Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Reproduction/developmental toxicity screening test in rats, OECD 421, GLP, oral gavage, doses: 0/80/240/600 mg/kg bw/d, NOAEL (parental systemic) = 240 mg/kg bw/day, NOAEL (offspring) = 600 mg/kg bw/day

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 Nov 2021 - Sep 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
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
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.
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
80 mg/kg bw/day
Dose / conc.:
240 mg/kg bw/day (nominal)
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
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)]
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: 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.
For detailed results, please refer to tables in the attached document
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
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
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
Critical effects observed:
no
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
For detailed results, please refer to tables in the attached document
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
Critical effects observed:
no
Reproductive effects observed:
no
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

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
600 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
good quality, as a study according to OECD TG 421 and GLP is available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 421, Pasics 2022


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


 


Supporting studies:


Sub-chronic Feed Studies


Rat


Methyl trans-styryl ketone was investigated for its repeated dose toxicity via the oral route (NTP, 2012a). Groups of 10 male and 10 female F344/N rats were fed diets containing 0%, 0.025%, 0.05%, 0.1%, 0.2%, or 0.4% methyl trans-styryl ketone (equivalent to average daily doses of approximately 18, 36, 72, 145, or 290 mg methyl trans-styryl ketone/kg body weight to males and 19, 38, 77, 150, or 300 mg/kg to females) for 14 weeks. Groups of 10 male and 10 female rats were fed the same concentrations for 24 days. All core study rats survived to the end of the study. Final mean body weights of males and females receiving 0.4% and mean body weight gains of males receiving 0.4% were significantly less than those of the controls. Feed consumption by exposed groups was similar to that by the controls. Clinical findings included diarrhoea and hyperactivity in males and females. Results of sperm motility and vaginal cytology evaluations indicated methyl trans-styryl ketone is unlikely to be a reproductive toxicant in male rats; however, it exhibits potential for reproductive toxicity in female rats based upon an increased probability of extended diestrus at the highest exposure concentration. In all exposed groups of males, there were treatment-related increased incidences of goblet cell hyperplasia of the respiratory epithelium of the nose and nephropathy of the kidney. In females, there was an increased incidence of goblet cell hyperplasia of the respiratory epithelium of the nose in the group receiving 0.4%. Goblet cell hyperplasia of the respiratory epithelium of the nose is likely due to inhalation of methyl trans-styryl ketone that volatilized from feed.


Based on the results of sperm motility and vaginal cytology evaluations, the reproductive organ weights, and the histopathology of the reproductive organs, NOEALs for reproductive toxicity is considered to be 290 mg/kg bw, the highest dose level tested, for males and 150 mg/kg bw for females.


Mouse


Methyl trans-styryl ketone was investigated for its repeated dose toxicity via the oral route in mice (NTP, 2012a). Groups of 10 male and 10 female B6C3F1 mice were fed diets containing 0%, 0.025%, 0.05%, 0.1%, 0.2%, or 0.4% methyl trans-styryl ketone (equivalent to average daily doses of approximately 55, 110, 220, 400, or 750 mg/kg to males and 50, 100, 200, 350, or 600 mg/kg to females) for 14 weeks. There were no treatment-related deaths in either sex (one male receiving 0.2% died following blood collection, and one control female was found dead). Mean body weights of males and females receiving 0.4% were significantly less than those of the controls. Feed consumption by exposed groups was similar to that by the controls. Hyperactivity in both sexes was the only treatment-related clinical finding. Results of sperm motility and vaginal cytology evaluations indicated methyl trans-styryl ketone is unlikely to be a reproductive toxicant in male mice; however, it exhibits potential for reproductive toxicity in female mice based upon an increased probability of extended diestrus at the lowest and the highest exposure concentrations. There were significantly increased incidences of olfactory epithelial atrophy of the nose in males and females receiving 0.4%. Goblet cell hyperplasia of the respiratory epithelium of the nose is likely due to inhalation of methyl trans-styryl ketone that volatilized from feed.


Based on the results of sperm motility and vaginal cytology evaluations, the reproductive organ weights, and the histopathology of the reproductive organs, NOAEL for reproductive toxicity is considered to be 750 mg/kg bw, the highest dose level tested, for males. No NOAEL could be identified for females due to the extended diestrus at the dose levels that were chosen for the evaluation of reproductive toxicity.


 


Sub-chronic Dermal Studies


Rat


Groups of 10 male and 10 female rats were dermally administered 0, 22, 44, 87.5, 175, or 350 mg methyl trans-styryl ketone/kg body weight in 95% ethanol, 5 days per week for 14 weeks (NTP, 2012a). Groups of 10 male and 10 female clinical pathology rats were administered the same doses for 23 days. All rats survived to the end of the study. Mean body weights of 175 and 350 mg/kg males were significantly less than that of the vehicle controls. Clinical findings in groups administered 175 or 350 mg/kg included dermal irritation, thickened skin, and ulceration at the site of application. Results of sperm motility and vaginal cytology evaluations indicated methyl trans-styryl ketone is unlikely to be a reproductive toxicant in male or female rats at the doses used in this study. There were no significant differences in any of the reproductive organ weights. Histologically, there were significantly increased incidences of epidermal hyperplasia, hyperkeratosis, chronic active inflammation, epidermal necrosis, and sebaceous gland hypertrophy in the skin at the site of application in males and/or females. There were significantly increased incidences of goblet cell hyperplasia of the nose in 350 mg/kg males and 22, 175, and 350 mg/kg females.


Based on the results of this study, NOAEL of 350 mg/kg bw for rats is considered for reproductive toxicity.


Mouse


Groups of 10 male and 10 female mice were dermally administered 0, 87.5, 175, 350, 700, or 1,400 mg methyl trans-styryl ketone/kg body weight in 95% ethanol, 5 days per week for 13 weeks (NTP, 2012a). All mice in the 700 and 1,400 mg/kg groups were sacrificed moribund before the end of the study. The final mean body weights of surviving groups of dosed males and females were similar to those of the vehicle controls; however, the mean body weight gains of the 175 mg/kg groups were significantly less than those of the vehicle controls. Clinical findings at the site of application included dermal irritation in 350 mg/kg males and crust formation in all 700 and 1,400 mg/kg mice except one female. Results of sperm motility and vaginal cytology evaluations indicated methyl trans-styryl ketone is unlikely to be a reproductive toxicant in male or female mice at the doses used in this study. There were no significant differences in any of the reproductive organ weights. There were treatment-related increased incidences of epidermal hyperplasia, hyperkeratosis, chronic active inflammation, epidermal necrosis, sebaceous gland hypertrophy, and hair follicle hyperplasia in the skin at the site of application in males and females. There were increased incidences of olfactory epithelial atrophy of the nose in groups of males and females administered 350 mg/kg or greater. Based on the results of this study, NOAEL of 350 mg/kg bw for mice is considered for reproductive toxicity.



