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

Toxicity to reproduction

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting 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:

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

Details on route of administration:

The oral route was selected since it is a route of administration which is recommended by the Regulatory Authorities for this type of study.
The dose formulations were administered by gavage using a plastic syringe fitted with a plastic gavage tube, once a day, at approximately the same time.

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

TType 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.
Diluted analytical samples prepared from 1.6 mg/mL and 240 mg/mL dose formulations in corn oil were found to be stable for 2 days when they were stored at room temperature and protected from light.

Duration of treatment / exposure:

In the males:
- 2 weeks before mating,
- during the mating period,
- until eutnanasia (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 per sex per dose

Control animals:

yes, concurrent vehicle

Positive control:

no (not required)

Observations and examinations performed and frequency:

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).

Sacrifice and pathology:

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).

Statistics:

yes

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 as they were of low magnitude and/or 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

Description (incidence and severity):

Decreased absolute and relative-to-body adrenal weights were noted in males treated at 300 or 1000 mg/kg/day and correlated with microscopic cortical atrophy, although seen in only 1/5 examined males.
There was a trend towards increase in liver weights in females treated at 1000 mg/kg/day in relative-to-body weights (+15%; p<0.01). This correlated with microscopic hepatocellular hypertrophy observed in all 5/5 animals.
There were also higher absolute and relative-to-body liver weights in males treated at 300 mg/kg/day but that trend was not seen in males at 1000 mg/kg/day. The relationship to test item administration was considered to be likely although there was no hypertrophy at this dose-level.
The other differences were considered not to be related to test item administration since they were of low magnitude, not dose related and with no gross/microscopic correlates.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Unscheduled deaths
One female given 1000 mg/kg/day was prematurely euthanized on Day 1 p.p. and had enlarged left horn with two dead fetuses.

At the end of the treatment period
No gross test item-related changes were noted in surviving animals.

The only changes reported, were considered to be unrelated to the test item administration since they were not dose-related, of low incidence and/or part of the spontaneous background in the untreated rats.

Neuropathological findings:

not examined

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
Non-adverse Mminimal 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
Non-adverse mMinimal 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
Non-adverse mMinimal 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
Non-adverse mMinimal 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. This finding was not associated with any degenerative or necrotic lesions and was of low magnitude. Thus, it was 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

Key result

Dose descriptor:

NOAEL

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

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.

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 concentrationsin 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 1 3p.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 1p.p.and by counting the number of nipples and areolae in male pups on Day 1 2p.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 determinedin 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 13p.p.andsubmitted 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 4,4'-isopropylidenediphenol, ethoxylated and methacrylic acid

EC Number:

935-411-2

Cas Number:

not available

Molecular formula:

C23H24O4 (C2H4O)n

IUPAC Name:

Reaction products of 4,4'-isopropylidenediphenol, ethoxylated and methacrylic acid

Test material form:

liquid

Test animals

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.

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

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)

Examinations

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))

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

1

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

2

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

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

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

2

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

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

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

Applicant's summary and conclusion

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.