4-phenylbutenone

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 Nov 2021 - Sep 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted 29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-phenylbutenone

EC Number:

204-555-1

EC Name:

4-phenylbutenone

Cas Number:

122-57-6

Molecular formula:

C10H10O

IUPAC Name:

4-phenylbut-3-en-2-one

Test material form:

solid

Remarks:

Solid mass, yellowish green

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Han:WIST of Wistar origin

Details on species / strain selection:

The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility and availability of historical control data at the Test Facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90. Hungary
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) Males 77–84 d; Females 80–85 d
- Weight at study initiation: (P) Males: 323-377 g; Females: 210-257 g
- Housing: Type III polypropylene/polycarbonate
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Mated females: individually
Males after mating: 2 animals / cage

- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 20 d
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): >10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

vegetable oil

Remarks:

Sunflower oil (Helianthi annui oleum raffinatum)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle (sunflower oil) in concentrations of 20, 60 and 150 mg/mL. Formulations were prepared in the formulation laboratory of Test Facility not longer than three days beforehand and stored at 5 ± 3°C until use.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is not soluble in distilled water and carboxy methyl cellulose at the intended concentrations. Therefore, sunflower oil was used for preparation of the formulations administered to the animals.
- Concentration in vehicle: 0, 20, 60, 150 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg body weight

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: until copulation occurred or 14 days had elapsed
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged (how): single

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations (verification of concentrations and homogeneity) was performed in the Analytical Laboratory of Test Facility twice during the study. Five aliquots of 10 mL of each formulation and five aliquots of 10 mL control substance (vehicle) were taken and analyzed.
Date of sampling: November 16 and December 14, 2021
Date of analysis: November 17 and December 15, 2021
Concentration of the test item in the dosing formulations varied in the range of 107 and 109 % of the nominal values at both analytical occasions.
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. The recovery of the test item from the vehicle was within the acceptance criteria – 100 and 107 % relative to nominal concentrations of 1 mg/mL and at 200 mg/mL, respectively. 4-Phenyl-3-buten-2-one proved to be stable in sunflower oil at the intended concentrations at room temperature for one day and at 5 ± 3°C for three days.

Duration of treatment / exposure:

41 days treatment/ observation period for male animals (pre-mating, mating and post-mating)
51 - 64 days treatment/ observation period for dams (pre-mating, mating, gestation and lactation)
40- or 42-days treatment/ observation period (pre-mating, mating and post-mating) for non-pregnant female

Frequency of treatment:

daily

Details on study schedule:

The experimental period involved 20 days of acclimatization (including 14 days for examination of estrous cycle) and 41 days treatment/ observation period for male animals (pre-mating, mating and post-mating), 51-64-days treatment/ observation period for dams (pre-mating, mating, gestation and lactation), 40- or 42-days treatment/ observation period (pre-mating, mating and post-mating) for nonpregnant female animals and necropsy days.
For F1 offspring, investigations included 4-, 13- or 14-16-day observation period and euthanasia or necropsy day depending on the endpoints (blood sampling on post-natal days 4 and 13 or terminal necropsy on post-natal days 14-16).
The study was terminated on Day 64.
The day of first treatment was considered as day 0 of examination.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

80 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

240 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

600 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen on the basis of the results of preliminary repeated dose toxicity studies in rats already available for the test item. The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals.
The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.
- Rationale for animal assignment (if not random): All parental (P) male and female animals were sorted according to body weight and divided to weight groups aided by a computerized calculation. There was an equal number of animals from each weight group in each of the experimental groups assigned by randomization to ensure that the mean weight of animals from all test groups was as uniformly as practicable. Grouping was aided by SPSS/PC+ software, verifying the homogeneity and variability between the groups according to the actual body weight.

Positive control:

none

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly starting from first day of dosing

FOOD CONSUMPTION: Yes
- The food consumption was determined weekly by weighing the given and non-consumed diet with an accuracy of 1 g during the treatment period except mating phase (pre-mating days 0, 7, 13, and post-mating days 20, 27, 34 and 40 for male animals; pre-mating days 0, 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for dams).

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

The estrous cycle was investigated in all female animals being considered for study by examining vaginal smears before the treatment started for two weeks.
Animals exhibiting typical 4-5 days cycles were used in the study preferably. However, two female animals showing irregular cycle were also included because of the high number of animals that failed to exhibit typical cycle. Including these animals had no influence on the outcome of the study. Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of copulation.
Vaginal smear also was prepared on the day of the necropsy.
Vaginal smears were stained with 1 % aqueous methylene blue solution. The smears were examined with a light microscope.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on Day 41
- Maternal animals: All surviving pregnant animals on Lactation days 14-16 (Days 50-64), non-pregnant animals on Day 40, 42


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

HISTOPATHOLOGY / ORGAN WEIGHTS
Detailed histological examinations were performed on the ovaries, testes, epididymides – with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure – in the animals in the control and high dose groups. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Full histological processing and evaluation of organs and tissues were performed in the dead female animal (1/12 at 600 mg/kg bw/day).
In addition, the following organs were processed and examined histologically based on the macroscopic findings or on the thyroid hormone levels:
- stomach: 12/12 control male, 1/12 male at 240 mg/kg bw/day, 12/12 male and 1/12 female at 600 mg/kg bw/day
- kidneys: 2/12 male in the control group, 2/12 male and 2/12 female at 80 mg/kg bw/day, 3/12 female at 240 mg/kg bw/day, 2/12 male and 1/12 female at 600 mg/kg bw/day
- skin: 1/12 female at 80 mg/kg bw/day
- lymph node: 1/12 male at 600 mg/kg bw/day
- thymus: 1/12 male control
- thyroid glands: 12/12 male at 600 mg/kg bw/day
The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome (at a thickness of 2-4 µm), placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.

At the time of termination, body weight, brain weight, weight of the testes, epididymides and prostate as well as seminal vesicles with coagulating glands as a whole, of adult male animals were determined. Paired organs were weighed together. Absolute organ weight was recorded. Relative (to body and brain weight) organ weights were calculated and reported. The thyroid weights were determined in all parental male animals based on findings in thyroid hormone levels.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
Pups selected for thyroid gland preservation (one male and one female pup per litter) were subjected to gross macroscopic observations.Pups euthanized on post-natal day 13 were carefully examined externally for gross abnormalities. Selected organs and tissues were excised, trimmed of any adherent tissue, as appropriate, weighed

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

Statistics:

The statistical evaluation of appropriate data (see below lists) was performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out.
If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.

-Lists of parameters that statistical analysis was performed
Body weight, Body weight gain, Food consumption, Percentage of fertile males, Male copulatory index, Thyroid hormone (FT3, FT4 and TSH) level, Organ weights (absolute and relative to the body and brain weights), Female copulatory index, Female fertility index, Gestation index, Duration of pregnancy (days), Number of implantations / dams, Post-implantation mortality, Litter weight on postnatal days 0, 4 and 13, Mean body weight gain per litter between postnatal days 0-4, 4-13 and for overall postnatal days, Number of live births per litter, and number of viable pups per litter on postnatal days 0, 4 and 13, Survival Index of pups on postnatal day 13, Sex ratio % (on postnatal days 0 and 13), Normalized anogenital distance, Number of nipples/areolae in male pups and Thyroid hormone (FT3, FT4 and TSH) level of pups on postnatal day 13

Reproductive indices:

Copulatory index: Measure of animals’ ability to mate
Males: [(No. of males with confirmed mating)/(Total no. of males cohabited)]*100
Females: [(No. of sperm positive females)/(Total no. of females cohabited)]*100

Fertility index: Measure of male’s ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant.
Males: [(No. of males impregnating a female)/(Total no. of males with confirmed mating)]*100
Females: [(No. of pregnant females)/(No. of sperm positive females)]*100

