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Key value for chemical safety assessment

Effects on fertility

Description of key information

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test with Hydroxyacetone in Wistar Rats according to OECD 422 and GLP; m/f, 12/sex/dose, 0, 150, 450, 1000 mg/kg bw/d in water; no adverse effects in adults or offspring noted; NOAEL = 1000 mg/kg (systemic toxicity), NOAEL = 1000 mg/kg (reproductive toxicity), NOEAL = 1000 mg/kg (F1 Offspring);

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018/11/28 - 2019/05/09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
OGYÉI (21.04.2016)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Premating exposure duration for parental (P0) animals : animals were dosed 14 days prior to mating
- Basis for dose level selection : range finding study
- Route of administration: orally (by gavage)
- Other considerations: 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.
Species:
rat
Strain:
Wistar
Remarks:
Han:WIST
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.
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) 73-75 days
- Weight at study initiation: (P) Males: 293-339 g; Females: 178-210 g
- Fasting period before study: none
- Housing: Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Pregnant females: individually
Males after mating: 2 animals / cage
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H complete diet for rats and mice - breeding and maintenance, ad libitum
- Water (e.g. ad libitum): tap water, as for human consumption, ad libitum
- Acclimation period: 20 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): Above 10 air-exchanges/ hour by a central air-condition system
- Photoperiod (hrs dark / hrs light): Artificial light, from 6 a.m. to 6 p.m.
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Distilled water - Aqua purificata
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Hydroxyacetone was formulated in the vehicle (distilled water) in concentrations of 30, 90 and 200 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility beforehand not longer than for seven days and stored at room temperature until use. Hydroxyacetone proved to be homogenous mixable with water and stable in distilled water at the intended concentrations at room temperature for at least 7 days.

VEHICLE
- Concentration in vehicle: 30, 90, 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): 2582-0718 and 181001
Details on mating procedure:
- M/F ratio per cage: 1/ 1
- Length of cohabitation: 1 - 5 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing formulations (control of concentration) was performed in the Analytical Laboratory of Test Facility three times during the study. Five aliquots of 5 mL of each formulation and five aliquots of control substance (vehicle) were taken and analyzed. The samples were stored at room temperature until analysis. Concentration of the test item in the dosing formulations varied between the range of 101 % and 108 % in comparison to the nominal values. The suitability of the analytical method and 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 (relative to nominal concentrations: 98 % at ca. 1 mg/mL and 102 % at ca. 200 mg/mL).
Hydroxyacetone proved to be homogenous mixable with water and stable in distilled water at the intended concentrations at room temperature for at least 7 days.
Duration of treatment / exposure:
49-54 days treatment/observation period (depending on the effectiveness of mating)
Frequency of treatment:
daily
Details on study schedule:
Age at mating of the mated animals in the study: 10 weeks
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
450 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen on the basis of the results of a preliminary dose range finding study with Hydroxyacetone in rats. The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals and not exceeding the recommended limit dose by the respective OECD guideline. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.
- Fasting period before blood sampling for clinical biochemistry: approximately 16 hours (overnight)
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes (general clinical observations)
- Time schedule: once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first exposure and once weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed on the first day of dosing (Day 0) and weekly thereafter and on the day of the necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE :
The food consumption was determined weekly by reweighing the given and non-consumed diet

OTHER:
EXAMINATION OF PLACENTAL SIGN
All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the gestational day 13. If the test was negative on day 13, the examination was repeated on day 14 of gestation.

CLINICAL PATHOLOGY (hematology and clinical chemistry)
conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy). Three samples were taken from each animal: one for hematology, one for determination of blood clotting times and the third one to obtain serum samples for clinical chemistry. Parameters examined for hematology, blood coagulation and clinical chemistry are refererred to in table 1, 2 and 3. In addition, serum samples were separated for determination of serum levels of thyroid hormones (FT4 and TSH).
Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears each day before the treatment started from each animal being considered for study for two weeks. Estrous cycle was evaluated and considered at randomization. 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 mating.
Vaginal smears were prepared and evaluated in each female animal on the day of necropsy. Vaginal smears were stained with 1 % aqueous methylene blue solution then were examined with a light microscope.
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations:
testis weight, epididymis weight, other:activity of spermatogenesis in testes, storage of mature spermatozoa in epididymides
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, weight gain, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, gross abnormalities, other: FT4 and TSH (on post-partal day 13): Blood samples were collected from 2-7 pups per litter on post-natal day 4 (if it was feasible; samples were pooled by litter), from 6-7 pups per litter on post-partal/post-natal day 13;

