 weeks) and gestation (3 weeks) until Day 13 post-partum (p.p.) inclusive.         

 

F0 animals:

No test item-related deaths occurred during the study. Ptyalism was observed with dose-related increased incidences. This sign, commonly noted when a test item is administered by gavage, was not considered to represent an adverse effect. The functional observation battery and motor activity were unaffected by the test item treatment. Body weight and food consumption were not impaired by the test item treatment. The estrous cycle was not impacted by the test item treatment. The mating, fertility and delivery data were unaffected by the test item treatment.

At laboratory investigations, higher cholesterol level was noted in males given 1000 mg/kg/day (1.9-foldvs.controls) with the same tendency at 100 and 300 mg/kg/day (+26 to +28%vs.controls). This isolated finding was considered to be non-adverse. Hematology and urinary parameters were not impacted by the test item treatment. Thyroid hormone analysis did not reveal any disturbances in F0 males at sacrifice.

At pathology, no gross test item-related changes were noted. Decreased adrenal weights were noted in males treated at 300 or 1000 mg/kg/day and correlated with microscopic non-adverse cortical atrophy at 1000 mg/kg/day. There was a trend towards increase in liver weights in males treated at 300 mg/kg/day and in females treated at 1000 mg/kg/day that correlated with non-adverse microscopic hepatocellular hypertrophy. Non-adverse microscopic hepatocellular hypertrophy was also observed in males at 1000 mg/kg/day.  Indeed, at microscopic examination, non-adverse tubular vacuolation was noted in the kidneys from one male and one female treated at 1000 mg/kg/day, together with non-adverse tubular basophilia in males treated at = 100 mg/kg/day. Non-adverse sinusoidal erythrocytes (i.e.hemorrhage) were noted in the mesenteric lymph node from males treated at = 300 mg/kg/day and from females treated at = 100 mg/kg/day.

 

Pups: Observations of the pups from birth to Day 13 p.p.did not show any effects on mortality, viability, clinical signs, sex ratio or anogenital distance. Lower body weight gain (-12 and -13%, respectively,on Days 4-13 p.p.) and non-statistically lower body weight (-9% on Day 13 p.p.in both sexes) were observed in male and female pups at 1000 mg/kg/day. The same tendency was noted at 100 and 300 mg/kg/day, but with lower magnitude (Day 13p.p.body weight was 4 to 5% lower than that of controls) and with poor dose relationship. Presence of areolae was observed in three and five male pups at 300 and 1000 mg/kg/day, respectively. Thyroid hormone analysis did not reveal any disturbance in pups on Day 13 p.p.

 

Based on the experimental conditions of this study:

. the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day in males and females based on the absence of adverse findings at this dose level,

. the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day based on the absence of effects on mating or fertility at this dose level,

. the NOEL for toxic effects on progeny was considered to be 100 mg/kg/day given the dose-related retention of areolae in male pups from 300 mg/kg/day on Day 12p.p.

Effects on developmental toxicity

Description of key information

A developmental toxicity study in rat (OECD 414, 2020) was performed on Reaction products of 4,4'-isopropylidenediphenol, ethoxylated and methacrylic acid. No adverse parental or developmental toxicity were observed up to the highest dose of 1000 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August - September 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The test was initiated based on a request for information in a final ECHA decision (CCH-D-2114376213-53-01/F).

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

RjHan:SD (CD®), SPF

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, le Genest-Saint-Isle, France
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 242g to 342g
- Fasting period before study: No
- Housing: Individual housing in polycarbonate cages with stainless steel lids and containing autoclaved sawdust. Each cage contained nylabone and rat hut for environmental enrichment.
- Diet: Free access to SSNIFF rat/mouse pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water: Free access to bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: 4 or 5 days

CONTAMINANT ANALYSES
The batches of diet and sawdust were analyzed by the Suppliers for composition and contaminant levels. Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants were present in the diet, drinking water or sawdust at levels which could be expected to interfere with, or prejudice, the outcome of the study.

ENVIRONMENTAL CONDITIONS (SET CONDITIONS)
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 12 August 2019 To: 05 September 2019

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The formulations were prepared according to Citoxlab France/Study No. 46148 AHS (homogeneity testing) describing the preparation procedure for a range of concentrations covering the lowest and highest used in this study. The formulations were freshly prepared on the days of treatment. The dose formulations were maintained under delivery conditions (at room temperature and protected from light) throughout the administration procedure. They were stirred for at least 15 minutes before administration and were maintained under continuous magnetic stirring throughout the dosing procedure. The test item dose formulations were administered within 4 hours after the end of their preparation.

A constant dosage volume of 5 mL/kg bw/day was used (concentrations 0, 20, 60 and 200 mg/mL).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose formulation analyses were done using a validated method (HPLC/UV).
Determination of test item concentrations in dose formulations was done twice in the study (Day 5 and Day 20 p.c.).
Acceptance criterion: Measured concentration = nominal concentration ± 10%

Details on mating procedure:

Females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as Day 0 p.c.

Duration of treatment / exposure:

16 days (from Day 5 to Day 20 post-coitum (p.c.) inclusive)

Frequency of treatment:

Once daily

Duration of test:

females were sacrificed Day 21 post-coitum

Dose / conc.:

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

Remarks:

Low dose group

Dose / conc.:

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

Remarks:

Mid dose group

Dose / conc.:

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

Remarks:

High dose group

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected on the basis of the results of a previous OECD 422 study (Citoxlab France/Study No. 46151 RSR), in which the test item was administered by gavage at 100, 300 or 1000 mg/kg bw/day to male and female rats from 2 weeks before mating, through mating and, for females, through gestation until Day 13 post-partum (p.p.). The No Observed Adverse Effect Level (NOAEL) for parental toxicity and the No Observed Effect Level (NOEL) for reproductive performance were established at 1000 mg/kg bw/day due to the absence of adverse effects at this high dose. The NOEL for toxic effects on progeny was considered to be 100 mg/kg/ bwday based on the retention of areola in male pups at 300 and 1000 mg/kg bw/day.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day before the treatment period and three times a day during the treatment period for mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day

BODY WEIGHT: Yes
- Time schedule for examinations: recorded on Days 2, 5, 7, 9, 12, 15, 18 and 21 p.c.

FOOD CONSUMPTION: Yes
- The quantity of food consumed by each female was recorded for the following intervals: Days 2-5, 5-7, 7-9, 9-12, 12-15, 15-18 and 18-21 p.c.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Gross macroscopy
- Weight of the gravid uterus was determined
- Thyroids with parathyroids were weighed after fixation, microscopic examination was performed on the thyroids with parathyroids from all animals.
- For apparently non pregnant females, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.
- A gross evaluation of placentas was also undertaken. The placenta weight was recorded for each fetus and the fetal weight/placental weight ratio was calculated.

OTHER:
Blood samples were taken from all females at termination to determine the levels of the thyroid hormones (T4 and T3) and Thyroid Stimulating Hormone (TSH).

Ovaries and uterine content:

The ovaries and uterus of the females were examined to determine:
- number of corpora lutea,
- number and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of uterine scars,
- number and distribution of implantation sites.

The following classification was used to record:
- uterine scar: uterine implantation without implant,
- early resorption: evidence of implant without recognizable embryo,
- late resorption: dead embryo or fetus with degenerative changes,
- dead fetus: dead fetus with no degenerative changes.

Fetal examinations:

- External examinations: Yes: all per litter
The body weight of each fetus was recorded. The sex of each fetus was determined at the time of hysterectomy by measurement of the anogenital distance (AGD) and was confirmed by internal examination of sexual organs at detailed dissection of the soft tissues or at evisceration.
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

Data on body weight, food consumption and litter data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).
Overview of the strategy used for the statistical analyses of hormone and anogenital distance data and organ weights is attached below.

Historical control data:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No relevant clinical signs were observed during the study.
Ptyalism was noted in 19/24 females at 1000 mg/kg bw/day, starting at the earliest on Day 8 p.c. and lasted generally until the end of the treatment period. This sign, also reported in one female at 100 mg/kg bw/day and one female at 300 mg/kg bw/day, is commonly observed when a test item is administered by gavage and was not considered to represent an adverse effect.
The other clinical signs recorded in pregnant females were considered to be unrelated to the test item treatment as they were isolated findings (chromodacryorrhea in one female at 1000 mg/kg bw/day) and/or are commonly observed in pregnant females of this strain and age (brownish vaginal discharge in one female at 300 mg/kg bw/day from Day 15 p.c. and in one female at 1000 mg/kg bw/day on Day 20 p.c.).

Description (incidence and severity):

n/a

Mortality:

no mortality observed

Description (incidence):

No unscheduled deaths occurred during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effects were noted on mean body weights or mean body weight changes at any dose level.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No effects were noted on mean food consumption at any dose level

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):

The mean thyroid hormone levels (T3, T4 and TSH) were considered to be unaffected by the test item treatment in the absence of statistically significant changes and dose-related changes.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No effects on mean gravid uterus weights, placenta weights, carcass weights or mean net body weight changes (g) were observed at any dose level. No test item-related changes in the thyroid weights were recorded.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related macroscopic post-mortem findings were observed at necropsy of the dams at any dose level.
The only macroscopic observation, i.e. enlarged placenta, was not attributed to the test item treatment as it was recorded both in control and test item-treated females with the same incidence.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test item-related microscopic findings were observed.

Histopathological findings: neoplastic:

not examined

Details on results:

At hysterectomy on Day 21 p.c., 20/24, 24/24, 21/24 and 24/24 females were pregnant with live fetuses in the groups treated at 0, 100, 300 and 1000 mg/kg bw/day, respectively. In the control group, 4 non-pregnant females were found and two females in the mid dose group were non-pregnant. One female in the mid dose group was found with no live fetuses and uterine scars.

Number of abortions:

no effects observed

Description (incidence and severity):

No effects on hysterectomy data were observed at any dose level, a table summarizing the results is included below.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

The statistically lower mean number of early resorptions in females at 1000 mg/kg bw/day, when compared with controls, was considered to be not relevant due to the direction of change. The higher number of implantation scars at 300 mg/kg bw/day was considered to be unrelated to the test item treatment as this finding was due to the contribution of a single female and was not observed in the high dose group.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: No effects observed up to and including the highest dose tested (1000 mg/kg bw/day)

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Mean fetal body was unaffected by the test item treatment at any dose level (mean fetal weights of 5.91g, 5.87g, 5.97g and 5.85g for the fetuses in the control, low, mid and high dose, respectively). The fetal body weight/ placental weight ratio was comparable between the groups (8.26, 8.58, 8.69 and 8.26 for the control, low, mid and high dose, respectively).

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was unaffected by the test item treatment at any dose level (mean percentage of males of 49.5%, 49.9%, 49.5% and 44.6% for the fetuses in the control, low, mid and high dose, respectively). .

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No test item-related external variations or malformations were observed at any dose level. In controls, an anasarca (generalized oedema) was observed in one fetus, a finding commonly observed in this species and strain.

Skeletal malformations:

no effects observed

Description (incidence and severity):

The incidences of fetuses and litters incidences with skeletal variations and malformations are attached. No test item-related skeletal variations were observed at any dose level. When compared with controls, higher incidence of fetuses and litters with unossified or incompletely ossified bones was noted among test item-treated animals. These findings, not statistically significant, were either poorly or not dose-related or reported with a low incidence, with no effects on mean fetal body weight. A relationship to the test item treatment was therefore considered to be unlikely.

Visceral malformations:

no effects observed

Description (incidence and severity):

No visceral malformations were observed at any dose level. The incidences of fetuses and litters with soft tissues variations are attached. No test item-related visceral variations were observed at any dose level. The few findings recorded were considered to be incidental as they were noted both in control and test item-treated animals and/or are common findings in pregnant rats of this strain and age.
No test item-related skeletal cartilage effects were observed at any dose level.
No test item-related skeletal malformations were observed at any dose level. The few findings recorded were considered to be incidental as they were noted both in control and test item-treated animals and/or were noted in isolated fetuses and remained within the range of the historical data range of the test facility.

Other effects:

no effects observed

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed up to and including the highest dose tested (1000 mg/kg bw/day)

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

CHEMICAL ANALYSES OF THE DOSE FORMULATIONS

The test item concentrations in the administered dose formulations analyzed on Day 5 p.c.and Day 20 p.c. remained within an acceptable range of variations (-3.3% to -0.2%) when compared to the nominal values (± 10% of the nominal concentrations).

No test item was detected in the control dose formulation.

Conclusions:

Based on the results of a prenatal developmental study, performed with Reaction products of 4,4'-isopropylidenediphenol, ethoxylated and methacrylic acid according to OECD/EC guidelines and GLP principles, the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 1000 mg/kg bw/day, the No Observed Effect Level (NOEL) for embryo-fetal development was considered to be 1000 mg/kg bw/day.

Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item, Reaction products of 4,4'-isopropylidenediphenol, ethoxylated and methacrylic acid, on the pregnant female rat and on the developing organism, following daily oral administration (gavage) from Day 5 to Day 20 post-coitum (p.c.) inclusive.

Three groups of 24 time-mated female Sprague-Dawley rats received the test item, Reaction products of 4,4'-isopropylidenediphenol, ethoxylated and methacrylic acid, once daily by the oral route (gavage) at dose levels of 100, 300 or 1000 mg/kg/day from Day 5 to Day 20 p.c. inclusive. A control group of

24 time-mated females received the vehicle (corn oil), under the same experimental conditions. A constant dosage volume of 5 mL/kg/day was used. The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. At termination, thyroid hormones (TSH, T3 and T4) were determined for all females. On Day 21 p.c., the females were euthanized and a macroscopic post-mortem examination was performed. Thyroids with parathyroids were weighed and examined microscopically. Hysterectomies were performed and the numbers of corpora lutea, implantation sites, early and late resorptions, and live and dead fetuses

were recorded. Placentas were observed and weighed. The fetuses were weighed, examined for external abnormalities and sexed by internal examination of the gonads. The anogenital distance was measured. The fetuses were subjected to detailed external, soft tissue and skeletal examinations.