Gestation index: Measure of pregnancy that provides at least one live pup
[(No. of females with live born pups)/(No. of pregnant females)]*100

Offspring viability indices:

Post-implantation/ pre-natal mortality (intrauterine mortality):
[(No. of implantations - No. of liveborns)/(No. of implantations)]*100

Post-natal mortality /Extra-uterine mortality:
[(No. of liveborns - No. of live pups on PND13)/(No. of liveborns)]*100

Survival Index:
[(No. of live pups on PND13)/(No. of pups born)]*100

Sex ratio
[(No. of pups examined-No. of males(females))/(No. of pups examined)]*100

Normalized anogenital distance
[(Absolute anogenital distance)/(Body weight)]

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item related clinical signs (decreased activity and/or nuzzling up the bedding material) were detected with variable incidence and duration in male and in female animals at 600 mg/kg bw/day from Days 3 or 4 up to the end of the treatment period.
Some male and female animal at 600 mg/kg bw/day salved immediately before the treatment – presumably due to a reflexive process or gastric lesions.
In the dead female animal (1/11 mated female at 600 mg/kg bw/day, no. 425) nuzzling up the bedding material was observed on the mating day and on gestation days 0 and 1 before the death. This animal died before the implantation occurred, therefore, pregnancy could not be proven and disposition of dead animal was not feasible.
Parental animals (male and female) exhibited predominantly normal behavior and physical condition with no abnormalities in the control and at 80 and 240 mg/kg bw/day at the daily clinical observations. Alopecia on the skin of hips was noted for one female animal (1/12) at 80 mg/kg bw/day from gestation day 20 up to and including lactation day 15. Alopecia is a common finding in experimental rats of this strain with similar age occurring also in non-treated animals.
In male animals at 600 mg/kg bw/day, decreased activity (12/12), nuzzling up the bedding material (12/12), salivation before the treatment (3/12), left leaning posture and circling if holding upright down at tail (1/12) were observed.
Similarly, female animals at 600 mg/kg bw/day showed decreased activity, nuzzling up the bedding material and salivation before the treatment during the pre-mating, post-mating, gestation and lactation periods as follows:
- decreased activity: 12/12 pre-mating, 1/1 post-mating, 5/10 gestation
- nuzzling up the bedding material: 12/12 pre-mating, 1/1 post-mating, 10/10 gestation, 10/10 lactation
- salivation before the treatment: 5/12 pre-mating, 1/1 post-mating, 1/10 gestation, 1/10 lactation
Transient neuro-behavioral changes (abnormal posture and circling, from Day 34 up to Days 35/ 36) in one male animal were toxicologically not relevant and were probably individual ones considering the low incidence and short duration.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female animal died during the course of the study (1/12 at 600 mg/kg bw/day) one day after successful mating on Day 16. There were no preceding clinical signs, however, the body weight decreased from Day 13 up to Day 15 (13 g reduction between Days 13 and 15) in this animal.
Based on histopathology findings, the cause of the death was presumably related to gastric lesions induced by the test item.
There was no mortality in the control, 80, 240 or 600 mg/kg bw/day groups in male animals or in the control, 80 or 240 mg/kg bw/day in female animals during the entire observation period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight development was not adversely influenced in male or female animals at 80, 240 or 600 mg/kg bw/day during the entire treatment period.
Slight reduction of body weight gain in male animals at 600 mg/kg bw/day resulted in only slight changes in the mean body weight (≤6 % reduction relative to control) and therefore this change was considered to be a non-adverse effect of no toxicological relevance.
There were no statistically significant differences between the control and test item-administered male animals at 80 and 240 mg/kg bw/day in the mean body weight and body weight gain from Day 0 up to and including Day 40 as well as in female animals at 80, 240 and 600 mg/kg bw/day during the premating, gestation and lactation periods.
Statistical significance with respect to the control was observed at the lower mean body weight gain of male animals at 600 mg/kg bw/day between Days 0-7 and if summarized (between Days 0 and 40). As a result, the mean body weight was lowered with respect to the control at a similar degree on Days 7, 13 and 20.
The body weight gain of male animals was only transiently lowered at 600 mg/kg bw/day during week 1 and was similar to the control from Day 7 up to the termination of the study. The difference with respect to the control in the mean body weight gain of these animals improved but not ceased by the end of the treatment period (-93 % up to -21 % relative to control, on week 1 and if summarized, respectively).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item-related adverse changes in the food consumption of male or female animals at any dose level (80, 240 or 600 mg/kg bw/day).
A slight reduction of the mean daily food consumption was transient and was in full accordance with body weight changes in male animals at 600 mg/kg bw/day during week 1. Therefore, this minor change was judged to have no toxicological relevance.
The mean daily food consumption was similar in the control and 80 or 240 mg/kg bw/day groups during the entire observation period (pre-mating and post-mating in male animals, pre-mating, gestation and lactation periods in female animals).
Statistical significance with respect to the control was observed at the slightly lower mean daily food consumption in male and female animals at 600 mg/kg bw/day between Days 0 and 7 and at the higher mean food consumption in male animals at 600 mg/kg bw/day between Day 27-34.
This minor although statistically significant change in the mean daily food consumption was judged to be toxicologically not relevant due to the low degree and transient occurrence at starting of the treatment.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