GROSS EXAMINATION OF DEAD PUPS
yes, for external and internal abnormalities, dead pup was subjected to necropsy by macroscopic examination on postnatal day 1.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving parental animals were euthanized and subjected to gross pathology one day after the last treatment. Not mated female animal was subjected to necropsy on Day 35.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
- The tissues indicated in Table 4 were prepared for microscopic examination and weighed, respectively.
Histopathology examination was performed on ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups (male or female)
Five dams and their male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology and blood coagulation, clinical chemistry, gross necropsy, organ weighing and histopathology examination.
The body weight, brain weight and weight of the testes, epididymides, prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. In addition, for five males and females randomly selected from each group, adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed.
Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose groups. In addition, organs showing macroscopic findings at the necropsy were processed and examined histologically in animals of the low and mid dose groups.
Postmortem examinations (offspring):
SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: on postnatal day 13 or shortly thereafter

GROSS NECROPSY
- Pups euthanized on day 13 post-partum, or shortly thereafter, were carefully examined for gross abnormalities. Gross necropsy consisted of determination of macroscopic changes of organs or tissue.

HISTOPATHOLOGY / ORGAN WEIGTHS
- observation of kidney of two pups
Statistics:
The statistical evaluation of appropriate data 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.
Reproductive indices:
Copulatory index: Measure of animals’ ability to mate; Fertility index: Measure of male’s ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant; Gestation index: Measure of pregnancy that provides at least one live pup, for formulas please refer to 'Any other information on material and methods'
Offspring viability indices:
Post-implantation mortality (intrauterine mortality); Post-natal mortality; Survival Index; Sex ratio, for formulas please refer to 'Any other information on material and methods'
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The body weight development was undisturbed in male and female animals at 150, 450 and 1000 mg/kg bw/day during the entire treatment period.
The mean body weight was comparable in the control and at 150, 450 and 1000 mg/kg bw/day groups in male animals during the pre-mating, mating and post-mating periods.
In the female animals at 150 mg/kg bw/day, statistical significance with respect to the control was detected at the lower mean summarized body weight gain during the pre-mating period (between Days 0 and 13).
The mean body weight gain exceeded that of the control group in female animals at 150 mg/kg bw/day during the gestation period reaching statistical significance between gestation days 14 and 21 and if summarized (between gestation days 0 and 21). This slight change in the body weight gain resulted in statistically significantly higher mean body weight in female animals at 150 mg/kg bw/day by the end of gestation period (gestation day 21).
There were no statistically or biologically significances in the body weight or body weight gain of female animals in the control and 150 mg/kg bw/day groups during the lactation period.
The mean body weight was comparable in the control at 450 and 1000 mg/kg bw/day groups in female animals during the pre-mating, gestation and lactation periods.
The sporadic changes in the body weight and body weight gain of female animals at 150 mg/kg bw/day were of minor degree and independent from the administration of the test item as similar findings were not detected at the higher dose groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean daily food consumption was similar in the control and 150, 450 or 1000 mg/kg bw/day groups during pre-mating and post mating periods in male animals and during the pre-mating, gestation and lactation periods in female animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological evaluation did not reveal test item related changes in the examined parameters at 150, 450 or 1000 mg/kg bw/day (male and female).
Statistically significant difference with respect to their control was noted for some red blood cell parameter in male animals at 150 mg/kg bw/day (lower mean corpuscular hemoglobin content – MCHC), at 450 mg/kg bw/day (lower mean corpuscular volume – MCV; lower mean corpuscular hemoglobin – MCH; and lower mean MCHC) and at 1000 mg/kg bw/day (lower mean MCHC).
In the female animals, the mean platelet count (PLT) was slightly above the control value at 450 and at 1000 mg/kg bw/day.