Short description of key information:
There was no indication based upon the sperm motility and vaginal cytology evaluation results, reproductive organ weights, and histopathology of the reproductive organs that methyl trans-styryl ketone has any potential to be a reproductive toxicant in male or female rats or mice when applied dermally. Since significant amounts of the test chemical were absorbed dermally in rats and in mice, the dermal route of administration would provide a sufficient amount of chemical systemically available to assess possible toxicity in tissues, including in reproductive organs (Sauer et al., 1977; NTP, 2012a).

Effects on developmental toxicity

Description of key information

Read-Across: Pre-natal developmental toxicity in rats, OECD 414, GLP, oral gavage, doses: 0/125/250/500 mg/kg bw/d, NOAEL (maternal general tox) = 125 mg/kg bw/d, NOAEL (developmental tox) = 250 mg/kg bw/d (Chaudhari 2020)

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to Read-Across Statement attached in Section 13
Reason / purpose for cross-reference:
read-across source
Species:
rat
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related clinical signs or symptoms were observed in any animal up to 250 mg/kg bw/d. Symptoms of hypothermia, lethargy, prostration and excessive salivation were observed in all animals of the 500 mg/kg bw/d group starting from the first day of dosing until the termination. These clinical signs were evident from 1 h after dosing until the evening observation. However, no clinical signs were observed at the morning observation. This indicates that these signs were reversible in nature. No treatment-related clinical signs were observed in nesting/pregnancy behaviour in any of the animals.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One animal from the high dose group was found dead during morning observation on GD9 Although gross lesions on lung and stomach were observed in this animal, the exact cause of death could not be determined.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight of pregnant and non-pregnant females remained camparable on GD 0, 5, 8, 11, 14, 17 and 20 for the control, 125 and 250 mg/kg bw/d group. The mean body weights of pregnant females on GD17 was significantly reduced in the 500 mg/kg bw/d group as compared to the control (p<0.05), whereas it remained comparable to the control on GD 0, 5, 8, 11 and 14. The % change in body weights with respect to GD0 was also statistically comparable among all the dose groups of pregnant and non-pregnant females, except for the decreased body weight gain in the 500 mg/kg bw/d group as compared to the control on GD17 (p<0.05). A trend of decrease in body weight and body weight gain was observed in pregnant females of the 500 mg/kg bw/ d group compared to the control on GD20. Although it remained statistically insignificant due to high variation in the groups, the change in body weight and body weight gain at 500 mg/kg bw/d was considered as an effect of the test item.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumed per day was significantly reduced in the 125 and 500 mg/kg bw/d groups on GD8 and GD11 when compared to the control group, while it remained statistically comparable among the groups at all other time points. Although the observed decrease in the food consumption of the 500 mg/kg bw/d group was considered to be an effect of the test item administration, it lacks toxicological significance due to its transient occurrence (significant reduction only on GD8 and GD11 and not on GD14, 17 and 20). On the contrary, the significant change observed in the 125 mg/kg bw/d group lacked dose-dependency and was therefore considered to be an inconsequential effect.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Thyroid hormones (T3 and T4) and thyroid stimulating hormone (TSH) levels in the sera were found to be statistically comparable among the control and treatment groups.
Endocrine findings:
no effects observed
Description (incidence and severity):
Thyroid hormones (T3 and T4) and thyroid stimulating hormone (TSH) levels in the sera were found to be statistically comparable among the control and treatment groups. No significant difference in the absolute and relative weight of thyroid and parathyroid between the treatment groups and the control group were found. Three females in the 500 mg/kg bw/d group exhibited unilateral and bilateral congenital cysts in the thyroid gland. These were considered to be spontaneous in nature and not attributable to the test item
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were observed in nesting/pregnancy behaviour in any of the animals.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significant difference in the absolute and relative weight of uterus, ovary, thyroid and parathyroid between the treatment groups and the control group were found.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No gross findings were observed at 0 or 125 mg/kg. At 250 mg/kg, two animals showed pathological alterations in stomach which included white spots of muscular portion, increased size/swelling of muscular portions, and swelling of glandular portion. At 500 mg/kg, a total of 15 female animals showed pathological alterations in stomach which included white spots of muscular tissue, increased size of muscular portion, and increased size/swelling of glandular portion and red discolouration of the lung.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):