only thyroid hormones analysed (please refer to 'Endocrine findings')

Endocrine findings:

no effects observed

Description (incidence and severity):

The thyroid hormone (FT3, FT4 and TSH) levels were not adversely affected in parental male animals or in PND13 offspring at any dose levels.
No statistically significant differences with respect to the control were observed in the concentrations of FT3, FT4 and TSH of PND13 offspring at 80, 240 and 600 mg/kg bw/day.
In the parental male animals, lowered mean FT3 and FT4 levels were detected at 600 mg/kg bw/day when compared to the control. However, the mean and individual FT3 and FT4 values were within the min and max values reported in the HCD for this rat strain. The mean TSH concentrations were slightly above the mean control value in male animals independently from doses. However, there was a big variation in the individual values in all dose groups with individual values exceeding the historical control ranges in all dose groups including the concurrent controls – 1/12 control, 4/12 at 80 mg/kg bw/day, 1/12 at 240 mg/kg bw/day and 2/12 at 600 mg/kg bw/day – independent from doses. Also, the mean TSH value in the 80 and 600 mg/kg bw dose group very slightly exceeded the historical control range. The higher TSH values are sporadic and lack any dose-response relationship. Also, they do not correlate with lower FT3/FT4 values in individual animals. Therefore, this is not considered to be a specific effect of the test item.
To confirm this and exclude any effects on the thyroid of the test item, the thyroids of the male animals of the high dose group were weighted and examined histopathologically. There were no changes in the weight or cell morphology of thyroid glands in the high dose administered male animals. As there was no enlargement and no histological changes observed in the thyroid, a test item influence is considered as unlikely. Slight decreases in FT3 and FT4 levels along with a slightly elevated TSH concentration, partly with a wide variation and without any indications for histological changes, are considered to be a normal physiological reaction and of no toxicological relevance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Please refer to 'Clinical signs'

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological investigation did not reveal toxic or other test item-related lesions detectable by microscopic examination of the investigated reproductive organs of experimental male and female animals at 600 mg/kg bw/day.
Squamous cell hyperplasia was detected in the non-glandular area of stomach in all male animals at 600 mg/kg bw/day, in one male animal at 240 mg/kg bw/day and in two female animals at 600 mg/kg bw/day. Squamous cell hyperplasia was accompanied by ulceration and restorative inflammation in all high dose administered male animals only.
The cell morphology of thyroid glands of male animals in the control and 600 mg/kg bw/day groups was identical.