Statistically significant differences in these parameters were considered to be of no biological significance as the values correlated well with the historical control values (MCV, MCH, MCHC, PLT) or did not reveal a dose-response (light changes in MCV, MCH were detected at the lower doses but not in the high dose treated animals). Statistical significance at PLT was due to the relatively low value of the actual control group compared to the historical control ranges.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related alterations in the examined clinical chemistry parameters at 150, 450 or 1000 mg/kg bw/day (male or female).
The examined clinical chemistry parameters were similar to their control in male animals at 150, 450 and 1000 mg/kg bw/day.
In the female animals, statistical significance with respect to the control was detected at higher mean concentration of albumin (ALB) at 1000 mg/kg bw/day.
The statistically significant differences in ALB+ in female animals were considered to have little or no toxicological importance because values – mean and individual – corresponded well to the historical control value.
The thyroid hormone (free T4 and TSH) levels were not affected by the test item in parental male animals (150, 450 and 1000 mg/kg bw/day) and in offspring sampled on postnatal day 13.
There were no statistically or biologically significant differences between the control and test item administered groups in the serum level of thyroid hormones /FT4 or TSH).
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation battery did not show any alterations in the behavior or reactions to different type of stimuli of selected male or female animals in the control, 150, 450 or 1000 mg/kg bw/day groups at the end of the treatment period.
There were no changes in the physical condition, behavior or in reactions to different types of stimuli in the male or female animals of control and test item treated groups in the examined parameters during the course of the functional observations.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histopathological examinations did not reveal any toxic or other test item related lesions in the investigated reproductive organs of male and female animals at 1000 mg/kg bw/day.
Histological examination revealed in one male animal (1/7) of the 1000 mg/kg bw/day group moderate chronic progressive nephropathy (CPN), which is an important spontaneous renal disease of the commonly used strain of laboratory rat because it is a serious confounding factor in experimental pathology and toxicology studies. Taking into account the incidence and historical control values, CPN found in one animal only was considered as an individual finding.
Minimal or mild alveolar emphysema in the lungs in male animals (1/5 control, 1/5 at 1000 mg/kg bw/day) were detected sporadically. The pulmonary emphysema was considered as a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure and not test item related.
Acute hemorrhage was seen in the thymus in some male (2/5) and female (2/5) animal at 1000 mg/kg bw/day. This finding was considered to be a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination. In addition, this is a common finding and the frequency noted here has been seen in other studies in control animals too.
Hyperplasia of bronchus associated lymphoid tissue (BALT) in the lungs in minimal or mild degree in some animal (2/5 control male; 1/5 male at 1000 mg/kg bw/day) is a physiological immune-morphological phenomenon, occurring also in not treated rats and it is of no toxicological relevance.
Focal subcapsular fibrosis in the liver was in connection with the mechanical irritation due to the diaphragmatic hernia in one control female animal.
One or both sides pyelectasia was observed in some male animals (2/6 control, 1/1 at 150 mg/kg bw/day; 1/1 at 450 mg/kg bw/day; 1/7 at 1000 mg/kg bw/day) and in single female animal in the control (1/5) and 450 mg/kg bw/day (1/1) groups without other histopathological lesions (degeneration, inflammation, fibrosis): Pyelectasia – without inflammatory or other pathological lesions – is a common finding in experimental rats of this strain with similar age.
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
A test item influence on the estrous cycle was not detected at any dose level (150, 450 or 1000 mg/kg bw/day).
Statistical significance was detected for a slightly lower mean number of the days in diestrous in female animals at 450 mg/kg bw/day during the pre-mating period. This finding was evaluated as an accidental finding and not related to the treatment with the test item as a similar change was not detected in the high dose group and all values were well within the historical control range
There were no statistically or biologically significant differences between the control and test item treated groups (150, 450 and 1000 mg/kg bw/day) in any other examined parameters of estrous cycle: all animals showed regular cycles, the mean number of cycles, the mean length of cycles, mean number of days in pro-estrous or estrous were similar in all groups and there were no animals with prolonged estrous or diestrous during the pre-mating period.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
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 morphologically in the testes of investigated control and treated animals.
Reproductive performance:
no effects observed
Description (incidence and severity):
