2-hydroxy-3-phenoxypropyl acrylate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test is available on 2-Hydroxy-3-phenoxypropyl acrylate.

In this screening toxicity study, no adverse effects on reproductive performance (mating, fertility and delivery) and on progeny were observed at the highest dose of 100 mg/kg/day. This highest dose was selected based on the local effects observed.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 July 2018 - 15 October 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 at study initiation: approximately 10 weeks old for males and 11 weeks for females
- Mean body weight: at the beginning of the treatment period, the males had a mean body weight of 409 g (range: 387 g to 430 g) and the females had a mean body weight of 263 g (range: 241 g to 292 g)
- Fasting period before study: no
- Housing: F0 animals were individually housed, except during mating (males + females) and lactation (females + pups), in polycarbonate cages with stainless steel lids and containing autoclaved sawdust. Individual housing of F0 animals was chosen as group housing for pregnant animals can adversely affect gestation and lactation, and to avoid aggressive behaviour around mating
- 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): about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 21 August 2018 to 15 October 2018.

Route of administration:

oral: gavage

Vehicle:

other:

Remarks:

0.5% (w/v) methylcellulose aqueous solution

Details on exposure:

PREPARATION OF DOSING FORMULATIONS: Emulsion in the vehicle
Suitable formulation in the selected vehicle.

- Concentration in vehicle: 2, 6 and 20 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: the actual test item concentrations in the analyzed dose formulations (i.e. in Weeks 1, 3 and 6) remained within an acceptable range of variation (-3.6% to +9.8%) when compared with the nominal values (nominal concentration ± 15% required).
The dose formulations containing the test item and prepared at 1 mg/mL, 5 mg/mL and 200 mg/mL in 0.5% methylcellulose in water treated by reverse osmosis (w/v) were found to be homogeneous and stable after 14 days (1 mg/mL) and 10 days (5 and 200 mg/mL) at room temperature and protected from light.

Duration of treatment / exposure:

In the males:
- 2 weeks before mating,
- during the mating period (up to 4 days),
- until euthanasia (i.e. after 30 days of treatment),

In the females:
- 2 weeks before mating,
- during the mating period (up to 4 days),
- during pregnancy,
- during lactation until Day 13 post-partum inclusive,
- until euthanasia for females with no evidence of mating or no delivery.

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

100 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, basis on the results of a previous study performed in the same species, in which the test item was administered daily by gavage to five males and five females at dose levels of 0,100, 300 or 1000 mg/kg/day for 2 weeks.
The dose level of 1000 mg/kg/day was associated, with mortality in males and females, thin appearance, half-closed eyes, hypoactivity, ventral recumbency, staggering gait, soiled mouth, ptyalism, loud breathing and/or dyspnea in both sexes, body weight loss (males and females) leading to a lower terminal body weight in males, moderately to markedly reduced food consumption in both sexes, organ weight changes reaching statistical significance in the heart (absolute weight slightly decreased in males), the kidneys (relative weight moderately increased in males) and the liver (absolute and relative weights slightly to moderately increased in males and/or females) and macroscopic changes for both sexes in the stomach (gelatinous, white deposit, thickened and/or gelatinous wall), forestomach (white discoloration and/or thickened wall), intestine (distension with gas), portal and/or mandibular lymph nodes (enlargement) and/or seminal vesicles (reduced in size). At microscopic examination, adverse ulcerative, hyperplastic, degenerative and inflammatory findings were recorded in the forestomach and stomach of males and females.

The dose level of 300 mg/kg/day was associated with ptyalism in males and females and/or soiled mouth in females, lower body weight gain in males and macroscopic changes for both sexes in the stomach (females only: thickened and/or gelatinous wall), forestomach (white discoloration and/or thickened wall), intestine (distension with gas) and/or portal and/or mandibular lymph nodes (enlargement). At microscopic examination, adverse ulcerative, hyperplastic, degenerative and inflammatory findings were recorded in the forestomach and stomach from males.

The dose level of 100 mg/kg/day was associated with ptyalism in one isolated male and macroscopic changes in the forestomach of males (thickened wall). At microscopic examination, hyperplasia and/or hyperkeratosis were noted in forestomach from all males and females.

The Maximal Tolerable Dose (MTD) was established at 300 mg/kg/day for the males and 1000 mg/kg/day for the females. The high-dose level of 100 mg/kg/day was selected for the current study based on the microscopic results of the previous study. The low-dose and mid-dose were selected using a ratio representing approximately a 3-fold interval (i.e. 10 and 30 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 and 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 on the first day of treatment (Day 1), then once a week until euthanasia.
The body weight of each female was recorded on the first day of treatment (Day 1), then once a week until mated (or until euthanasia for the female with no evidence of mating), on Days 0, 7, 14 and 20 post coitum (p.c.) (and on the day of euthanasia for females which did not deliver), and on Days 1, 4, 8 and 13 p.p.
One prematurely euthanized female from group 3 was weighed before euthanasia.

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 intervals Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval 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 first five males and lactating females from each group euthanized as scheduled were evaluated with a Functional Observation Battery once at the end of the treatment period. For females, this was performed 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 five lactating females from each group euthanized as scheduled, on the day of euthanasia.

COAGULATION:
- Time schedule: the parameters were determined from the first five males and five lactating females from each group euthanized as scheduled, on the day of euthanasia.

CLINICAL CHEMISTRY:
- Time schedule: the parameters were determined from the first five males and five lactating females from each group euthanized as scheduled, on the day of euthanasia.

THYROID HORMONES:
- Time schedule:
* at termination on Day 4 p.p. from at least two culled pups/litter,
* at termination on Day 13 p.p. from at least 2 pups/litter,
* at termination on Day 14 p.p. from all F0 females,
* at termination from all F0 males.
The levels of the thyroid hormone (T4) and thyroid stimulating hormone (TSH) were determined respectively by LC-MS/MS or Luminex MAP® technology for pups sampled on Day 13 p.p. and for F0 males sampled at termination.

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:
- during the 2 weeks of the pre-treatment period (including for the two supernumerary females per group),
- from the beginning of the treatment period during the pre-mating and mating periods, until the females were mated,
- on Day 14 p.p. before euthanasia, to allow correlation with histopathology of reproductive organs.