Microscopic findings at 250 mg/kg included focal hyperkeratosis in stomach in one animal (minimal in severity). At 500 mg/kg, microscopic findings included focal/multifocal hyperkeratosis in stomach; focal, erosion and oedema of muscular tissue in stomach, lymphocyte infiltration in muscular tissue in stomach, diffuse degeneration of glandular tissue in stomach and focal alveolar haemorrhage in lung in 7 animals. Minimal to moderate hyperkeratosis of stomach observed in most of the animals from the 500 mg/kg bw/d group were considered to be test item-related. This can be due to local irritation of the gastric mucosa.
Three females in the 500 mg/kg bw/d group exhibited unilateral and bilateral congenital cysts in the thyroid gland. These were considered to be spontaneous in nature and not attributable to the test item.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The number of implantation sites and the pre-and post-implantation loss (%) remained unchanged in treatment groups when compared to control.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No significant changes in the number of resorptions were observed in any dose groups.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The early and late resorptions were found to be comparable among all the treatment groups and control group.
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead foetuses were observed in any dose groups.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
At termination 18/25, 24/25, 17/25 and 13/25 females were found to be pregnant from the control 125 mg/kg bw, 250 mg/kg bw, and 500 mg/kg bw dose groups, respectively. Pregnancy rates were calculated as 72%, 96%, 68% and 52% for the control, 125 mg/kg bw, 250 mg/kg bw/day, and 500 mg/kg dose groups, respectively. This observation indicates a statistically significant and dose-dependent effect of the test item on the pregnancy rate.
Other effects:
no effects observed
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
changes in number of pregnant
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
changes in number of pregnant
Dose descriptor:
LOAEL
Remarks:
local
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg, a significant decrease in female foetal weight (by 8.2%) and a significant decrease in sex-combined foetal weight (by 6.4%) were observed as compared to the control group. No other significant changes in foetal weight were observed.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No reduction in number of live offspring was observed in any dose group when compared to control.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratios were comparable in all groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No significant differences in litter size were observed in any groups.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
No significant differencein AGD were observed in any groups.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Gross external observation of foetuses revealed that 2.09, 3.79, 5.00 and 6.60 percentage of foetuses from control (G1), 125 (G5), 250 (G2) and 500 (G3) mg/kg bw/d, respectively, showed malformations/variations. The variation included haemorrhage:1.57 (G1), 2.07 (G5), 3.89 (G2), 4.72 (G3) % of foetuses and the malformations include: retarded growth (Runt): 0.52 (G1), 1.38 (G5), 0.56 (G2) and 1.89 (G3) % of foetuses; dome-shaped head: 1.03 (G5) and 0.94 (G3) % of foetuses; Anury (absence of tail): 0.56 (G2) % of foetuses. A significant correlation was found for the doses of test item and the total % of fetuses with malformations/variations (P<0.05). Further, when individual malformations/variations were compared, a significant increase in incidences of haemorrhage was observed in fetuses of the 500 mg/kg bw/d group as compared to the control group (P<0.05). However, no significant correlation was observed when litter was used as the unit for comparing the malformations/variations or when data of individual malformations/variations (other than haemorrhage) were compared. These observations were found to be independent of the treatment and rather seem to be spontaneous in nature.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A number of skeletal variations/malformations were observed, however none of them were attributed to the test chemical since they lacked dose dependency and (or) were considered common to developing foetuses.
The following skeletal malformations/variations were observed in the skull: incomplete ossification of Frontal and Parietal observed in 1 /57 foetus at 500 mg/kg, dome-shaped Frontal and Parietal found in 1/150 foetus at 125 mg/kg, 1/57 at 500 mg/kg, flattened Parietal observed in 1/150 at 125 mg/kg and 1/57 at 500 mg/kg, fused Nasal, Premaxilla and Maxilla found in 1/150 at 250 mg/kg, elongated and bipartite Interparietal in 1/94 foetus at 250 mg/kg, small triangular-shaped Interparietal observed in 1/150 at 125 mg/kg, absence of Zygomatic in 2/94 at 250 mg/kg, absence of Hyoid in 2/100 foetuses at 0 mg/kg. Fused ribs were observed in 1/94 foetuses at 250 mg/kg, branched ribs in 1/57 foetuses at 500 mg/kg, misaligned ribs at 1/94 at 250 mg/kg and 2/57 foetuses at 500 mg/kg. Misaligned ribs were seen in 2/57 foetuses at 500 mg/kg, absent Thoracic, Lumbar, Sacral and caudal Vertebrae in 1/94 foetuses at 250 mg/kg.
Visceral malformations:
no effects observed
Description (incidence and severity):
No variations/malformations were observed during visceral and head razor examinations in any of the groups.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
A significant difference in crown to rump length was observed in the 125 mg/kg bw/d group when compared to the control (P<0.001). Such change was not observed in any other of the dose group and therefore it was considered to be an incidental finding.
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 1. Table for Maternal Evaluation


 








































































































Groups



Control



Low Dose



Mid Dose



High Dose



Dose (mg/kg body weight)



0



125



250



500



Initial animals per group



25



25



25



25



Confirmed Pregnancy at necropsy



18



24



17



13



Pregnancy rate (%)



72



96



68



52



No. of Corpora lutea



14.50 ± 2.62



13.33 ±1.97



13.94 ± 2.73



13.15 ± 2.34



No. of implantation sites



12.44 ± 3.17



12.92 ± 1.84



12.24 ± 3.17



11.08 ± 3.80



No. of resorptions



1.83 ± 2.81



0.83 ± 1.01



1.65 ± 2.26



1.92 ± 3.48



Pre-implantation loss (%)



13.15 ± 20.98



2.91 ± 5.41



12.82 ±15.23



16.66 ± 25.53



Post-implantation loss (%)



14.11 ± 22.04



6.19 ± 6.77



15.23 ± 22.21



18.66 ± 34.90



Litter size



10.61 ± 3.81



12.08 ± 1.69



10.59 ± 4.18



8.83 ± 4.95



No of live foetuses



10.61 ± 3.81



12.08 ± 1.69



10.59 ± 4.18



8.83 ± 4.95



No of dead foetuses



0



0



0



0



Live Foetuses (%)



100



100



100



100



Values are represented as Mean± SD.


Pre-implantations loss = [(no. of corpora lutea − no. of implantations)/ no. of corpora lutea] x 100


Post-implantation loss = [(no. of resorptions)/ no. of total implantations] x 100


 


Table 2. Table for Body Weight


 
























































































































































Pregnant Females


 



Group



Dose (mg/kg)



 


 



Day of gestation and body weight in grams



0



5



8



11



14



17



20



Control



0



Mean



215.56



236.39



245.11



257.50



272.56



298.06



332.00



SD



13.26



13.14



13.53



14.36



14.97



17.06



25.65



N



18



18



18



18



18



18



18



 Low Dose



125



Mean



214.25



234.92



243.00



256.83



273.13



298.08



334.58



SD



12.81



9.66



10.44



10.87



12.16



14.10



15.37



N



24



24



24



24



24



24



24



  Mid Dose



250



Mean



213.94



237.00



244.94



258.06



272.41



294.35



330.18



SD



12.23



14.83



16.37



17.83



21.72



25.19



32.90



N



17



17



17



17



17



17



17



 High Dose



500



Mean



215.54



237.54



241.69



247.67



263.83



279.08↓



309.00



SD



11.64



15.23



13.93



10.80



13.03



21.94



30.36



N



13



13



13



12



12



12



12



↓: Decreased as compared to control (P<0.05)


 
























































































































































Non-Pregnant Female


 



Group



Dose (mg/kg)



 



Day of gestation and body weight in grams



0



5



8



11



14



17



20



 Control



0



Mean



215.14



233.57



240.29



244.29



244.14



246.14



250.14



SD



11.96



12.22



10.27



17.04



16.93



17.14



20.45



N



7



7



7



7



7



7



7



 Low Dose



125



Mean



211.00



219.00



219.00



223.00



225.00



230.00



238.00



SD



./.