Dead animal:
Squamous cell hyperplasia accompanied with ulceration and restorative inflammation in the forestomach (non-glandular area) was seen in female animal found dead at 600 mg/kg bw/day. Inflammation was accompanied with infiltration of inflammatory cells and fibroblast proliferation in the lamina propria in dead animal. In addition, congestion in the lungs (in connection with agony) and atrophy (lymphocyte depletion) in the spleen occurred in dead animal. No toxic or other histological lesions were detectable in the liver, adrenal glands, kidneys, ovaries or in the wall of abdominal aorta, regarding the macroscopic findings.

Surviving animals:
The investigated organs of reproductive system (testes, epididymides) were histologically normal and characteristic on the sexually mature organism in the male animals in control (12/12) and in 600 mg/kg bw/day (12/12) groups. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa); representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphology in the testes of investigated control and treated animals. The histological picture of epididymides was normal in all cases as well.
In the female animals in the control (12/12) and 600 mg/kg bw/day groups (12/12, including dead and non-pregnant females), the ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle in the most cases. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
The histological structure and the cellularity of pituitary with special attention on the cytomorphology and proportion of acidophilic and basophilic cells in the adenohypophysis were the same in the control and treated male and female animals.
Focal squamous cell hyperplasia and ulceration accompanied with restorative inflammation were seen in the mucous membrane of non-glandular stomach (forestomach) in several animals:
- Focal squamous cell hyperplasia: 1/1 male at 240 mg/kg bw/day, 12/12 male at 600 mg/kg bw/day and 1/1 female at 600 mg/kg bw/day
- ulceration accompanied with restorative inflammation: 12/12 male at 600 mg/kg bw/day
No dysplasia and down growths of the hyperplastic basal cells (suspected neoplasm formation) were detectable in the altered areas.
No inflammatory or hyperplastic lesions were seen in the glandular stomach of the affected animals.
Hyperplasia is characterized by thickening of the epithelial layer that often forms a broad-based lesion with marked hyperkeratosis and infiltration of inflammatory cells in the lamina propria, accompanied with fibroblast proliferation.
Necrosis and ulceration of the non-glandular stomach mucosa is rarely seen except in treated rats. If focal necrosis of superficial epithelium affects a sufficient number of cells, erosion or ulceration may be present, depending on the depth of the lesion (Uehara et al., 2018).
The high concentration of the test item in the formulation administered via gavage likely plays a dominant role in the development of these local lesions.
Histological examination did not reveal pathological lesion (atrophy degeneration, pigmentation, mineralization, amyloidosis, inflammation, proliferation, follicular hyperplasia, C-cell hyperplasia, adenoma, carcinoma) in the investigated thyroid glands of experimental animals belonging to the control or high dose groups. (The most important lesions of thyroid gland are the atrophy, degeneration, pigmentation, mineralization, amyloidosis, inflammatory lesion, proliferation, follicular cell hyperplasia, C-cell hyperplasia, adenomas and carcinomas.)
Hyperplasia of mesenteric lymph node was an individual finding in single male animal at 600 mg/kg bw/day (1/1) presumably in accordance with gastric lesion.
One or both sided pyelectasia was detected in male (2/2, 2/2 and 2/2, in the control, 80 and 600 mg/kg bw/day, respectively) and in female (2/2, 3/3. 1/1, at 80, 240 and 600 mg/kg bw/day, respectively). Pyelectasia without degenerative, inflammatory or other histological (fibrotic etc.) lesions is considered as a common finding in laboratory rats and is judged to be of no toxicological significance.
Atrophy of hair follicles accompanied with multifocal alopecia occurred in single female animal (1/1 female) at 80 mg/kg bw/day. This is a common finding in laboratory rats and is considered as individual disease.
No morphological evidence of test item-related acute or subacute injury (degeneration, inflammation, necrosis etc.) of the liver, the pancreas, the cardiovascular system, the respiratory system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the central, or peripheral nervous system, the eyes, the integumentary system, the reproductive system was observed.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The examined parameters of estrous cycle – percentage of animals with regular/ irregular cycle, mean number of cycles, mean length of cycles, mean number of days in pro-estrous, estrous or diestrous – were comparable in animals appointed for the control and test item dosed groups during the pre-treatment period and mostly during pre-mating period.
There were no significant differences between the control and test item-treated animals in the estrous cycle at 80, 240 and 600 mg/kg bw/day during the two weeks pre-mating period.
Statistical significance with respect to the control was only detected at the lower mean number of days in proestrus at 600 mg/kg bw/day.
Overall, there was no test item-related adverse or biologically relevant impairment of estrous cycle during the two weeks pre-mating period.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