Sperm parameters (parental animals):

Parameters examined in males of parental generation:
- weighing and microscopic examination: see Tissue Procedure Table below
- special emphasis paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

STANDARDISATION OF LITTERS:
On Day 4 p.p., the size of each litter was adjusted by randomly culling extra pups to obtain as nearly as possible four males and four females per litter. Whenever necessary, partial adjustment (for example five males and three females) was permitted. No cross-fostering was performed.

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,
- weight gain,
- clinical signs,
- anogenital distance (AGD),
- presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS:
- external abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
- Males: after the end of the pairing period (at least 4 weeks of treatment in total),
- Females: on Day 14 p.p.

The following F0 females were euthanized by the same way without overnight fasting:
- females which did not deliver: on Day 26 p.c. (after a body weight recording to check for a possible un-noticed delivery),
- female with no evidence of mating: 25 days after the end of the mating period (after a body weight recording).

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) (including females that did not mate or conceive, and pregnant females that did not deliver) 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.

GROSS NECROPSY:
A complete macroscopic post-mortem examination was performed on all F0 animals including one prematurely euthanized female (group 3, difficulties to deliver). 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 reproductive organs.
The numbers of corpora lutea and implantation sites were recorded for females euthanized as scheduled on Day 14 p.p. and for two females (one from group 3 and one from group 4) euthanized on Day 26 p.c. due to no delivery.
For apparently 3 non-pregnant females (one from group 1, one from group 2 and one from group 3), 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).
Thyroids with parathyroids of the selected pups/litter euthanized on Day 13 p.p. were preserved in 10% buffered formalin.

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,
- reproductive organs from animals that did not mate or conceive, or from pregnant females that did not deliver, to investigate possible causes i.e. one male and one female (group 1), one male and one female (group 2), two females and one male (group 3), another female (group 3) and one female (group 4).

Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Based upon the results of the microscopic examination of the high-dose group, the stomach of the low- and intermediate-dose groups was examined in the first five euthanized as scheduled males and the first five females euthanized on Day 14 p.p.

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 the thyroids were sampled:
- 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 prematurely euthanized because of dying mother were euthanized by an intraperitoneal injection of sodium pentobarbital.
Found dead and prematurely euthanized pups were submitted to a detailed external examination (including orifices and buccal cavity), after euthanasia when applicable. Particular attention was paid to the external genital organs and to whether the pups had been fed (e.g. presence of milk in the stomach) when possible. Then, they were discarded without any further examination.
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.
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 euthanized on Day 13 p.p. was recorded before euthanasia.

Statistics:

Body weight, food consumption and reproductive 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, hormones, motor activity, sex ratio, anogenital distance, nipples/aerolae and post-implantation loss 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) / 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 live born 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))

AGD/cube root of body weight ratio (calculated with Excel):

AGD
______________
³vBody weight

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see table 2.
Ptyalism was observed mainly in males given 100 mg/kg/day and in some test item-treated females during the premating period. This clinical sign was still observed in some females at 100 mg/kg/day during pregnancy but was no longer noted during the lactation period. This sign, commonly noted when a test item is administered by gavage, was not considered to represent an adverse effect.
At 30 mg/kg/day, piloerection, round back and emaciated appearance were noted in 1/7 females on Day 4 p.p. As these clinical signs were not dose-related and/or were most probably due to parturition conditions (this female lost 55 g over the first 4 days of the lactation period), a test item relationship was considered to be unlikely.
All the other clinical signs (i.e. areas of hair loss, cutaneous lesions, scabs, chromodacryorrhea, abnormal growth of teeth, and/or placenta in the bedding) were considered to be unrelated to the test item as they were present in control animals, were not dose-related, were reported sporadically in only a few animals, are commonly observed findings in this species and strain and/or resulted from a logical sequence of parturition.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

see table 1.
Males
There were no unscheduled deaths in males.

Females
. At 0 mg/kg/day
One female was euthanized on Day 26 p.c. for absence of delivery. No remarkable clinical signs were observed prior to euthanasia. A slight body weight loss of 11 g was noted between Days 14 and 20 p.c. At necropsy, no macroscopic post-mortem findings were observed. This female was non-pregnant. At microscopic examination, no relevant changes were observed in the reproductive organs of this female and its corresponding male.

. At 10 mg/kg/day
One female was euthanized on Day 26 p.c. for absence of delivery. No remarkable clinical signs were observed prior to euthanasia. A slight body weight loss of 7 g was noted between Days 14 and 20 p.c. At necropsy, no macroscopic post-mortem findings were observed. This female was non-pregnant. At microscopic examination, no relevant changes were observed in the reproductive organs of this female and its corresponding male.

. At 30 mg/kg/day
One female was euthanized on Day 53. This female did not mate and was therefore not pregnant. No remarkable clinical signs were observed prior to euthanasia. At necropsy, no macroscopic post-mortem findings were observed. At microscopic examination, no relevant changes were observed in the reproductive organs of this female. Its corresponding male had unilateral seminal vesicle and epididymis agenesis, and severe unilateral tubular atrophy/hypoplasia of the testes. These changes unrelated to test item administration, were the likely cause of absence of mating.

One female was euthanized on Day 26 p.c. for absence of delivery. No remarkable clinical signs were observed prior to euthanasia. A slight body weight loss of 20.8 g was noted between Days 20 and 26 p.c. At necropsy, no macroscopic post-mortem findings were observed. This female was pregnant with 14 implantation sites. At microscopic examination, no relevant changes were observed in the reproductive organs of this female.

Another female was euthanized on Day 24 p.c. due to difficulties to deliver. Signs of poor clinical condition (i.e. pallor of eyes and extremities, piloerection, round back, hypoactivity and abdominal breathing) were observed prior to euthanasia. Eleven live pups and three dead or cannibalized pups were found in the bedding, and 15 implantation scars and one dead fetus were noted in the uterus. The subcutaneous tissue of the thoracic region and the thymus (which was also reduced in size) had a gelatinous appearance, the kidneys, stomach and the wall of intestines showed tan discoloration and/or irregular color, and the periphery of the lungs was whitish. Macroscopic findings observed in kidneys and thymus correlated microscopically with marked necrosis and marked lymphoid atrophy, respectively. In the absence of similar changes in other test item-treated animals, including at the high-dose, these changes are considered to be related to dystocia and stress and not directly test item-related.