./.



./.



./.



./.



./.



./.



N



1



1



1



1



1



1



1



 Mid Dose



250



Mean



222.00



240.38



242.25



249.88



247.75



250.25



253.00



SD



6.72



16.10



10.65



11.21



9.38



9.91



11.30



N



8



8



8



8



8



8



8



 High Dose



500



Mean



223.33



242.58



242.50



245.33



242.83



242.25



248.33



SD



15.20



22.44



19.64



20.83



15.80



15.78



16.74



N



12



12



12



12



12



12



12



 


Table 3. Table for Body Weight Change


 











































































































































Pregnant Females


 



Group



Dose (mg/kg)



 



Gestation Day and body weight change (%)



5



8



11



14



17



20



 Control



0



Mean



9.74



13.80



19.61



26.62



38.48



54.15



SD



2.76



3.32



5.27



5.92



6.98



9.50



N



18



18



18



18



18



18



 Low Dose



125



Mean



9.86



13.64



20.12



27.74



39.43



56.56



SD



5.12



5.43



6.03



6.64



7.85



9.72



N



24



24



24



24



24



24



 Mid Dose



250



Mean



10.80



14.51



20.64



27.32



37.55



54.29



SD



3.85



4.48



5.23



7.07



8.19



12.13



N



17



17



17



17



17



17



 High Dose



500



Mean



10.19



12.14



15.92



23.48



30.60↓



44.72



SD



3.27



2.70



2.86



4.04



8.72



14.17



N



13



13



12



12



12



12



↓: Decreased as compared to control (P<0.05)


 











































































































































Non-Pregnant Females


 



Group



Dose  (mg/kg)



 



Gestation Day and body weight change (%)



5



8



11



14



17



20



 Control



0



Mean



8.66



11.85



13.66



13.51



14.43



16.27



SD



4.35



5.23



7.35



5.65



5.52



7.20



N



7



7



7



7



7



7



 Low Dose



125



Mean



3.79



3.79



5.69



6.64



9.00



12.80



SD



./.



./.



./.



./.



./.



./.



N



1



1



1



1



1



1



 Mid Dose



250



Mean



8.19



9.12



12.55



11.60



12.73



13.95



SD



4.42



3.38



3.52



2.32



3.06



3.15



N



8



8



8



8



8



8



 High Dose



500



Mean



8.88



8.92



10.15



9.00



8.65



11.34



SD



9.71



9.58



9.78



7.38



5.89



5.80



N



12



12



12



12



12



12



 


Table 4. Table for External Gross Evaluation of Foetuses


 

















































































































































































































Group



 Control



 Low Dose



 Mid Dose



 High Dose



Dose level (mg/kg body weight)



0



125



250



500



Total No. of Litters with live foetuses



18



24



17



11



Total No. of live Foetuses



191



290



180



106



Total No. of Male Foetuses



98



133



89



61



Total No. of Female Foetuses



93



157



91



45



Weight of foetuses (g)



3.74 ± 0.59



3.69 ± 0.35



3.57 ± 0.38



3.50 ± 0.37↓



Weight of male foetuses (g)



3.82 ± 0.63



3.82 ± 0.30



3.64 ± 0.35



3.62 ± 0.32



Weight of female foetuses (g)



3.65± 0.54



3.57 ± 0.35



3.51 ± 0.39



3.35 ± 0.40↓



Weight of Placenta (g)



0.47 ± 0.09



0.49 ±0.07



0.50 ± 0.09



0.45 ± 0.09



Crown to rump length (cm)



3.68 ± 0.23



3.41 ± 0.31↓↓↓



3.68 ± 0.28



3.67 ± 0.28



AGD of Male foetuses (mm)



3.20 ± 0.33



3.10 ± 0.25



3.17 ± 0.26



3.10 ± 0.28



AGD of Female foetuses (mm)



1.30 ± 0.21



1.28 ± 0.16



1.30 ± 0.35



1.27 ± 0.14



Normalised AGD of Male foetuses



2.06 ± 0.23



1.98 ± 0.18



2.06 ± 0.16



2.02 ± 0.16



Normalised AGD of Female foetuses



0.85 ± 0.14



0.84 ± 0.10



0.86 ± 0.27



0.85 ± 0.10



Male/Female sex ratio (per dam)



1.31 ± 1.28



0.94 ± 0.45



1.25 ± 1.03



1.53 ± 1.28



External observations



Total no. of Foetuses with No Abnormality Detected



187



279



171



99



Total no. of Foetuses with abnormalities



4



11



9



7



Foetuses with abnormalities (%)



2.09



3.79



5.00



6.60 ↑



Litters with abnormalities (%)



5.56



33.33



35.29



27.27


 

Abnormalities



Haemorrhage (V)



3(1)



6(5)



7(5)



5(3)



Haemorrhage (%)



1.57(5.56)



2.07(20.83)



3.89(29.41)



4.72(27,27) ↑



Runt (M)



1(1)



4(4)



1(1)



2(2)



Runt (%)



0.52(5.56)



1.38(16.67)



0.56(5.88)



1.89(18.18)



Anury (M)



0(0)



0(0)



1(1)



0(0)



Anury (%)



0(0)



0(0)



0.56(5.88)



0(0)



Dome-shaped Head (M)



0(0)



3(3)



0(0)



1(1)



Dome-shaped Head (%)



0(0)



1.03(12.50)



0(0)



0.94(9.09)



Values are represented as Mean± SD; AGD: Anogenital distance; Normalised AGD: AGD/ Body weight(1/3); the incidence of the individual defect is presented as a number/ Percent of foetuses (number/ percent of litters); Abnormalities: includes malformations and variations; M: Malformations; V: Variations ↓:Decreased as compared to control (P<0.05); ↓↓↓:Decreased as compared to control (P<0.001); ↑:Increased as compared to control (P<0.05)


 


Table 5. Table for Skeletal Anomalies of the Foetuses


 
























































































































































































































































Groups



 Control



 Low Dose



 Mid Dose



 High Dose



Dose (mg/kg body weight)



0



125



250



500



 



No.