The reproductive performance was not influenced by the test item at 80, 240 or 600 mg/kg bw/day in male or female animals based on the examined parameters.
Statistical significance with respect to the control was detected at the slightly higher fertility index (higher percentage of fertile male animals) at 80 and 240 mg/kg bw/day and lower fertility index of male animals at 600 mg/kg bw/day.
Similarly, the fertility index (percentage of pregnant female animals) exceeded the control at 80 and 240 mg/kg bw/day.
The percentage of pregnant females and of females with live offspring, the pre-coital interval, number of conceiving days, copulatory and gestation indices were comparable in the control and 80, 240 and 600 mg/kg bw/day.
These minor differences with respect to the control were indicative of biological variation and the data met well historical control values even at lowered fertility index at 600 mg/kg bw/day. In the absence of related findings in the reproductive parameters, this minor difference was considered to be toxicologically not relevant.

Details on results (P0)

For detailed results, please refer to tables in the attached document

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive performance

2

Dose descriptor:

NOAEL

Effect level:

240 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

gross pathology

histopathology: non-neoplastic

3

Dose descriptor:

NOEL

Effect level:

80 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

gross pathology

Remarks on result:

other: Thickening of gastric mucosa at cardia (forestomach) in a single male animal at 240 mg/kg bw/day

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Test item-related clinical signs were not identified in the offspring between post-natal days 0 and 13.
The percentage of offspring with clinical signs was not related to dosing in the control, 80, 240 and 600 mg/kg bw/day groups. Cold, not suckled, smaller than others, dead and missing offspring were detected at the clinical observations in control, 240 or 600 mg/kg bw/day groups mainly shortly after birth.
The percentage of cold pups was highest in the control group.
These signs and random differences with respect to the control were considered to be toxicologically not relevant as the signs were transient and were not associated with depression of the development of the offspring.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test item-related effect on offspring’s extra uterine mortality at 80, 240 or 600 mg/kg bw/day.
The mean number of live pups per litter and the mean number of viable pups per litter were similar in all groups on post-natal days 0, 4 and 13.
The survival indices were comparable in the control and test item-treated (80, 240 or 600 mg/kg bw/day) groups on post-natal days 4 and 13.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight development of the offspring was undisturbed during the observation period from birth to post-natal day 13.
The mean weights (litter and pup) and mean weight gains (litter and pup) were mostly similar in the control and in all test item-treated groups (80, 240 and 600 mg/kg bw/day) on post-natal days 0, 4 and 13.
Statistical significance was detected at the higher mean litter weight on post-natal day 4 and at the higher mean litter weight gain compared to the control between post-natal days 0-4 at 80 mg/kg bw/day.
The mean pup weight was slightly lowered when compared to control at 600 mg/kg bw/day on postnatal day 0 and was comparable to the control thereafter (PND4, PND8).
When male and female pups were evaluated separately, statistical significance with respect to the control was detected at the higher mean body weight of male pups at 80 mg/kg bw/day and of female pups at 80 and 240 mg/kg bw/day as well as at the slightly lower mean body weight of male pups at 600 mg/kg bw/day on post-natal day 4. However, as these statistical differences occurred only initially, improved over the course of the study and the deviating values met well the individual ranges of the historical control data, these findings were considered to be toxicologically not relevant.
The mean litter weights and litter weight gain were comparable in the control and at 240 and 600 mg/kg bw/day and the mean pup weight and pup weight gain were comparable in the control and at 80 and 240 mg/kg bw/day on post-natal days 0, 4 and 13.
Overall, the body weight data were considered to be not adversely impaired.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No statistically significant differences with respect to the control were observed in the concentrations of FT3, FT4 and TSH of PND13 offspring at 80, 240 and 600 mg/kg bw/day.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The anogenital distances (absolute and normalized) were not adversely affected in male or female offspring at 80, 240 or 600 mg/kg bw/day.
In male offspring, statistical significance was observed at the shorter mean normalized anogenital distance at 80 mg/kg bw/day and at the longer mean normalized anogenital distance at 600 mg/kg bw/day when compared to the control.
In the female offspring, the absolute anogenital distance was slightly shorter at 600 mg/kg bw/day, the mean normalized anogenital distance was slightly shorter at 80 mg/kg bw/day when compared to the control.
However, the changes in anogenital distances were of minor degree – i.e., were within or close to the historical controls – therefore these were considered as neither biologically relevant nor toxicologically adversely impaired.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There was no nipple in male offspring at 80, 240 or 600 mg/kg bw/day on PND13. Nipples/areoles were not visible in any of the examined male offspring in the control or 80, 240 or 600 mg/kg bw/day groups on post-natal day 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination after post-natal day 13.
There were no macroscopic findings in dead or stillborn pups on post-natal days 0 – 2:
- 1 dead male and 1 stillborn male in control group
- 3 dead pups (2 male and 1 female) and 1 stillborn female pup at 240 mg/kg bw/day. Female stillborn pup was cannibalized by mother (head was missing)
Terminally (on post-natal days 14-16), the organs and tissues were normal in pups subjected to necropsy observations: 22/22 control, 24/24 at 80 mg/kg bw/day, 24/24 at 240 mg/kg bw/day, 20/20 at 600 mg/kg bw/day.