. At 100 mg/kg/day
One female was euthanized on Day 26 p.c. for absence of delivery. Piloerection was observed prior to euthanasia and a body weight loss of 43.8 g was noted between Days 20 and 26 p.c. This female was pregnant with three implantation sites. At necropsy, agenesis of the right kidney, ureter and uterine horn was noted. This was considered as being the likely cause of non delivery. Enlarged corpora lutea were observed in the ovary (considered normal given the ongoing pregnancy).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see table 5.
There were no statistically significant differences on mean body weight or mean body weight change at any dose level in males or in females during the premating, gestation and lactation periods.

Lower mean body weight gain was noted in females at 30 or 100 mg/kg/day between Days 1 and 4 p.p. (+2 g and + 6 g, respectively, vs. +17 g in controls, not statistically significant) and then between Days 8 and 13 p.p. in females at 100 mg/kg/day (+9 g vs. +15 g in controls), leading to a lower mean body weight gain at the high-dose level over the whole lactation period (+32 g vs. +43 g in controls).
At 30 mg/kg/day, this effect was mainly due to the body weight loss of one female at parturition and was therefore considered to be incidental.
At 100 mg/kg/day, these differences were considered to be of minor toxicological importance as they were of slight magnitude, isolated and/or with no effects on the mean body weight.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

see table 6.
There were no relevant effects on mean food consumption in males or females at any dose level.
Differences between control and test item-treated animals consisted of slight lower mean food consumption between Days 8 and 15 of the premating period in females at 30 or 100 mg/kg/day (-16% and -12% vs. controls, respectively).
A relationship with the test item was considered to be unlikely as these variations were not dose-related and mainly due to the high value of one control female (43 g/animal/day).

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):

There were no statistically significant test item-related effects on hematology parameters at any dose level.

Test item-related differences from controls consisted of low mean neutrophil (-18% and -13% in males and females at 100 mg/kg/day, respectively), eosinophil (-17% and -13% in males and females at 100 mg/kg/day, respectively) and basophil (-20% in males at 30 mg/kg/day and -20% and -33% in males and females at 100 mg/kg/day, respectively) counts, high mean lymphocyte (+16% in females at 100 mg/kg/day) and monocyte (+26% in males at 100 mg/kg/day) counts and/or low mean reticulocyte count (-13% in females at 100 mg/kg/day).
As these variations were of slight magnitude, not statistically significant, within or close to the range of the Historical Control Data, marginal, without any relationship to microscopic findings and/or poorly dose related, they were considered to be of no toxicological importance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see table 10.
At 100 mg/kg/day and when compared with controls, statistically significant, dose-related, higher mean chloride level was noted in males (+2%) and lower mean inorganic phosphorus level was recorded in females (-17%) compared to controls. As mean values were within or close to the range of the Historical Control Data, in the absence of any correlates with microscopic observations on kidneys and/or in view of their slight magnitude, these differences were considered to be of minor toxicological importance.
The other differences from controls consisted of low mean inorganic phosphorus level (females at 10 or 30 mg/kg/day), low mean urea level (females from 10 mg/kg/day), high mean albumin level (females at 10 mg/kg/day), low mean cholesterol level (males at 100 mg/kg/day), high mean triglyceride level (males and females from 10 mg/kg/day), high mean alkaline phosphatase activity (females from 10 mg/kg/day), low mean aspartate aminotransferase activity (males from 10 mg/kg/day and females from 30 mg/kg/day) and/or low mean alanine aminotransferase activity (males from 10 mg/kg/day). In view of the slight magnitude and the direction of the changes, in the absence of effects in the other sex and/or relationships to microscopic findings, and/or as mean values were not dose-related and/or within the range of the Historical Control Data, and/or differences resulted from higher mean control values, these findings were not considered as toxicologically important.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

see tables 3 and 4.
Motor activity
There were no relevant differences between the test item-treated and control groups in motor activity (horizontal movements and rearing) in males and females.

At 10 mg/kg/day in males and females, differences from controls were noted in the mean number of horizontal movements (males: -24%) and rearing movements (males: -27% and females: +49%). Females given 100 mg/kg/day also showed variations in the mean number of rearing movements (+20% vs. controls). These values were considered to be of no toxicological importance as they were of opposite trend, not statistically significant, poorly dose-related and/or as values remained close to or within the standard deviation of control values.

Functional Observation Battery
There were no test item treatment-related neurologic abnormalities.

An absence of urination was recorded in 4/5 males at 30 or 100 mg/kg/day versus 2/5 control males and in 3/5 females at 10 or 100 mg/kg/day versus 1/5 control females. Absence of grooming was noted in 5/5 males at 30 or 100 mg/kg/day versus 3/5 control males and in 4/5 females at 100 mg/kg/day versus 2/5 in control females. Higher mean landing foot splay was recorded in males (102 mm vs. 68 mm in controls) and in females (108 mm vs. 94 mm in controls) at 100 mg/kg/day and higher mean rectal temperature (38.2°C vs. 37.4 in controls) was observed in males at 100 mg/kg/day. These findings were considered to be of no toxicological importance as they were within the range of physiological values and only observed in one sex (rectal temperature), poorly dose-related (urination) and/or did not correlate with each other, and/or with any other findings (urination, grooming, landing foot splay and rectal temperature).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see table 12.
Dose-related hyperplasia of the squamous epithelium of the stomach was observed at all dose levels, except in males dosed at 10 mg/kg/day. It was accompanied by hyperkeratosis.
This finding is most probably related to local irritancy of the test item. It was not accompanied by noticeable inflammation and clinical signs and was therefore considered non adverse.
Implantation sites in the uterus and lactation in the mammary gland were not recorded, since they represent expected changes in all females in this type of study.
All other observations belonged to the spectrum of spontaneous changes in rats of this age and strain and bore no relationship to test item administration. 4/5 control and 5/5 high dose females had mucification of the vagina. The remaining control female was in diestrus. Vaginal mucification is expected for lactating females (pups starting to eat solid food from Day 14 p.p.). During this transitory period, diestrus stages are also expected to be observed in a few dams that have started cycling. The testicular morphology was similar in control and high dose animals, with no evidence of disruption of the spermatogenic cycle.