%



No.



%



No.



%



No.



%



Foetuses examined



100



150



94



57



No abnormality detected



83



134



86



41



Total and % Foetuses with abnormalities



17



17.00



16



10.67



8



8.51



16



28.07



 



Skull



Incomplete ossification of Frontal & Parietal (V)



0



0



0



0



0



0



1



1.75



Dome-shaped frontal and Parietal (M)



0



0



1



0.67



0



0



1



1.75



Flattened Parietal (M)



0



0



1



0.67



0



0



0



0



Fused Nasal, Premaxilla & Maxilla (M)



0



0



1



0.67



0



0



0



0



Elongated, Bipartite Interparietal (M)



0



0



0



0



1



1.06



0



0



Small Triangular-shaped Interparietal (M)



0



0



1



0.67



0



0



0



0



Absence of Zygomatic (M)



0



0



0



0



2



2.13



0



0



Absence of Hyoid (M)



2



2



0



0



0



0



0



0



 



Ribs



Fused (M)



0



0



0



0



1



1.06



0



0



Branched (M)



0



0



0



0



0



0



1



1.75



Misaligned (M)



0



0



0



0



1



1.06



2



3.51



Absent (M)



0



0



0



0



0



0



0



0



 



Sternebrae



Unossified/Incompletely ossified Sternebrae (1 or more) (V)



13



13.0



12



8.0



5



5.32



13



22.81



Misaligned (M)



1



1



0



0



0



0



2



3.51



 



Vertebrae



Unossified Caudal vertebrae (V)



1



1



0



0



0



0



0



0



Thoracic, Lumbar, Sacral and caudal Vertebrae absent (M)



0



0



0



0



1



1.06



0



0



The incidence of the individual defect is presented as a number and percentages of litters


Abnormalities: includes malformations and variations; M: Malformations; V: Variations


 


Table 6. Table for Skeletal Anomalies of the Foetuses(with respect to Litters)


 













































































































































































































































Groups



 Control



 Low Dose



 Mid Dose



 High Dose



Dose (mg/kg body weight)



0



125



250



500



 



No.



%



No.



%



No.



%



No.



%



Litters examined



18



24



17



11



No abnormality detected



7



11



13



2



Total and % Litters with abnormalities



11



61.11



13



54.17



4



23.53



9



81.82



 



Skull



Incomplete ossification of Frontal & Parietal (V)



0



0



0



0



0



0



1



9.09



Dome-shaped frontal and Parietal (M)



0



0



1



4.17



0



0



1



9.09



Flattened Parietal (M)



0



0



1



4.17



0



0



0



0



Fused Nasal, Premaxilla & Maxilla (M)



0



0



1



4.17



0



0



0



0



Elongated, Bipartite Interparietal (M)



0



0



0



0



1



5.88



0



0



Small Triangular-shaped Interparietal (M)



0



0



1



4.17



0



0



0



0



Absence of Zygomatic (M)



0



0



0



0



2



11.76



0



0



Absence of Hyoid (M)



2



11.11



0



0



0



0



0



0



 



Ribs



Fused (M)



0



0



0



0



1



5.88



0



0



Branched (M)



0



0



0



0



0



0



1



9.09



Misaligned (M)



0



0



0



0



1



5.88



2



18.18



 



Sternebrae



Unossified/Incompletely ossified Sternebrae (1 or more) (V)



9



50.0



11



45.83



3



17.65



6



54.55



Misaligned (M)



1



5.56



0



0



0



0



2



18.18



 



Vertebrae



Unossified Caudal vertebrae (V)



1



5.56



0



0



0



0



0



0



Thoracic, Lumbar, Sacral and caudal Vertebrae absent (M)



0



0



0



0



1



5.88



0



0



The incidence of the individual defect is presented as a number and percentages of litters


Abnormalities: includes malformations and variations; M: Malformations; V: Variations


 


Table 7. Table for Absolute Organ Weight


 

















































































































Pregnant



Group



Dose (mg/ Kg)



 



Body weight



Uterus



Ovaries



Right



Left



Control



0



Mean



332.00



61.73



0.0878



0.0818



SD



25.65



20.64



0.0152



0.0208



N



18



18



18



18



Low dose



125



Mean



334.58



66.61



0.0791



0.0828



SD



15.37



8.57



0.0197



0.0145



N



24



24



24



24



Mid dose



250



Mean



330.18



62.60



0.0860



0.0854



SD



32.90



25.27



0.0138



0.0183



N



17



17



17



17



High dose



500



Mean



309.00



49.70



0.0861



0.0753



SD



30.36



24.55



0.0202



0.0112



N



12



12



12



12



 

















































































































Non-Pregnant




Group



Dose (mg/ Kg)



 



Body weight



Uterus



Ovaries



Right



Left



Control



0



Mean



250.14



0.87



0.0634



0.0706



SD



20.45



0.14



0.0053



0.0083



N



7



7



7



7



Low dose



125



Mean



238.00



0.82



0.0655



0.0539



SD



./.



./.



./.



./.



N



1



1



1



1



Mid dose



250



Mean



253.00



0.88



0.1388



0.0712



SD



11.30



0.14



0.2047



0.0074



N



8



8



8



8



High dose



500



Mean



248.33



1.10



0.07



0.08



SD



16.74



0.41



0.0102



0.0100



N



12



12



12



12



 


Table 8. Table for Relative Organ weight to Body weight


 

















































































































Pregnant



Group



Dose (mg/ Kg)



 



Body weight



Uterus



Ovaries



Right



Left



Control



0



Mean



332.00



18.41



0.0264



0.0249



SD



25.65



5.63



0.0042



0.0073



N



18



18



18



18



Low dose



125



Mean



334.58



19.88



0.0237



0.0248



SD



15.37



2.13



0.0059



0.0043



N



24



24



24



24



Mid dose



250



Mean



330.18



18.68



0.0261



0.0259



SD



32.90



7.16



0.0040



0.0050



N



17



17



17



17



High dose



500



Mean



309.00



15.57



0.0278



0.0245



SD



30.36



7.21



0.0053



0.0035



N



12



12



12



12



 

















































































































Non Pregnant



Group



Dose (mg/ Kg)



 



Body weight



Uterus



Ovaries



Right



Left



Control



0



Mean



250.14



0.35



0.0255



0.0283



SD



20.45



0.07



0.0027



0.0033



N



7



7



7



7



Low dose



125



Mean



238.00



0.35



0.0275



0.0226



SD



./.



./.



./.



./.



N



1



1



1



1



Mid dose



250



Mean



253.00



0.35



0.0534



0.0282



SD



11.30



0.06



0.0761



0.0030



N



8



8



8



8



High dose



500



Mean



248.33



0.44



0.0277



0.0317



SD



16.74



0.16



0.0045



0.0048



N



12



12



12



12



 


Table 9. Table for Gross Pathology Observation


 




















































































































































































Pregnant Females



Group



Control



Low dose



Mid dose



High dose



Dose (mg/kg)



0



125



250



500



Total No. of TS Animals Observed



18



24



17



13



Organ & Lesion



No. of animals with abnormalities



0



0



0



7



No. of animals with no abnormalities



18



24



17



6



Stomach: Muscular portion



X



X



X


 

White Spot



X



X



X


 

Minimal



X



X



X



1



 Mild



X



X



X



1



Moderate



X



X



X



1



Severe



X



X



X


 

Increased size / swollen



X



X



X


 

Minimal



X



X



X



1



Mild



X



X



X



1



Moderate



X



X



X



1



Glandular portion



X



X



X


 

Increased size / Swollen



X



X



X


 

Moderate



X



X



X



1



Red Spot



X



X



X


 

Minimal



X



X



X



1



Lung: Red discoloration



X



X



X


 

Minimal



X



X



X



1



Mild



X



X



X


 

Moderate



X



X



X


 

Note:All organs/tissues as plan were observed for study


 




















































































































































































Non-Pregnant Females



Group



Control



Low dose



Mid dose



High dose



Dose (mg/kg)



0



125



250



500



Total No. of TS Animals Observed



7



1



8



12



Organ & Lesion



Abnormality Detected



0



0



2



8



No Abnormality Detected



7



1



6



4



Stomach: Muscular portion



X



X


  

White Spot



X



X


  

Minimal



X



X


  

 Mild



X



X



1



1



Moderate



X



X


 

4



Severe



X



X


 

1



Increased size / swollen



X



X


  

Minimal



X



X


  

Mild



X



X


 

1



Moderate



X



X



1


 

Glandular portion



X



X


  

Increased size / Swollen



X



X


  

Moderate



X



X



1



1



Red Spot



X



X


  

Minimal



X



X


 

1



Lung: Red discoloration



X



X


  

Minimal



X



X


 

1



Mild



X



X


  

Moderate



X



X


  

Note:All organs/tissues as plan were observed for study


 


Table 10. Table for Microscopic Observation


 



























































































































































































Pregnant Females



Group



Control



Low dose



Mid dose



High dose



Dose (mg/kg)



0



125



250



500



Total No. of TS Animals Observed



18



24



17



13



Organ & Lesion



Abnormality Detected



0



0



0



7



No Abnormality Detected



18



24



17



6



Stomach:Muscular Stomach


 

 


  

Hyperkeratosis


 

 


  

Focal Minimal



X



X



X



1



Focal Mild



X



X



X



2



Multifocal Mild



X



X



X



2



Diffuse Minimal



X



X



X



1



Erosion


 

 


  

Focal Mild



X



X



X



1



Focal Moderate



X



X



X



1



Edema


 

 


  

Diffuse minimal



X



X



X



1



Lymphocyte Infiltration


 

 


  

Focal Minimal



X



X



X



1



Glandular Stomach


 

 


  

Degeneration


 

 


  

Diffuse minimal



X



X



X



1



Lung: Haemorrhage Alveolar


 

 


  

Focal Minimal



X



X



X



1



Thyroid:Congenital Cyst 


 

 


  

Unilateral



X



X



X



1



 

















































































































































Non-Pregnant Females



Group



Control



Low dose



Mid dose



High dose



Dose (mg/kg)



0



125



250



500



Total No. of TS Animals Observed



7



1



8



12



Organ & Lesion



Abnormality Detected



0



0



1



8



No Abnormality Detected



7



1



1



4



Stomach:Muscular Stomach


 

 


  

Hyperkeratosis


 

 


  

Focal Minimal



X



X



1



1



Focal Mild



X



X


 

1



Focal moderate



X



X


 

1



Multifocal Minimal



X



X


 

1



Multifocal Mild



X



X


 

3



Diffuse Minimal



X



X


 

1



Glandular Stomach


 

 


  

Infiltration lymphocyte Mucosal


 

 


  

Focal Mild



X



X


 

1



Thyroid:Congenital Cyst 


 

 


  

   Bilateral



X



X


 

1



Unilateral



X



X


 

1



 


Table 11. Table for Head Razor and Visceral Observation Record of Foetuses


 









































Group



Control



Low dose



Mid dose



High dose



Dose (mg/kg)



0



125



250



500



Total No. of Foetuses Observed



63



132



86



50



Abnormality Detected



00



00



00



00



No Abnormality Detected



63



132



86



50


Conclusions:
Based on the results obtained under the experimental conditions used in this study, it is concluded that the No Observed Adverse Effect Level (NOAEL) and Lowest Observed Adverse Effect Level (LOAEL) of Cinnamaldehyde (CAS No. 104-55-2) were 125 mg/kg bw/day and 250 mg/kg bw/day, respectively for general maternal toxicity. At 500 mg/kg bw/day of cinnamaldehyde (CAS No. 104-55-2) significant reductions were observed in pregnancy rate and mean foetal body weights. Therefore, the NOAEL and LOAEL of cinnamaldehyde (CAS No. 104-55-2) were 250 mg/kg bw/day and 500 mg/kg bw/day, respectively for developmental toxicity for both maternal and F1 generations.