The examined parameters of reproductive performance were not affected by the test item at 150, 450 or 1000 mg/kg bw/day in male or female animals.
There was no difference between the control and test item treated groups in the gestation or copulatory indices, in the mean pre-coital interval or mean conceiving days. The noted statistical significances with regard to the higher fertility indices at 150, 450 and 1000 mg/kg bw/day (male and female) are indicative for a biological variation, as all values were within the historical control range. In addition, this finding is most likely caused by the failure of mating in one control pair.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
other: systemic toxicity
Remarks on result:
not determinable due to absence of adverse toxic effects
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Test item related clinical signs were not detected in the offspring between postnatal days 0 and 13. The percentage of cold and not suckled – no milk in the stomach – offspring was slightly above the control at 1000 mg/kg bw/day. The values were also slightly higher than in the historical control studies, however these signs were only transient (observed shortly after the born only) and did not influence the offspring’s development.
Other clinical signs (hemorrhage on the nose, paleness, cyanotic skin, smaller than normal, missing) were observed with low incidence in the 150, 450 or 1000 mg/kg bw/day groups.
These signs are commonly observed in offspring of not treated dams of this strain or showed no dose dependency in this study. Therefore, these observations were considered to be not related to the treatment of mothers and have no toxicological relevance.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
There was no test item related effect on offspring’s extra uterine mortality.
There were no dead pups on postnatal day 0 in the control, 150, 450 or 1000 mg/kg bw/day groups. Some pups were missing at the daily observations (2/140, 1/132 and 3/143, at 150, 450 and 1000 mg/kg bw/day, respectively) during the study. These findings corresponded with the historical control therefore, no test item influence was assumed.
The mean number of live pups per litter and the mean number of viable pups per litter were similar in all groups on postnatal days 0, 4 and 13. There were no significant differences between the control and test item treated (150, 450 or 1000 mg/kg bw/day) groups in the survival indices.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The body weight development of the offspring was undisturbed during the observation period.
The mean litter weights and mean pup weights as well as the mean litter weight gains and mean pup weight gains were similar in the control and in all test item treated groups (150, 450 and 1000 mg/kg bw/day) on postnatal days 0, 4 and 13.
Statistical significance was observed for the slightly higher mean body weight of male and female pups at 150 mg/kg bw/day comparing to their control on postnatal day 4. These differences with respect to the control were minor and a similar finding was not detected at the evaluation of the body weight of male and female pups together. Thus, this finding was considered to be not toxicologically relevant.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related macroscopic findings in pups subjected to necropsy.
Three offspring were necropsied on postnatal day 0. The lung flotation test was negative for all the three pups – stillborns – and there were no macroscopic changes in the organs or tissues.
At the terminal necropsy, both sides pyelectasia was observed in two pups (1 female at 150 mg/kg bw/day; 1 male at 450 mg/kg bw/day).
Histopathological findings:
no effects observed
Description (incidence and severity):
Histological examinations did not reveal any pathological lesions in the kidneys of these offspring.
Other effects:
no effects observed
Description (incidence and severity):
Sex distribution: There were no test item related differences between the control and all test item treated groups in the ratio or in the litter means of genders on postnatal days 0, 4 or 13.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
Remarks on result:
not determinable due to absence of adverse toxic effects
Reproductive effects observed:
no
Conclusions:
Under the conditions of the present OECD 422 study, Hydroxyacetone administered at 150, 450 or 1000 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) or gave any indication to cause endocrine effects in parental male and female Han:WIST rats as far as investigated in this study.
There were also no signs of systemic toxicity in the selected male or female animals at 150, 450 or 1000 mg/kg bw/day.
The development of the F1 offspring was not impaired from birth to post-natal day 13 nor indication for an endocrine effect as far as investigated in this study was noted after repeated oral administration of dams at 150, 450 or 1000 mg/kg bw/day.
Based on these observations the No Observed Adverse Effect Levels (NOAEL) by active ingredient were determined as follows:
NOAEL for reproductive performance of male/ female rats: 1000 mg/kg bw/day
NOAEL for systemic toxicity of male/ female rats: 1000 mg/kg bw/day
NOAEL for F1 Offspring: 1000 mg/kg bw/day
Executive summary:

The objective of this study was to obtain information on the toxic potential of test item and on the possible effects of the test item on reproduction and development as well as on some endocrine parameters when repeatedly administered orally (by gavage) to rats at doses of 150, 450 and 1000 mg/kg bw/day by compared to control animals according to OECD 422 and under GLP compliance.

As a screening test, it was intended to provide initial information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time and on the possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 13 post-partum associated with administration of repeated maternal doses. In addition, selected parameters indicative for potential endocrine effects were investigated.

Hydroxyacetone was administered orally (by gavage) once daily at 0 (vehicle only), 150, 450 and 1000 mg/kg bw/day doses to four groups of Han:WIST rats consisting of 12 animals per sex per group in concentrations of 30, 90 and 200 mg/mL corresponding to a 5 mL/kg bw dosing volume. A group of vehicle (distilled water) treated animals (n= 12/sex) served as a control.

The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Hydroxyacetone was stable in the vehicle in concentrations of 1 mg/mL and 200 mg/mL at room temperature for at least 7 days.

The concentration of the test item in the dosing formulations administered to the animals was checked three times during the study. Hydroxyacetone concentrations in the dosing formulations varied within the range of 101 % and 108 % (in comparison to the nominal values) and confirming the proper preparation of the dosing formulations.

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 49 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 51, 54 days). Not mated female animal was administered for 35 days. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period and during the mating period until evidence of copulation. Vaginal smears were prepared on the day of the necropsy for each dam. Not mated female animal was subjected to necropsy on Day 35.

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.

Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4 and TSH) from 2-7 pups per litter (where it was feasible: litters with ≥ 10 offspring) on post-natal day 4, from all dams and from 6-7 pups per litter at termination on post-partum/post-natal day 13 and from all parent male animals at termination. FT4 and TSH was determined in the samples of parental male animals and PN 13 offspring.

As no changes were detected, the measurement of samples of dams and PND4 was not required and not performed.

Five dams and their male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology and blood coagulation, clinical chemistry, gross necropsy, organ weighing and histopathology examination.

All parental animals were euthanized and subjected to gross pathology one day after the last treatment. The body weight, brain weight and weight of the testes, epididymides, prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. In addition, for five males and females randomly selected from each group, adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed.

Thyroid gland was preserved from all adult males and females and one male and one female pup per litter for the intended subsequent histopathological examination.

Histopathology examination was performed on ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups (male or female).

Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose groups. In addition, organs showing macroscopic findings at the necropsy were processed and examined histologically in animals of the low and mid dose groups.

The results of this study were summarized as follows:

Mortality

There was no test item related mortality at any dose level (150, 450 and 1000 mg/kg bw/day).

Clinical and functional observation

Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor at the detailed weekly clinical observations or at the functional observations. The behavior and physical condition of the animals was not impaired at any dose level (150, 450 or 1000 mg/kg bw/day) during the entire treatment period.

Body weight and body weight gain

The body weight development was not adversely affected by the test item in male or in female animals at 150, 450 or 1000 mg/kg bw/day during the entire treatment period (pre-mating and post-mating period for male animals; pre-mating, gestation and lactation periods for female animals).

Food consumption

The mean daily food consumption was very similar in male or female animals in control and at 150, 450 and 1000 mg/kg bw/day during the entire study.

Estrous cycle

A test item influence on the estrous cycle was not found at any dose level (150, 450 and 1000 mg/kg bw/day).

Reproductive performance

There were neither significant nor dose related differences between the control and test item treated male or female animals in the examined parameters of reproductive performance or in the delivery parameters of dams (150, 450 and 1000 mg/kg bw/day).

Hematology and blood coagulation

Hematological and blood coagulation investigation did not reveal test item related changes in the examined parameters at 150, 450 or 1000 mg/kg bw/day.

Clinical chemistry

There were no test item related effects on the examined clinical chemistry parameters at 150, 450 or 1000 mg/kg bw/day (male or female).

Serum thyroid hormones

There were no test item related changes in the serum thyroid hormone (FT4 and TSH) levels at any dose (parental male or 13-day offspring).

Necropsy

Macroscopic findings related to the effect of the test item were not found in male and female animals at 150, 450 or 1000 mg/kg bw/day.

Organ weight

There were no test item related changes in the weights (absolute and relative to body or brain weights) of brain, testes, epididymides and prostate, seminal vesicle with coagulating gland as a whole, of male animals at any dose level.

The weights of organs of selected animals were comparable in the control and test item treated groups (male and female).

Histopathology

There were no toxic or other test item related lesions detectable by histological examination in the investigated reproductive organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) and the thyroid of male or female animals administered with 1000 mg/kg bw/day.

Histopathological examinations did not indicate any toxic or other test item related lesions in the investigated organs of selected male and female animals at 1000 mg/kg bw/day.

Offspring

The offspring’s development was undisturbed at 150, 450 and 1000 mg/kg bw/day from birth to post-natal day 13. No effect on the mortality, clinical signs, body weight development, anogenital distance (male and female) or nipple retention (male) were detected.

Conclusion

Under the conditions of the present OECD 422 study, Hydroxyacetone administered at 150, 450 or 1000 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) or gave any indication to cause endocrine effects in parental male and female Han:WIST rats as far as investigated in this study.

There were also no signs of systemic toxicity in the selected male or female animals at 150, 450 or 1000 mg/kg bw/day.

The development of the F1 offspring was not impaired from birth to post-natal day 13 nor indication for an endocrine effect as far as investigated in this study was note after repeated oral administration of dams at 150, 450 or 1000 mg/kg bw/day.

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

NOAEL for reproductive performance of male/ female rats: 1000 mg/kg bw/day

NOAEL for systemic toxicity of male/ female rats: 1000 mg/kg bw/day

NOAEL for F1 Offspring: 1000 mg/kg bw/day

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
There is a Klimisch 1 OECD TG 422 study available, showing no test item related effects up to 1000 mg/kg when administered repeatedly. Hence, the database is of high quality.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Toxicity to reproduction_Szakonyiné, 2019

The objective of this study was to obtain information on the toxic potential of test item and on the possible effects of the test item on reproduction and development as well as on some endocrine parameters when repeatedly administered orally (by gavage) to rats at doses of 150, 450 and 1000 mg/kg bw/day by compared to control animals according to OECD 422 and under GLP compliance.

As a screening test, it was intended to provide initial information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time and on the possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 13 post-partum associated with administration of repeated maternal doses. In addition, selected parameters indicative for potential endocrine effects were investigated.

Hydroxyacetone was administered orally (by gavage) once daily at 0 (vehicle only), 150, 450 and 1000 mg/kg body weight (mg/kg bw/day) doses to four groups of Han:WIST rats consisting of 12 animals per sex per group in concentrations of 30, 90 and 200 mg/mL corresponding to a 5 mL/kg bw dosing volume. A group of vehicle (distilled water) treated animals (n= 12/sex) served as a control.

The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Hydroxyacetone was stable in the vehicle in concentrations of 1 mg/mL and 200 mg/mL at room temperature for at least 7 days.

The concentration of the test item in the dosing formulations administered to the animals was checked three times during the study. Hydroxyacetone concentrations in the dosing formulations varied within the range of 101 % and 108 % (in comparison to the nominal values) and confirming the proper preparation of the dosing formulations.

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 49 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 51, 54 days). Not mated female animal was administered for 35 days. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period and during the mating period until evidence of copulation. Vaginal smears were prepared on the day of the necropsy for each dam. Not mated female animal was subjected to necropsy on Day 35.

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.

Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4 and TSH) from 2-7 pups per litter (where it was feasible: litters with ≥ 10 offspring) on post-natal day 4, from all dams and from 6-7 pups per litter at termination on post-partum/post-natal day 13 and from all parent male animals at termination. FT4 and TSH was determined in the samples of parental male animals and PN 13 offspring.

As no changes were detected, the measurement of samples of dams and PND4 was not required and not performed.

Five dams and their male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology and blood coagulation, clinical chemistry, gross necropsy, organ weighing and histopathology examination.

All parental animals were euthanized and subjected to gross pathology one day after the last treatment. The body weight, brain weight and weight of the testes, epididymides, prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. In addition, for five males and females randomly selected from each group, adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed.

Thyroid gland was preserved from all adult males and females and one male and one female pup per litter for the intended subsequent histopathological examination.

Histopathology examination was performed on ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups (male or female).

Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose groups. In addition, organs showing macroscopic findings at the necropsy were processed and examined histologically in animals of the low and mid dose groups.

The results of this study were summarized as follows:

Mortality

There was no test item related mortality at any dose level (150, 450 and 1000 mg/kg bw/day).

Clinical and functional observation

Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor at the detailed weekly clinical observations or at the functional observations. The behavior and physical condition of the animals was not impaired at any dose level (150, 450 or 1000 mg/kg bw/day) during the entire treatment period.

Body weight and body weight gain

The body weight development was not adversely affected by the test item in male or in female animals at 150, 450 or 1000 mg/kg bw/day during the entire treatment period (pre-mating and post-mating period for male animals; pre-mating, gestation and lactation periods for female animals).

Food consumption

The mean daily food consumption was very similar in male or female animals in control and at 150, 450 and 1000 mg/kg bw/day during the entire study.