One female, which did not deliver, had agenesis of one uterus horn. This was considered as being a possible cause of no delivery. One male, which did not mate, had unilateral seminal vesicle and epididymis agenesis, and severe unilateral tubular atrophy/hypoplasia of the testes. These changes unrelated to test item administration, were the likely cause of absence of mating. Reproductive organs in other animals which were not pregnant, did not mate or did not deliver were unremarkable.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

see table 11.
Thyroid hormones
The thyroid hormone levels were considered to be unaffected by the test item in F0 males.
When compared with controls, there was a statistically significant, lower mean T4 concentration at 10 mg/kg/day and there were lower mean TSH concentrations from 10 mg/kg/day reaching statistically significance from 30 mg/kg/day. These variations were considered to be fortuitous as they did not correlate between each other, were not dose-related, remained within the range of the Historical Control Data and had no histopathological correlates on thyroid and pituitary glands.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

see table 7.
There were no effects on mean length of the estrous cycle or mean number of cycles.
A trend towards an increase in mean number of days of diestrus was observed in females at 100 mg/kg/day. This was considered to be of no toxicological importance in the absence of statistical significance and in the absence of relevant differences in estrous cycles at microscopic examination of the genital tract. In addition, such a difference was already observed during the pre-treatment period (5.1 vs. 4.1 in controls).

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Pairing, mating and fertility
see table 8.
There were no effects on mating, fertility or gestation index.
Low percentages in the gestation index at 30 and 100 mg/kg/day were not dose-related and/or were within the range of reference data. They were therefore not considered to be test item-related.

Delivery data
see table 9.
There were no effects on the mean duration of gestation, numbers of corpora lutea and implantation sites, numbers of pups delivered or percentages of pre-/post-implantation losses.
The high mean post-implantation losses recorded in test item-treated animals were not dose-related or were within the range of the Historical Control Data (16.7 % - 22.5%). They were therefore not considered to be test item-related.

Table 1: Mortality

Dose level (mg/kg/day) 0 10 30 100
Humane reasons 0 0 1 (pg) 0
Did not mate 0 0 1 (npg) 0
No delivery 1 (npg) 1 (npg) 1 (pg) 1(pg)
Total surviving animals 9/10 9/10 7/10 9/10
pg: pregnant

Table 2: Clinical signs

Sex Male Female
Dose level (mg/kg/day) 0 10 30 100 0 10 30 100
Pre-mating (females) or whole study (males)
Ptyalism 0 1 0 9 0 3 2 3
Total affected animals 0/10 1/10 0/10 9/10 0/10 3/10 2/10 3/10
Gestation
Ptyalism - - - - 0 0 0 5
Total affected animals - - - - 0/9 0/9 0/7 5/9
Lactation
Piloerection - - - - 0 0 1 0
Round back - - - - 0 0 1 0
Emaciated appearance - - - - 0 0 1 0
Total affected animals - - - - 0/9 0/9 1/7 0/9
-: not applicable.

Table 3: Motor activity

Sex Male Female
Dose level (mg/kg/day) 0 10 30 100 0 10 30 100
Horizontal movements 744 569 678 679 370 333 321 408
SD 302.8 193.4 174.8 253.0 211.9 221.0 86.4 187.6
% from controls - -24 -9 -9 - -10 -13 +10
Rearing 171 124 174 196 82 122 86 98
SD 56.4 47.1 50.8 103.0 43.1 85.3 48.3 35.2
% from controls - -27 +2 +15 - +49 +5 +20
-: not applicable.
Statistical analysis: no significance.

Table 4: Functional observation battery

Sex Male Female
Dose level (mg/kg/day) 0 10 30 100 0 10 30 100
Grooming
. absence 3 3 5 5 2 2 3 4
. presence 2 2 0 0 3 3 2 1
Urination
. absence 2 2 4 4 1 3 2 3
. presence 3 3 1 1 4 2 3 2
Landing foot splay (mm) 68 67 61 102 94 92 89 108
SD 28 18 22 22 24 26 41 32
% from controls - -1 -10 +50 - -2 -5 +15
Rectal temperature (°C) 37.4 37.4 37.6 38.2 38.7 38.4 38.2 38.4
SD 0.4 0.3 0.6 0.6 0.6 0.6 0.4 0.2
% from controls - 0 +1 +2 - -1 -1 -1
-: not applicable.

Table 5: Body weight and body weight change

Sex Male Female
Dose level
(mg/kg/day) 0 10 30 100 0 10 30 100
Pre-mating period (females) and whole study (males)
. Day 1 408 410 409 411 258 262 268 263
% from controls - 0 0 +1 - +2 +4 +2
. Day 8 453 461 457 458 266 268 271 270
% from controls - +2 +1 +1 - +1 +2 +2
. Day 15 487 500 494 496 275 279 280 278
% from controls - +3 +1 +2 - +1 +2 +1
. Day 29 526 542 538 541 - - - -
% from controls - +3 +2 +3 - - - -
. Days 1 - 8 +45 +51 +49 +48 +8 +6 +3 +8
. Days 8 - 15 +34 +39 +37 +38 +8 +11 +9 +8
. Days 1 - 15 +79 +91 +85 +85 +16 +17 +12 +16
. Days 15 - 29 +40 +42 +43 +45 - - - -
. Days 1 - 29 +118 +132 +129 +131 - - - -
Gestation
. Day 0 p.c. - - - - 281 281 278 283
% from controls - - - - - 0 -1 +1
. Day 20 p.c. - - - - 434 430 435 446
% from controls - - - - - -1 0 +3
. Days 0 - 20 p.c. - - - - +153 +149 +157 +163
Lactation
. Day 1 p.p. - - - - 334 335 327 349
% from controls - - - - - 0 -2 +4
. Day 13 p.p. - - - - 377 384 368 380
% from controls - - - - - +2 -2 +1
. Days 1 - 4 p.p. - - - - +17 +10 +2 +6
. Days 4 - 8 p.p. - - - - +12 +18 +22 +16
. Days 8 - 13 p.p. +15 +22 +17 +9
. Days 1 - 13 p.p. - - - - +43 +49 +40 +32
-: not applicable.
Statistical analysis: no significance.