Based on the read-across approach, a comparable result is expected for the target substance.
Executive summary:

The present study aimed to provide information on general and developmental toxicological endpoints associated with the repeated oral administration of graduated doses of Cinnamaldehyde (CAS No.104-55-2) to Wistar rats. For this purpose, the OECD test guideline 414 was followed. The study was performed in compliance with GLP.
A total of 125 pregnant female rats were randomised into 5 experimental groups each containing 25 animals. The groups, viz., G1, G5, G2, G3 and G4 received 0, 125, 250, 500 and 1000 mg/kg bw/day, respectively. The initial doses of 0, 250, 500 and 1000 mg/kg bw (G1, G2, G3 and G4) were selected based on the available data in the literature and a limited dose range finding study. The additional dose level of 125 mg/kg bw (G5) was included due to unexpected mortality at 1000 mg/kg bw (G4). Dose administration was through an oral gavage and the vehicle used in this study was corn oil. All groups were administered with dose formulation once daily from gestation day (GD) 5 to 19 (one day prior to scheduled sacrifice).
To ensure the accurate administration of the test item, the concentrations and homogeneity of the dose formulation were verified through formulation analyses in the first and last weeks of treatment. The results of these were found within the acceptable limits in both occasions.
Observations on the animals encompassed mortality/morbidity, onset of any clinical signs/symptoms, body weight, body weight changes, feed consumption, pregnancy observations, foetal developmental parameters, serum hormone levels, gross pathology and histopathology.
The results showed that Cinnamaldehyde (CAS No. 104-55-2) does not cause mortality up to doses of 250 mg/kg bw/d when administered orally to pregnant rats. However, mortality was observed for all animals treated at 1000 mg/kg bw/d and for one animal treated at 500 mg/kg bw/d.
No clinical signs or symptoms were observed in animals up to doses of 250 mg/kg bw/d (G2) throughout the duration of the experiment. At least one of the following symptoms was observed in all animals treated at 500 mg/kg bw (G3) from GD 5, 6 or 7 and onwards: hypothermia, lethargy, prostration and excessive salivation. At 1000 mg/kg bw (G4) at least one of the following symptoms were observed in most of the animals from GD 5 and onwards: abdominal breathing, abnormal gait, bradycardia, hypothermia, lethargy, prostration and excessive salivation. The mean body weight and percent change in body weight with respect to GD 0 remained comparable for G1, G5, G2 and G3 for all the time-points recorded with the exception of GD 17 wherein significant reductions in both the parameters were observed for G3 (500 mg/kg) with respect to the control group. Also, a trend of decrease in body weight and body weight gain was observed in pregnant females of G3 (500 mg/kg bw) as compared to G1 (Control) group on GD 20. Significant decrease in body weight and body weight gain on GD 8 was observed for G4 (1000 mg/kg bw) when compared with G1 (control). Further, feed consumption was reduced significantly on GD 8 for G5 (125 mg/kg bw), G3 (500 mg/kg bw) and G4 (1000 mg/kg bw) and on GD11, it was reduced significantly in G5 (125 mg/kg bw) and G3 (500 mg/kg bw) groups as compared to G1 (control) group. It remained statistically comparable among the experimental groups at every other point of observation. The changes in body weight, body weight gain and feed consumption were neither consistent nor did they seem dose-dependent for the dose levels up to 250 mg/kg bw and hence presume to be toxicologically insignificant. However, the significant difference observed in body weight and body weight gain in G3 (500 mg/kg body weight) and G4 (1000 mg/kg body weight) group was considered to be an effect of test item administration.
Thyroid hormones (T3 and T4) and thyroid stimulating hormone (TSH) levels remained comparable across the groups for pregnant and non-pregnant females.
At termination 18/25, 24/25, 17/25, 13/25 and 9/25 females were found to be pregnant from G1 (control), G5 (125 mg/kg bw), G2 (250 mg/kg bw), G3 (500 mg/kg bw) and G4 (1000 mg/kg bw) group respectively. This observation indicates statistically significant and dose-dependent adverse effect of Cinnamaldehyde (CAS No. 104-55-2) on the pregnancy rate at 500 mg/kg bw and 1000 mg/kg bw. However, other maternal evaluation parameters including numbers of corpora lutea, implantation sites, resorptions and percentage of pre- and post-implantation loss remained comparable between all the treatment groups with control group. Further, no significant difference was observed for mean litter size or sex ratio between control and treatment groups.
No treatment-related changes were observed in absolute or relative weights of uterus, ovary, thyroid and parathyroid of pregnant and non-pregnant females.
The weights of placenta also remained comparable amongst all the groups. A statistically significant change, which was observed in crown-rump lengths of G5 (125 mg/kg bw) foetuses, was not found to be dose-dependent or treatment-related. However, significant decrease observed in the weights of female foetuses and combined weights of foetuses (male and female) in G3 (500 mg/kg bw) group as compared to control group were deemed to be treatment-related. Anogenital distance (AGD) and normalised AGD remained comparable with control for both male and female foetuses up to 500 mg/kg bw. No treatment-related gross, skeletal, or visceral malformations or variations were observed in the study.
Gross pathological and microscopic examination of females revealed lesions of the stomach in G2 (250 mg/kg bw), G3 (500 mg/kg bw) and G4 (1000 mg/kg bw) group and of the lung in G3 (500 mg/kg bw) and G4 (1000 mg/kg bw) group. These lesions were found to be dose-dependent and hence, related to Cinnamaldehyde (CAS No. 104-55-2) exposure.

Based on the results obtained under the experimental conditions used in this study, it is concluded that the No Observed Adverse Effect Level (NOAEL) and Lowest Observed Adverse Effect Level (LOAEL) of Cinnamaldehyde (CAS No. 104-55-2) were 125 mg/kg bw/day and 250 mg/kg bw/day, respectively for general maternal toxicity. At 500 mg/kg bw/day of cinnamaldehyde (CAS No. 104-55-2) significant reductions were observed in pregnancy rate and mean foetal body weights. Therefore, the NOAEL and LOAEL of cinnamaldehyde (CAS No. 104-55-2) were 250 mg/kg bw/day and 500 mg/kg bw/day, respectively for developmental toxicity for both maternal and F1 generations.