Histopathological findings:

not examined

Details on results (F1)

For detailed results, please refer to tables in the attached document

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

gross pathology

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of the present oral exposure study, the test item did not adversely affect reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats at 80, 240 and 600 mg/kg bw/day as far as investigated in this study. At 600 mg/kg bw/day, death (female), clinical signs (male and female) and local effects in the forestomach (macroscopic and microscopic lesions in all males and in two females) were observed. Local effects were seen in the forestomach at 240 mg/kg bw/day (macroscopically and microscopically observed, one male). There were no test item-related effects at 80 mg/kg bw/day (male and female). The development of the F1 offspring was not impaired from birth up to post-natal day 13 as far as investigated in this study (sex distribution, clinical signs, mortality, body weight, anogenital distance, nipple retention, necropsy) after repeated oral administration of dams at 80, 240 and 600 mg/kg bw/day.
Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL for systemic toxicity of male/ female rats: 240 mg/kg bw/day
NOEL for male rats: 80 mg/kg bw/day
NOEL for female rats: 240 mg/kg bw/day
NOAEL for reproductive performance of male/ female rats: 600 mg/kg bw/day
NOAEL for F1 Offspring: 600 mg/kg bw/day

Executive summary:

A Reproduction/Developmental Toxicity Screening Test according to OECD TG 421 and GLP was performed. The objective of this study was to obtain initial information on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 80, 240 and 600 mg/kg bw/day compared to control animals. Four groups of Han:WIST rats consisting of 12 animals per group and sex were administered orally (by gavage) once daily at 0 (vehicle only), 80, 240 and 600 mg/kg bw/day doses in concentrations of 20, 60 and 150 mg/mL corresponding to a 4 mL/kg bw dosing volume. A group of vehicle (sunflower oil) treated animals (n= 12/sex) served as a control. All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 41 days). Dams were additionally exposed through the gestation period and up to lactation days 13-15, i.e., up to the day before necropsy (altogether for 50-64 days). Non-pregnant female animals were administered for 40, 42 days – during the premating, mating and post-mating periods, for 24 or 25 days after positive vaginal smear was detected. The dams were allowed to litter and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter.

One female animal died during the course of the study at 600 mg/kg bw/day one day after successful mating on Day 16. There were no preceding clinical signs and the body weight decreased from Day 13 up to Day 16 in this animal. Based on histopathology findings, the cause of the death was related to gastric lesions induced by the test item. Test item related clinical signs (decreased activity and/or nuzzling up the bedding material) were detected with variable incidence and duration in male and in female animals at 600 mg/kg bw/day from Days 3 or 4 up to the end of the treatment period. The body weight development was not adversely influenced in male or female animals at 80, 240 or 600 mg/kg bw/day during the entire treatment period. Slight reduction of body weight gain in male animals at 600 mg/kg bw/day resulted in only slight changes in the mean body weight (≤6 % reduction relative to control) and therefore this change was considered to be a non-adverse effect of no toxicological relevance. There were no test item-related adverse changes in the food consumption of male or female animals at any dose level. There was no test item related adverse or biologically relevant impairment of the estrous cycles, pregnancies, deliveries and reproductive performance at any dose level. The mean weights of thyroid glands, testes, epididymides, seminal vesicle with coagulating gland or prostate as a whole (absolute and relative to body and brain weights) were comparable in the male animals in control and 600 mg/kg bw/day. The thyroid hormone (FT3, FT4 and TSH) levels were not adversely affected in parental male animals or in PND13 offspring at any dose levels. Minor variations with TSH and FT3/FT4 values above and below control levels were found. However, this was limited to few individual animals and lacked dose-response, as well as biological plausibility. Follow-up examinations on organ weight and histopathology of the thyroid found no changes in high dose administered male animals. Thus, these minor changes are not toxicologically relevant. Gross necropsy revealed test item related macroscopic findings in the stomach of several male animals and in two female animals at 600 mg/kg bw/day (thickened, hard mucous membrane, adhesion to stomach or diaphragm, or dilatation, thin wall and liquid accumulation in the stomach) and in one male animal at 240 mg/kg bw/day (thickening of gastric mucosa). Histopathological investigation did not reveal toxic or other test item-related lesions detectable by microscopic examination of the investigated reproductive organs of experimental male and female animals at 600 mg/kg bw/day.  Squamous cell hyperplasia was detected in the non-glandular area of stomach in all male animals at 600 mg/kg bw/day, in one male animal at 240 mg/kg bw/day and in two female animals at 600 mg/kg bw/day. Squamous cell hyperplasia was accompanied by ulceration and restorative inflammation in all high dose administered male animals only. There were no differences in the cell morphology (histology) of thyroid glands of male animals in the control and 600 mg/kg bw/day groups.

The offspring’s development was not impaired at 80, 240 or 600 mg/kg bw/day from birth up to post-natal day 13. No adverse effects on mortality, clinical signs, body weight, anogenital distance, nipple retention or necropsy were detected in the offspring terminated as scheduled.

 

Conclusion

Under the conditions of the present oral exposure study, 4-Phenyl-3-buten-2-one did not adversely affect reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats at 80, 240 and 600 mg/kg bw/day as far as investigated in this study.

At 600 mg/kg bw/day, death (female), clinical signs (male and female) and local effects in the forestomach (macroscopic and microscopic lesions in all males and in two females) were observed.

Local effects were seen in the forestomach at 240 mg/kg bw/day (macroscopically and microscopically observed, one male).

There were no test item-related effects at 80 mg/kg bw/day (male and female).

The development of the F1 offspring was not impaired from birth up to post-natal day 13 as far as investigated in this study (sex distribution, clinical signs, mortality, body weight, anogenital distance, nipple retention, necropsy) after repeated oral administration of dams at 80, 240 and 600 mg/kg bw/day.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

 

NOAEL for systemic toxicity of male/ female rats: 240 mg/kg bw/day

NOEL for male rats: 80 mg/kg bw/day

NOEL for female rats: 240 mg/kg bw/day

NOAEL for reproductive performance of male/ female rats: 600 mg/kg bw/day

NOAEL for F1 Offspring: 600 mg/kg bw/day