Table 6: Food consumption

Sex Male Female
Dose level
(mg/kg/day) 0 10 30 100 0 10 30 100
Pre-mating period (females) and whole study (males)
Days 1 - 8 35 36 36 35 22 22 21 22
% from controls - +3 +3 0 - 0 -5 0
Days 8 -15 35 37 37 35 25 24 21 22
% from controls - +6 +6 0 - -4 -16 -12
Gestation
Days 0 - 7 p.c. - - - - 26 27 25 27
% from controls - +4 -4 +4
Days 7 - 14 p.c. - - - - 27 28 27 28
% from controls - +4 0 +4
Days 14 - 20 p.c. - - - - 33 32 32 35
% from controls - -3 -3 +6
Lactation
Days 1 - 4 p.p. - - - - 39 37 37 41
% from controls - -5 -5 +5
Days 4 - 8 p.p. - - - - 54 55 53 55
% from controls - +2 -2 +2
Days 8 - 13 p.p. - - - - 67 70 68 70
% from controls - +4 +1 +4
-: not applicable.
Statistical analysis: no significance.

Table 7: Estrous cycle

Dose level (mg/kg/day) 0 10 30 100
Mean number of cycles ± SD 2.8 ± 0.4 3.0 ± 0.0 2.9 ± 0.3 2.9 ± 0.3
Mean cycle length (days) ± SD 4.3 ± 0.8 4.0 ± 0.0 4.0 ± 0.1 4.2 ± 0.7
Number of females having a mean average cycle of 4-5 days 9 10 10 8
Mean number of days of diestrus 4.8 ± 2.0 4.3 ± 1.3 4.6 ± 1.8 5.8 ± 1.5
Statistical analysis: no significance.

Table 8: Pairing, mating and fertility data

Dose level (mg/kg/day) 0 10 30 100 Ref. Data
Number of animals paired (M + F) 10 + 10 10 + 10 10 + 10 10 + 10 79 + 80
Number of males mated 10 10 9a 10 79
Number of females mated 10 10 9a 10 80
Mean number of days taken to mate
(pre-coital time) ± SD 2.8 ± 0.8 2.3 ± 1.2 2.8 ± 1.1 2.3 ± 0.7 [1.4;4.3]
Number of pregnant females 9 9 9 10 79
Number of females with live born pups 9 9 7 9 75
Mating index (%)
. male 100 100 90 100 100
. female 100 100 90 100 100
Fertility index (%)
. male 90 90 100 100 [90;100]
. female 90 90 100 100 [90;100]
Gestation index (%) 100.0 100.0 77.8 90.0 [80;100]
M: male; F: female.

Table 9: Delivery data

Dose level (mg/kg/day) 0 10 30 100
Number of pregnant females 9 9 9 10
Number of females which delivered 9 9 7 9
Mean duration of gestation (days) ± SD 22.0 ± 0.0 22.0 ± 0.0 22.0 ± 0.6 22.0 ± 0.0
Mean number of corpora lutea ± SD 15.4 ± 2.5 15.8 ± 3.2 16.6 ± 2.1 15.0 ± 2.2
% from controls / +3 +8 -3
Mean number of implantation sites ± SD 14.1 ± 3.3 14.3 ± 3.5 15.4 ± 1.4 14.6 ± 1.9
% from controls / +1 +9 +4
Mean pre-implantation loss (%) ± SD 8.4 ± 16.1 8.5 ± 15.1 5.9 ± 11.4 2.5 ± 7.4
% from controls / +1 -30 -70
Mean number of pups delivered ± SD 12.9 ± 4.1 11.7 ± 3.5 12.6 ± 2.6 12.7 ± 2.3
Mean post-implantation loss (%) ± SD 10.7 ± 12.6 18.8 ± 13.1 18.2 ± 17.5 13.4 ± 8.6
% from controls +76 +70 +25
/: not applicable.
Statistical analysis: no significance.


Table 10: Blood biochemistry

Sex Male Female
Dose level (mg/kg/day) 0 10 30 100 0 10 30 100
Chloride (mmol/L) 105.0 106.2 106.0 106.7* 102.0 101.3 101.8 102.6
HCD 104.3 (101.0-108.0) 102.2 (97.3-106.2)
Inorganic phosphorus (mmol/L) 2.22 2.35 2.28 2.31 2.98 2.67 2.60 2.48*
HCD 2.24 (1.86-2.61) 2.55 (2.01-3.23)
Statistically significant: *: p<0.05.
HCD: mean (minimum-maximum) Historical Control Data values.

Table 11: Thyroid hormones

Sex F0 males
Dose level (mg/kg/day) 0 10 30 100
T4 (ng/mL) 41.82 35.25* 38.93 40.40
HCD Mean: 37.3; Minimum: 24.6; Maximum: 54.2
Standard deviation 4.941 6.656 3.171 5.401
n 10 10 10 10
% from controls / -16 -7 -3
TSH (pg/mL) 2415 1670 1418** 1614*
HCD Mean: 2349; Minimum: 424; Maximum: 6935
Standard deviation 876.0 488.3 741.0 862.4
n 10 10 10 10
% from controls / -31 -41 -33
Statistically significant: *: p<0.05 and **: p<0.01.
HCD: Historical Control Data.
n: number of animals with available values; /: not applicable.

Table 12: Microscopic examination

Gender Males Females
Group 1 2 3 4 1 2 3 4
Dose level (mg/kg) 0 10 30 100 0 10 30 100
Concentration (mg/mL) 0 2 6 20 0 2 6 20
Number of animals 10 10 10 10 10 10 9 10
Number examined 5 5 5 5 5 5 5 5
Stomach
Hyperplasia, squamous epithelium
Minimal 0 0 2 0 0 0 3 1
Slight 0 0 1 4 0 1 0 3
Moderate 0 0 0 0 0 0 0 1

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

> 100 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:

NOEL

Remarks:

(local toxicity)

Effect level:

< 10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOEL

Remarks:

(reproductive performance)

Effect level:

> 100 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.
There were no test item treatment-related behavioral changes or external abnormalities at pup examination.
Clinical signs were not attributed to the test item as they were not dose-related, were reported in isolated animals and/or are routinely observed in rat pups of this strain and age.

Mortality / viability:

no mortality observed

Description (incidence and severity):

see table 1.
There were no effects on pup viability.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

see table 3.
There were no relevant effects on mean body weight or mean body weight change during the lactation period.
On Day 1 p.p. and when compared with controls, statistically significant higher mean body weight was recorded in female pups at 10 and 100 mg/kg/day (+8% and +10%, respectively).
A relationship with the test item was considered to be unlikely as these variations were not dose-related and of minor amplitude.

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):

see table 5.
There were no relevant test item-related effects on AGD and AGD/BW 1/3 in the pups.
At 30 mg/kg/day and when compared with controls, females had statistically significant higher mean AGD and AGD/BW1/3 ratios (+18% and +16%, respectively). These findings were not attributed to the test item treatment as the differences from controls were not dose-related and were within the range of the Historical Control Data.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples or areolae were observed in any male pups.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio
see table 4.
There were no test item-related effects on sex ratios (% of male pups).

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Table 1: Pup viability

Dose level (mg/kg/day) 0 10 30 100
Number of pups (litters) 116 (9) 105 (9) 88 (7) 114 (9)
Mean live birth index (%) ± SD 96.7 ± 6.67 99.1 ± 2.77 98.8 ± 3.14 99.2 ± 2.37
Number of pups found dead
or cannibalized (litters affected)
(from Days 1 to 13 p.p.) 6 (3) 1 (1) 3 (2) 2 (2)
Mean viability index ± SD
(Day 4 p.p. %) 95.9 ± 6.62 99.1 ± 2.77 98.8 ± 3.14 99.2 ± 2.37
Mean lactation index ± SD
(Day 13 p.p. %) 100 ± 0.0 100 ± 0.0 96.4 ± 9.45 98.6 ± 4.17
Statistical analysis: no significance.

Table 2: Pup clinical signs

Dose level (mg/kg/day) 0 10 30 100
Abnormal color of abdomen, n (L) 1 (1)
Generalized palor 1 (1)
Cold to the touch, n (L) 4 (1)
Emaciated appearance, n (L) 2 (1) 14 (1)a
Absence of milk in stomach, n (L) 5 (2) 1 (1) 2 (2)
Hematoma head, limbs n (L) 6 (2) 1 (1) 1 (1)
Scab neck, thorax, abdomen n (L) 1 (1) 2 (2)
Wound, abdomen n (L) 1 (1)
Thinning of hair 7 (1)
Total pups affected, n (L) 14 (4) 3 (3) 15 (2) 10 (3)
( ): litter incidence; n: number of pups; L: litter.
a: litter from one female showing signs of poor clinical condition after parturition.

Table 3: Pup body weight

Sex Male Female
Dose level (mg/kg/day) 0 10 30 100 0 10 30 100
Body weight (g)
. Day 1 p.p. 8.0 8.3 8.1 8.3 7.3 7.9* 7.7 8.0**
. Day 4 p.p. post-culling 11.6 12.0 11.3 11.9 10.6 11.4 11.0 11.5
. Day 8 p.p. 21.3 21.3 20.8 21.4 19.9 20.3 20.0 20.5
. Day 13 p.p. 34.9 34.9 34.4 35.3 33.2 33.4 33.6 34.1

Pup body weight change (g)
. Days 1 to 4 p.p. +3.6 +3.7 +3.3 +3.6 +3.3 +3.6 +3.3 +3.6
. Days 4 to 8 p.p. +9.7 +9.3 +9.5 +9.5 +9.2 +8.8 +9.1 +9.0
. Days 4 to 13 p.p. +23.3 +22.9 +23.1 +23.4 +22.6 +22.0 +22.6 +22.5
. Days 8 to 13 p.p. +13.6 +13.5 +13.6 +13.9 +13.3 +13.2 +13.5 +13.5
Statistical analysis: *: p<0.05 and **: p<0.01

Table 4: Sex ratio

Dose level (mg/kg/day) 0 10 30 100
. Day 1 p.p. (%) 54.6 ± 9.70 45.6 ± 20.60 47.6 ± 11.55 48.4 ± 10.27
. Day 13 p.p. (%) 52.5 ± 5.00 47.7 ± 12.87 51.8 ± 4.72 47.8 ± 4.53
Statistical analysis: no significance.

Table 5: Anogenital distance

Sex Males Females
Dose level (mg/kg/day) 0 10 30 100 0 10 30 100
Number of litters 9 9 7 9 9 9 7 9
AGD 4.58 4.61 4.86 4.75 2.50 2.67 2.94* 2.60
Standard deviation 0.08 0.26 0.33 0.30 0.231 0.264 0.402 0.238
HCD 5.23 (3.38-6.76)
n=256 pups 2.84 (1.59-4.71)
n=270 pups
BW on Day 1 p.p. 8.0 8.3 8.1 8.3 7.3 7.9* 7.7 8.0**
AGD/BW1/3 2.29 2.28 2.43 2.35 1.29 1.34 1.49* 1.30
Standard deviation 0.055 0.135 0.161 0.145 0.110 0.149 0.207 0.110
HCD 2.60 (1.68-3.29)
n=256 pups 1.43 (0.80-2.37)
n=270 pups
Statistical significance: *: p<0.05 and **: p<0.01.
HCD: mean (minimum-maximum) Historical Control Data values.