Based on the read-across approach, a comparable result is expected for the target substance.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Read-across from structurally similar substance
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Toxicity to reproduction: other studies

Additional information

Read-Across: Pre-natal developmental toxicity study acc to OECD Guideline 414 (Chaudhari 2020)


The present study aimed to provide information on general and developmental toxicological endpoints associated with the repeated oral administration of graduated doses of Cinnamaldehyde (CAS No.104-55-2) to Wistar rats. For this purpose, the OECD test guideline 414 was followed. The study was performed in compliance with GLP.
A total of 125 pregnant female rats were randomised into 5 experimental groups each containing 25 animals. The groups, viz., G1, G5, G2, G3 and G4 received 0, 125, 250, 500 and 1000 mg/kg bw/day, respectively. The initial doses of 0, 250, 500 and 1000 mg/kg bw (G1, G2, G3 and G4) were selected based on the available data in the literature and a limited dose range finding study. The additional dose level of 125 mg/kg bw (G5) was included due to unexpected mortality at 1000 mg/kg bw (G4). Dose administration was through an oral gavage and the vehicle used in this study was corn oil. All groups were administered with dose formulation once daily from gestation day (GD) 5 to 19 (one day prior to scheduled sacrifice).
To ensure the accurate administration of the test item, the concentrations and homogeneity of the dose formulation were verified through formulation analyses in the first and last weeks of treatment. The results of these were found within the acceptable limits in both occasions.
Observations on the animals encompassed mortality/morbidity, onset of any clinical signs/symptoms, body weight, body weight changes, feed consumption, pregnancy observations, foetal developmental parameters, serum hormone levels, gross pathology and histopathology.
The results showed that Cinnamaldehyde (CAS No. 104-55-2) does not cause mortality up to doses of 250 mg/kg bw/d when administered orally to pregnant rats. However, mortality was observed for all animals treated at 1000 mg/kg bw/d and for one animal treated at 500 mg/kg bw/d.
No clinical signs or symptoms were observed in animals up to doses of 250 mg/kg bw/d (G2) throughout the duration of the experiment. At least one of the following symptoms was observed in all animals treated at 500 mg/kg bw (G3) from GD 5, 6 or 7 and onwards: hypothermia, lethargy, prostration and excessive salivation. At 1000 mg/kg bw (G4) at least one of the following symptoms were observed in most of the animals from GD 5 and onwards: abdominal breathing, abnormal gait, bradycardia, hypothermia, lethargy, prostration and excessive salivation. The mean body weight and percent change in body weight with respect to GD 0 remained comparable for G1, G5, G2 and G3 for all the time-points recorded with the exception of GD 17 wherein significant reductions in both the parameters were observed for G3 (500 mg/kg) with respect to the control group. Also, a trend of decrease in body weight and body weight gain was observed in pregnant females of G3 (500 mg/kg bw) as compared to G1 (Control) group on GD 20. Significant decrease in body weight and body weight gain on GD 8 was observed for G4 (1000 mg/kg bw) when compared with G1 (control). Further, feed consumption was reduced significantly on GD 8 for G5 (125 mg/kg bw), G3 (500 mg/kg bw) and G4 (1000 mg/kg bw) and on GD11, it was reduced significantly in G5 (125 mg/kg bw) and G3 (500 mg/kg bw) groups as compared to G1 (control) group. It remained statistically comparable among the experimental groups at every other point of observation. The changes in body weight, body weight gain and feed consumption were neither consistent nor did they seem dose-dependent for the dose levels up to 250 mg/kg bw and hence presume to be toxicologically insignificant. However, the significant difference observed in body weight and body weight gain in G3 (500 mg/kg body weight) and G4 (1000 mg/kg body weight) group was considered to be an effect of test item administration.
Thyroid hormones (T3 and T4) and thyroid stimulating hormone (TSH) levels remained comparable across the groups for pregnant and non-pregnant females.
At termination 18/25, 24/25, 17/25, 13/25 and 9/25 females were found to be pregnant from G1 (control), G5 (125 mg/kg bw), G2 (250 mg/kg bw), G3 (500 mg/kg bw) and G4 (1000 mg/kg bw) group respectively. This observation indicates statistically significant and dose-dependent adverse effect of Cinnamaldehyde (CAS No. 104-55-2) on the pregnancy rate at 500 mg/kg bw and 1000 mg/kg bw. However, other maternal evaluation parameters including numbers of corpora lutea, implantation sites, resorptions and percentage of pre- and post-implantation loss remained comparable between all the treatment groups with control group. Further, no significant difference was observed for mean litter size or sex ratio between control and treatment groups.
No treatment-related changes were observed in absolute or relative weights of uterus, ovary, thyroid and parathyroid of pregnant and non-pregnant females.
The weights of placenta also remained comparable amongst all the groups. A statistically significant change, which was observed in crown-rump lengths of G5 (125 mg/kg bw) foetuses, was not found to be dose-dependent or treatment-related. However, significant decrease observed in the weights of female foetuses and combined weights of foetuses (male and female) in G3 (500 mg/kg bw) group as compared to control group were deemed to be treatment-related. Anogenital distance (AGD) and normalised AGD remained comparable with control for both male and female foetuses up to 500 mg/kg bw. No treatment-related gross, skeletal, or visceral malformations or variations were observed in the study.
Gross pathological and microscopic examination of females revealed lesions of the stomach in G2 (250 mg/kg bw), G3 (500 mg/kg bw) and G4 (1000 mg/kg bw) group and of the lung in G3 (500 mg/kg bw) and G4 (1000 mg/kg bw) group. These lesions were found to be dose-dependent and hence, related to Cinnamaldehyde (CAS No. 104-55-2) exposure.

Based on the results obtained under the experimental conditions used in this study, it is concluded that the No Observed Adverse Effect Level (NOAEL) and Lowest Observed Adverse Effect Level (LOAEL) of Cinnamaldehyde (CAS No. 104-55-2) were 125 mg/kg bw/day and 250 mg/kg bw/day, respectively for general maternal toxicity. At 500 mg/kg bw/day of cinnamaldehyde (CAS No. 104-55-2) significant reductions were observed in pregnancy rate and mean foetal body weights. Therefore, the NOAEL and LOAEL of cinnamaldehyde (CAS No. 104-55-2) were 250 mg/kg bw/day and 500 mg/kg bw/day, respectively for developmental toxicity for both maternal and F1 generations.


Based on the read-across approach, a comparable result is expected for the target substance.

Justification for classification or non-classification

Based on the available data, the registered substance does not meet criteria for classification and labelling according to the Regulation (EC) No 1272/2008.

Additional information