Estrous cycle

A test item influence on the estrous cycle was not found at any dose level (150, 450 and 1000 mg/kg bw/day).

Reproductive performance

There were neither significant nor dose related differences between the control and test item treated male or female animals in the examined parameters of reproductive performance or in the delivery parameters of dams (150, 450 and 1000 mg/kg bw/day).

Hematology and blood coagulation

Hematological and blood coagulation investigation did not reveal test item related changes in the examined parameters at 150, 450 or 1000 mg/kg bw/day.

Clinical chemistry

There were no test item related effects on the examined clinical chemistry parameters at 150, 450 or 1000 mg/kg bw/day (male or female).

Serum thyroid hormones

There were no test item related changes in the serum thyroid hormone (FT4 and TSH) levels at any dose (parental male or 13-day offspring).

Necropsy

Macroscopic findings related to the effect of the test item were not found in male and female animals at 150, 450 or 1000 mg/kg bw/day.

Organ weight

There were no test item related changes in the weights (absolute and relative to body or brain weights) of brain, testes, epididymides and prostate, seminal vesicle with coagulating gland as a whole, of male animals at any dose level.

The weights of organs of selected animals were comparable in the control and test item treated groups (male and female).

Histopathology

There were no toxic or other test item related lesions detectable by histological examination in the investigated reproductive organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) and the thyroid of male or female animals administered with 1000 mg/kg bw/day.

Histopathological examinations did not indicate any toxic or other test item related lesions in the investigated organs of selected male and female animals at 1000 mg/kg bw/day.

Offspring

The offspring’s development was undisturbed at 150, 450 and 1000 mg/kg bw/day from birth to post-natal day 13. No effect on the mortality, clinical signs, body weight development, anogenital distance (male and female) or nipple retention (male) were detected.

Conclusion

Under the conditions of the present OECD 422 study, Hydroxyacetone administered at 150, 450 or 1000 mg/kg bw/day by oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) or gave any indication to cause endocrine effects in parental male and female Han:WIST rats as far as investigated in this study.

There were also no signs of systemic toxicity in the selected male or female animals at 150, 450 or 1000 mg/kg bw/day.

The development of the F1 offspring was not impaired from birth to post-natal day 13 nor indication for an endocrine effect as far as investigated in this study was note after repeated oral administration of dams at 150, 450 or 1000 mg/kg bw/day.

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

NOAEL for reproductive performance of male/ female rats: 1000 mg/kg bw/day

NOAEL for systemic toxicity of male/ female rats: 1000 mg/kg bw/day

NOAEL for F1 Offspring: 1000 mg/kg bw/day

Effects on developmental toxicity

Description of key information

See above, Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422),no adverse effects in adults or offspring noted.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Based on lack of any adverse effects on reproductive toxicity (and general systemic toxicity), it is impossible to hypothesize a concrete mode of action.

No definitive human relevance framework can be described due to the lack of any other effects securing any postulation, and no conclusion on biological plausibility can be drawn.

Despite the fact that no mode of action analysis can be performed, no data gap was identified here. The tonnage-driven data requirements under REACH were fully met, and the lack of effects does also not indicate any high hazard for humans and so does not trigger any further examinations.

Justification for classification or non-classification

On the basis of the results of the Repeated dose / reproduction toxicity screening test on rats, the no observed adverse effect level (NOAEL) of the test item was determined. The oral (gavage) administration of the test item to Wistar rats, at dose levels of 150, 450 or 1000 mg/kg bw/day did not produce any test item related effects in adults or offspring. So, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day as well as the NOAEL for reproductive toxicity was considered to be 1000 mg/kg bw/day within the confines of this study.

According to Regulation 1272/2008, Table 3.7.1(a) Hazard categories for reproductive toxicants, a substance must be considered as reproductive toxicant under the following conditions: Suspected human reproductive toxicant: Substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, and where the evidence is not sufficiently convincing to place the substance in Category 1. If deficiencies in the study make the quality of evidence less convincing, Category 2 could be the more appropriate classification. Such effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects.

No effects on fertility were noted up to the limit dose of 1000 mg/kg, and no systemic toxicity was noted. Consequently the criteria for classification as reproductive toxicant (Cat. 2) are not met, the substance does not need to be classified according to Regulation 1272/2008.

Additional information