Table 6: Pup thyroids

Sex Pups, Day 13 p.p.
Dose level (mg/kg/day) 0 10 30 100
T4 (ng/mL) 35.78 35.87 36.12 36.46
Standard deviation 3.631 5.530 5.596 3.828
n 9 9 7 9
% from controls / 0 +1 +2
TSH (pg/mL) 1375 1653 1731 1619
Standard deviation 464.0 567.3 718.6 631.0
n 9 8 6 8
% from controls / +20 +26 +18
n: number of animals with available values; /: not applicable.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 100 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

Reproductive effects observed:

no

Conclusions:

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 p.p. at the dose levels of 10, 30 or 100 mg/kg/day.

Based on the experimental conditions of this study:
- the No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 100 mg/kg/day in the absence of adverse findings at this high-dose level,
- the No Observed Effect Level (NOEL) for parental local toxicity could not be determined as microscopic findings were observed in the stomach in females from 10 mg/kg/day and in males from 30 mg/kg/day,
- the No Observed Effect Level (NOEL) for reproductive performance (mating, fertility and delivery) was considered to be 100 mg/kg/day in the absence of any test item-related findings at this high-dose level,
- the No Observed Effect Level (NOEL) for toxic effects on progeny was considered to be 100 mg/kg/day in the absence of any test item-related findings at this high-dose level.

Executive summary:

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, through mating and, for the females, throughout gestation and until Day 13 post-partum (p.p.).

This study provides information on:

. the possible health hazards likely to arise from repeated exposure over a relatively limited period of time,

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

 

The study was performed according to OECD guideline No. 422 (29 July 2016) and in compliance with GLP regulations.

 

Methods

Three groups of ten male and ten female Sprague-Dawley rats received the test item.

The test item was administered daily by the oral route (gavage) at dose levels of 10, 30 or 100 mg/kg/day, under a constant dosage volume of 5 mL/kg/day.

Males were treated for an overall period of at least 4 weeks: 2 weeks before mating, during the mating period (up to 4 days) and until the day before euthanasia. Females were treated for an overall period of 7 to 8 weeks: 2 weeks before mating, throughout mating (up to 4 days) and gestation (3 weeks) until Day 13 post-partum (p.p.) inclusive. A control group of ten males and ten females received the vehicle only (0.5% methylcellulose aqueous solution) under the same experimental conditions.

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

The F0 animals were checked daily during the treatment period for mortality, morbidity and clinical signs. Detailed clinical observations were conducted weekly. Body weight and food consumption were recorded weekly during the premating, mating (food consumption was not recorded during the mating period), gestation and lactation periods. Estrous cycle stage was determined each morning from 2 weeks before the treatment period until the females had mated, and on Day 14 p.p. before euthanasia.

The F0 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 euthanized and discarded without further examination.

Pups were weighed on Days 1, 4, 8 and 13 p.p. and observed daily for clinical signs, abnormal behavior and external abnormalities. The physical development of pups was assessed by measuring the anogenital distance on Day 1 p.p. (all pups before culling) and by counting the number of nipples and areolae in male pups on Day 12 p.p.

A Functional Observation Battery (FOB) was performed on five F0 animals per sex and group at the end of the treatment period.

Laboratory investigations (hematology and blood biochemistry) were carried out on at least five F0 males and five F0 females from each group at the end of the study. Plasma thyroid hormone levels (TSH and T4) were determined at terminationin all F0 males and in pups euthanized on Day 13 p.p.

The F0 males were euthanized after completion of the mating period (i.e. after 30 days of treatment) and F0 females were euthanized on Day 14 p.p.

A full macroscopic post-mortem examination was performed, with 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 five males and lactating females in the control and high-dose groups, on all macroscopic lesions (all groups) and on the stomach from five males and five lactating females at the low- and intermediate-dose levels. Reproductive organs from pairs that did not mate or with females that were not pregnant or from females that did not deliver were also microscopically observed.

Pups were euthanized on Day 13 p.p. and a detailed external examination was performed, with particular attention to the external genital organs. Thyroids with parathyroids from one pup/sex/litter were preserved.

 

Results

Chemical analysis:

The actual test item concentrations in the analyzed dose formulations (i.e. in Weeks 1, 3 and 6) remained within an acceptable range of variation (-3.6% to +9.8%) when compared with the nominal values (nominal concentration ± 15% required).

 

F0 animals:

Mortality: there were no test item-related unscheduled deaths.

 

Clinical signs: ptyalism occurred in both sexes and in all test item-treated groups during the premating period, with the exception of males at 30 mg/kg/day, and persisted in some females at 100 mg/kg/day during the gestation period. This sign, commonly observed when a test item is administered by gavage, was not considered as an adverse effect.

 

Functional Observation Battery and motor activity: there were no relevant effects on Functional Observation Battery tests or motor activity data at any dose level.

 

Body weight and body weight change: there were no statistically significant differences on mean body weight or mean body weight change at any dose level in males or in females during the premating, gestation and lactation periods. At 100 mg/kg/day and when compared with controls, a few variations of body weight gain were noted in females during the lactation period. These differences were considered to be related to the test item but of minor toxicological importance in view of their slight magnitude and in the absence of effects on body weight.

 

Food consumption: there were no relevant effects on food consumption at any dose level.

 

Hematology: there were no statistically significant test item-related effects on hematology parameters at any dose level. Minor changes observed were considered to be of no toxicological importance as they did not correlate with microscopic findings, were of slight magnitude, poorly dose-related, and/or within or close to the range of the Historical Control Data.

 

Blood biochemistry: at 100 mg/kg/day and when compared with controls, blood biochemistry changes included increased chloride level in males (+2%, p<0.05) and decreased inorganic phosphorus level in females (-17%, p<0.05). In view of their magnitude, as mean values remained within the range of the Historical Control Data and in absence of histopathological findings on kidneys, these changes were considered to be of minor toxicological importance.

 

Thyroid hormones: there were no test item-related effects on T4 or TSH levels in F0 males or pups at any dose level.

Pathology: there were no relevant differences in organ weights between test item-treated and control groups. Dose-related hyperplasia of the squamous epithelium of the stomach, accompanied by hyperkeratosis, possibly related to a local irritant effect was observed in the stomach in 1/5 females at 10 mg/kg/day, 3/5 males and 3/5 females at 30 mg/kg/day and in 4/5 males and 5/5 females at 100 mg/kg/day. This change was not accompanied by noticeable inflammation and was considered non adverse.

