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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
4'-hydroxyacetophenone
EC Number:
202-802-8
EC Name:
4'-hydroxyacetophenone
Cas Number:
99-93-4
Molecular formula:
C8H8O2
IUPAC Name:
1-(4-hydroxyphenyl)ethanone
Test material form:
other: white solid

Test animals

Species:
rat
Strain:
other: Crl:WI(Han) outbread
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 11 wks (F0)
- Weight at study initiation: (P) Males: 316-321 g; Females: 202-205 g
- Housing: male: 5/cage; female: single housing
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24 °C
- Humidity (%): 40-70 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test substance.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at WIL Research Europe and on information provided by the Sponsor.
Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Males were exposed for 30 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 43-46 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
Frequency of treatment:
7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 40, 150, 600 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of a 14-day dose-range finding study.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: Mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FUNCTIONAL OBSERVATIONS: The following tests were performed on the selected animals:
- hearing ability *
- pupillary reflex *
- static righting reflex *
- grip strength *
(* Scoring: score 0 = normal/present, score 1 = abnormal/absent)
-locomotor activity. Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

GENERAL REPRODUCTION DATA: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
no data
Litter observations:
Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made for all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation


Postmortem examinations (parental animals):

CLINICAL CHEMISTRY. Yes
Blood samples were collected from selected animals under anaesthesia using isoflurane between 7.00 and 10.30 a.m. Furthermore, from the selected animals an additional blood sample (0.5 mL) was collected into serum tubes for possible future measurement of the thyroid hormones triiodothyronine (T3) and thyroxine (T4). The following parameter were analyzed: alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate (Inorg. Phos).

HEMATOLOGY: Yes
The following parameter were analyzed: White blood cells (WBC), differential leucocyte count: neutrophils, lymphocytes, monocytes, eosinophils, basophils, red blood cells, reticulocytes, red blood cell distribution width (RDW), haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelets, prothrombin time (PT), activated partial thromboplastin time (APTT),

PATHOLOGY. Yes
The numbers of former implantation sites and corpora lutea were recorded for all paired females. Samples of the following tissues and organs were collected from all animals and fixed in 10% buffered formalin: Adrenal glands, peyer's patches [jejunum, ileum] if detectable, brain - cerebellum, mid-brain, cortex, pituitary gland, caecum, preputial gland, cervix, prostate gland, clitoral gland, rectum, colon, coagulation gland, sciatic nerve, duodenum, seminal vesicles, epididymides, skeletal muscle, eyes (with optic nerve (if detectable) and harderian gland), spinal cord -cervical, midthoracic, lumbar, spleen, femur including joint, sternum with bone marrow, heart, stomach (forestomach and glandular stomach), ileum, testes, jejunum, thymus, kidneys, thyroid including parathyroid if detectable, trachea, liver, urinary bladder, lung, uterus, lymph nodes - mandibular, mesenteric, vagina, all gross lesions.

HISTOPATHOLOGY. Yes
The following organs were examinedt:
- The preserved organs and tissues of selected 5 animals/sex of Groups 1 and 4.
- The thymus and stomach (females), kidneys (males) and spleen (males and females) of selected animals of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
- The additional slides of the testes of all males of Groups 1 and 4 and all males suspected to be infertile to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs of all animals of Groups 1 and 4 and of all males that failed to sire and all females that failed to deliver healthy pups. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

ORGAN WEIGHTS:
The following organ weights were recorded: Adrenal glands, spleen, brain, testes, epididymides, thymus, heart, uterus (including cervix), kidneys, prostate, liver, seminal vesicles including coagulating glands, ovaries, thyroid including parathyroid.
Postmortem examinations (offspring):
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 3; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 4) was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test (Ref. 5) was applied to motor activity data to determine intergroup differences.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS) No mortality occurred during the study period. One female at 600 mg/kg bw/day was sacrificed within 24 hours after total litter loss was observed. No clinical signs of toxicity were noted during the observation period.
Salivation seen after dosing among most animals at 600 mg/kg bw/day was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to taste of the test substance.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS) No toxicologically relevant changes in body weights and body weight gain were noted. The statistically significant higher body weight gain of females at 150 mg/kg bw/day over Days 1-8 of the premating period showed no dose related trend and was minor in nature. No toxicological relevance was therefore ascribed to this change.

HAEMATOLOGY No toxicologically relevant changes occurred in haematological parameters of treated rats. Any statistically significant changes in haematological parameters were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution and/or remained within the range considered normal for rats of this age and strain. These changes consisted of lower red blood cell counts and haematocrit in males at 150 mg/kg bw/day, lower relative monocyte counts and haemoglobin level in females at 600 mg/kg bw/day, and lower red blood cell counts in females at 40 mg/kg bw/day.