At hysterectomy on Day 21 p.c., 20/24, 24/24, 21/24 and 24/24 females were pregnant with live fetuses in the groups treated at 0, 100, 300 and 1000 mg/kg/day, respectively. No unscheduled deaths occurred during the study. Ptyalism was observed especially in most females at 1000 mg/kg/day. This sign, commonly noted when a test item is administered by gavage, was not considered to represent an adverse effect.

Body weight, body weight change and food consumption were unaffected by the test item treatment. At necropsy of the dams, no test item-related macroscopic findings were observed. Gravid uterus weight, carcass weight, net body weight change and gestation parameters were not impacted by the test item treatment. Thyroid hormone analysis and thyroid gland examination did not reveal any disturbances at any

dose level. No effects on the fetal body weight, placental weight, sex ratio or anogenital distance were noted at any dose level. At external, soft tissue or skeletal examination of the fetuses, no variations or malformations attributable to the test item treatment were noted.

 

The test item, Reaction products of 4,4'-isopropylidenediphenol, ethoxylated and methacrylic acid, was administered daily to time-mated female Sprague-Dawley rats by the oral route (gavage) at dose levels of 100, 300 or 1000 mg/kg/day from Day 5 to Day 20 p.c., inclusive.

Based on the results obtained in this study:

. the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 1000 mg/kg/day,

. the No Observed Effect Level (NOEL) for embryo-fetal development was considered to be 1000 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study is considered as reliable.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental toxicity in rats (2020)

The objective of this study was to evaluate the potential toxic effects of the test item, Reaction products of 4,4'-isopropylidenediphenol, ethoxylated and methacrylic acid, on the pregnant female rat and on the developing organism, following daily oral administration (gavage) from Day 5 to Day 20 post-coitum (p.c.) inclusive (TG OECD 414).

Three groups of 24 time-mated female Sprague-Dawley rats received the test item, Reaction products of 4,4'-isopropylidenediphenol, ethoxylated and methacrylic acid, once daily by the oral route (gavage) at dose levels of 100, 300 or 1000 mg/kg/day from Day 5 to Day 20 p.c. inclusive. A control group of

24 time-mated females received the vehicle (corn oil), under the same experimental conditions. A constant dosage volume of 5 mL/kg/day was used. The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. At termination, thyroid hormones (TSH, T3 and T4) were determined for all females. On Day 21 p.c., the females were euthanized and a macroscopic post-mortem examination was performed. Thyroids with parathyroids were weighed and examined microscopically. Hysterectomies were performed and the numbers of corpora lutea, implantation sites, early and late resorptions, and live and dead fetuses

were recorded. Placentas were observed and weighed. The fetuses were weighed, examined for external abnormalities and sexed by internal examination of the gonads. The anogenital distance was measured. The fetuses were subjected to detailed external, soft tissue and skeletal examinations.

At hysterectomy on Day 21 p.c., 20/24, 24/24, 21/24 and 24/24 females were pregnant with live fetuses in the groups treated at 0, 100, 300 and 1000 mg/kg/day, respectively. No unscheduled deaths occurred during the study. Ptyalism was observed especially in most females at 1000 mg/kg/day. This sign, commonly noted when a test item is administered by gavage, was not considered to represent an adverse effect.

Body weight, body weight change and food consumption were unaffected by the test item treatment. At necropsy of the dams, no test item-related macroscopic findings were observed. Gravid uterus weight, carcass weight, net body weight change and gestation parameters were not impacted by the test item treatment. Thyroid hormone analysis and thyroid gland examination did not reveal any disturbances at any

dose level. No effects on the fetal body weight, placental weight, sex ratio or anogenital distance were noted at any dose level. At external, soft tissue or skeletal examination of the fetuses, no variations or malformations attributable to the test item treatment were noted.

 

The test item, Reaction products of 4,4'-isopropylidenediphenol, ethoxylated and methacrylic acid, was administered daily to time-mated female Sprague-Dawley rats by the oral route (gavage) at dose levels of 100, 300 or 1000 mg/kg/day from Day 5 to Day 20 p.c., inclusive.

Based on the results obtained in this study:

. the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 1000 mg/kg/day,

. the No Observed Effect Level (NOEL) for embryo-fetal development was considered to be 1000 mg/kg/day.

Justification for classification or non-classification

Based on the available reproduction data, no classification of Reaction products of 4,4'-isopropylidenediphenol, ethoxylated and methacrylic acid is required for reprotoxicity according to the Regulation EC n°1272/2008.