 

Estrous cycle: there were no test item-related effects on the mean length of the estrous cycle or the mean number of cycles at any dose level.

 

Pairing, mating and fertility data: there were no relevant effects on the mating, fertility or gestation index at any dose level.

 

Delivery data: there were no effects on the mean duration of gestation, numbers of corpora lutea and implantation sites, numbers of pups delivered or percentages of pre-/post-implantation losses at any dose level.

 

Pups

Mortality and viability: there were no effects on the live birth, viability or lactation indexes at any dose level.

 

Clinical signs and external abnormalities: there were no effects on behavioral changes or external abnormalities at pup examination.

 

Pups body weight: there were no relevant effects on mean body weight or mean body weight change in pups at any dose level.

 

Sex ratio: there were no effects on the mean percentage of male fetuses (sex ratio) at any dose level.

 

Development: there were no relevant effects on the anogenital distance and there were no nipples or areolae in male pups at any dose level.

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 p.p. at the dose levels of 10, 30 or 100 mg/kg/day.

 

Based on the experimental conditions of this study:

. the No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 100 mg/kg/day in the absence of adverse findings at this high-dose level,

. the No Observed Effect Level (NOEL) for parental local toxicity could not be determined as microscopic findings were observed in the stomach in females from 10 mg/kg/day and in males from 30 mg/kg/day,

. the No Observed Effect Level (NOEL) for reproductive performance (mating, fertility and delivery) was considered to be 100 mg/kg/day in the absence of any test item-related findings at this high-dose level,

. the No Observed Effect Level (NOEL) for toxic effects on progeny was considered to be 100 mg/kg/day in the absence of any test item-related findings at this high-dose level.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study is considered to be reliable with a klimish 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

Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (Papineau 2019)

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, through mating and, for the females, throughout gestation and until Day 13 post-partum (p.p.). The study was performed according to OECD guideline No. 422 (29 July 2016) and in compliance with GLP regulations.

Three groups of ten male and ten female Sprague-Dawley rats received the test item. The test item was administered daily by the oral route (gavage) at dose levels of 10, 30 or 100 mg/kg/day, under a constant dosage volume of 5 mL/kg/day.

Males were treated for an overall period of at least 4 weeks: 2 weeks before mating, during the mating period (up to 4 days) and until the day before euthanasia. Females were treated for an overall period of 7 to 8 weeks: 2 weeks before mating, throughout mating (up to 4 days) and gestation (3 weeks) until Day 13 post-partum (p.p.) inclusive. A control group of ten males and ten females received the vehicle only (0.5% methylcellulose aqueous solution) under the same experimental conditions.

There were no test item-related unscheduled deaths. Ptyalism occurred in both sexes and in all test item-treated groups during the premating period, with the exception of males at 30 mg/kg/day, and persisted in some females at 100 mg/kg/day during the gestation period. This sign, commonly observed when a test item is administered by gavage, was not considered as an adverse effect. There were no relevant effects on Functional Observation Battery tests or motor activity data at any dose level. There were no statistically significant differences on mean body weight or mean body weight change at any dose level in males or in females during the premating, gestation and lactation periods. At 100 mg/kg/day and when compared with controls, a few variations of body weight gain were noted in females during the lactation period. These differences were considered to be related to the test item but of minor toxicological importance in view of their slight magnitude and in the absence of effects on body weight. There were no relevant effects on food consumption at any dose level. There were no statistically significant test item-related effects on hematology parameters at any dose level. Minor changes observed were considered to be of no toxicological importance as they did not correlate with microscopic findings, were of slight magnitude, poorly dose-related, and/or within or close to the range of the Historical Control Data. At 100 mg/kg/day and when compared with controls, blood biochemistry changes included increased chloride level in males (+2%, p<0.05) and decreased inorganic phosphorus level in females (-17%, p<0.05). In view of their magnitude, as mean values remained within the range of the Historical Control Data and in absence of histopathological findings on kidneys, these changes were considered to be of minor toxicological importance. There were no test item-related effects on T4 or TSH levels in F0 males or pups at any dose level. There were no relevant differences in organ weights between test item-treated and control groups. Dose-related hyperplasia of the squamous epithelium of the stomach, accompanied by hyperkeratosis, possibly related to a local irritant effect was observed in the stomach in 1/5 females at 10 mg/kg/day, 3/5 males and 3/5 females at 30 mg/kg/day and in 4/5 males and 5/5 females at 100 mg/kg/day. This change was not accompanied by noticeable inflammation and was considered non adverse.

There were no test item-related effects on the mean length of the estrous cycle or the mean number of cycles at any dose level. There were no relevant effects on the mating, fertility or gestation index at any dose level. There were no effects on the mean duration of gestation, numbers of corpora lutea and implantation sites, numbers of pups delivered or percentages of pre-/post-implantation losses at any dose level.

There were no effects on the live birth, viability or lactation indexes at any dose level. There were no effects on behavioral changes or external abnormalities at pup examination. There were no relevant effects on mean body weight or mean body weight change in pups at any dose level. There were no effects on the mean percentage of male fetuses (sex ratio) at any dose level. There were no relevant effects on the anogenital distance and there were no nipples or areolae in male pups at any dose level.

Based on the experimental conditions of this study:

. the No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 100 mg/kg/day in the absence of adverse findings at this high-dose level,

. the No Observed Effect Level (NOEL) for parental local toxicity could not be determined as microscopic findings were observed in the stomach in females from 10 mg/kg/day and in males from 30 mg/kg/day,

. the No Observed Effect Level (NOEL) for reproductive performance (mating, fertility and delivery) was considered to be 100 mg/kg/day in the absence of any test item-related findings at this high-dose level,

. the No Observed Effect Level (NOEL) for toxic effects on progeny was considered to be 100 mg/kg/day in the absence of any test item-related findings at this high-dose level.

Effects on developmental toxicity

Description of key information

No developmental study is required.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available, no classification for reprotoxicity is required for 2 -hydroxy-3 -phenoxypropyl acrylate according to the Regulation EC n°1272/2008.