CLINICAL BIOCHEMISTRY No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats. Statistically significant changes in clinical biochemistry parameters consisted of a higher creatinine level in males and females at 600 mg/kg bw/day, higher glucose level in males at 600 mg/kg bw/day, lower cholesterol level in males at 600 mg/kg bw/day and higher potassium level in females at 600 mg/kg bw/day. Means of these changes were only minimally outside the range considered normal for rats of this age and strain, and there were no histopathological correlates. The higher bile acid level in females at 150 and 600 mg/kg bw/day were confined to high values of two females in each group, and again there were no morphological correlates. Overall, these variations in clinical biochemistry parameters were considered to be of no toxicological relevance.
Two females at 600 mg/kg bw/day (nos. 73 and 76) showed a low and high inorganic phosphate value, respectively. Given the variable nature of this group response, this was considered to be of no toxicological relevance. The statistically significant lower total bilirubin level, and higher sodium and chloride level in males at 150 mg/kg bw/day occurred in the absence of a dose-related trend and remained within the range considered normal for rats of this age and strain. These changes were therefore considered to be of no toxicological relevance.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS) No toxicologically relevant effects on reproductive parameters were noted.
Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.
A total of three females, one each at 40, 150 and 600 mg/kg bw/day, did not deliver live offspring and were found to be non-pregnant. The incidences of non-pregnancy did not show a dose-related trend and were within the expected range. Therefore, these differences were considered not to be of toxicological relevance.

ORGAN WEIGHTS (PARENTAL ANIMALS) No toxicologically relevant changes in organ weights were noted. Females at 150 and 600 mg/kg bw/day showed a statistically significant lower thymus weight and thymus to body weight ratio. The mean relative thymus weight was approximately 30 and 42% lower than the control mean at 150 and 600 mg/kg bw/day, respectively. However, the control mean was slightly higher than normal, and no concurrent changes in blood parameters or histopathological changes were noted, and the means at 150 and 600 mg/kg bw/day remained essentially within the range considered normal for rats of this age and strain. The statistically significant higher uterus weight and uterus to body weight ratio of females at 40 mg/kg bw/day, and the statistically significantly higher liver to body weight ratio of females at 150 and 600 mg/kg bw/day occurred in the absence of a clear dose-related trend or concurrent histopathological changes, and means remained essentially within the range considered normal for rats of this age and strain.

GROSS PATHOLOGY (PARENTAL ANIMALS): Necropsy did not reveal any toxicologically relevant alterations. The incidence of macroscopic findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, did not show a dose-related incidence trend, and occurred in the absence of treatment-related histopathological changes. These necropsy findings were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY (PARENTAL ANIMALS) No toxicologically relevant histopathological changes were noted.
In the kidneys a slightly increased incidence of hyaline droplets in cortical tubules were noted in males of all treated groups. Hyaline droplets in the kidneys were recorded at a slight degree in one male of Group 1 (0 mg/kg b.w./day), minimal or slight degree in three males at 40 and 150 mg/kg b.w./day (Group 2 respectively Group 3) and at minimal or slight degree in five males at 600 mg/kg b.w./day (Group 4). The hyaline droplets were considered to represent alpha2µglobulin, a normal protein in male rats which undergoes re-absorption in the proximal cortical tubules. A range of chemicals are known to increase hyaline droplet formation. This protein is not present in higher mammals, including man. As the increase was only minimal, was not accompanied by degenerative tubular alterations and the severities of minimal or slight may be seen as a background finding in male rats of this age and strain this finding was considered to be a non-adverse finding.
A minimal increased incidence/severity of hemopoietic foci, primarily erythropoiesis was recorded in the spleen of females of all treated Groups. Hemopoietic foci were recorded in 4/5 females of Group 1 (0 mg/kg b.w./day, 2: minimal, 1: slight, 1: moderate), in 5/5 females at 40 mg/kg b.w./day (3: slight, 2: moderate) and 5/5 females at 150 and 600 mg/kg b.w./day (3: slight, 1: moderate, 1: marked). As severities up to a marked degree can be seen as a background finding in (pregnant or lactating) female rats of this type of study and the increase of severity as seen in this study was only minimal and did not show a dose-related trend, this finding was considered not to be of toxicological relevance.
In one female at 600 mg/kg b.w./day findings were recorded in the forestomach consisting of minimal inflammation and slight ulceration, hyperkeratosis and parakeratosis. Given that this finding was only slight in nature and absent among other animals of the same group, this was considered not to be of toxicological relevance.
All above and remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in rats of this age and strain.
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis after test item treatment. No abnormalities were seen in the reproductive organs of the animals that failed to deliver healthy offspring.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
ca. 600 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING) One female at 600 mg/kg bw/day had total litter loss. As all other litters of the same group comprised live offspring and were of normal size, this incidental occurrence was considered to be of no toxicological relevance.

CLINICAL SIGNS (OFFSPRING) Incidental clinical symptoms of pups consisted of a wound on the abdomen and left hindleg, small appearance, a blue spot on the head, scabs on the hind leg and hypothermia. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

BODY WEIGHT (OFFSPRING) No toxicologically relevant changes in body weights of pups were noted.
The statistically significant lower pup body weight at 40 mg/kg bw/day (male and female pups combined) on Day 4 of lactation was slight in nature and showed not dose-related trend. No toxicological relevance was ascribed to this change.

SEXUAL MATURATION (OFFSPRING) Sex ratio was unaffected and no abnormal findings could be detected.

GROSS PATHOLOGY (OFFSPRING) Incidental macroscopic findings of pups consisted of a wound on the abdomen and left hindleg, small appearance and absence of milk in the stomach. The nature and incidence of these findings remained within the range considered normal for pups of this age. Findings among the two pups of female no. 80 with total litter loss consisted of missing hindlegs, right foot and/or tail apex (probably due to cannibalism), and absence of milk in the stomach.

DEVELOPMENTAL PARAMETERS
No developmental toxicity was observed up to the highest dose level tested (600 mg/kg bw/day).
No toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy)

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 600 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion