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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, 42 resp. 51-57 days, gavage; NOAEL = 1000 mg/kg

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
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
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
Details on route of administration:
The test item was administered orally via gavage. The route of application was selected in compliance with international guidelines. The oral route is the anticipated route of human exposure to the test item.
Vehicle:
water
Remarks:
Distilled water - Aqua purificata
Details on oral 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
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
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
Observations and examinations performed and frequency:
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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: randomly selected animals from each group one day after the last treatment (i.e. on the day of necropsy)
- Anaesthetic used for blood collection: Yes, Blood samples were harvested from the retro-orbital venous plexus under Isofluran CP® anesthesia.
- Animals fasted: Yes, Animals were food deprived for approximately 16 hours (overnight) prior to blood collection
- How many animals: 5 male and 5 female animals per group
- Parameters checked in table [1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: randomly selected animals from each group one day after the last treatment (i.e. on the day of necropsy)
- Animals fasted: Yes, Animals were food deprived for approximately 16 hours (overnight) prior to blood collection
- How many animals: 5 male and 5 female animals per group
- Parameters checked in table [3] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during last exposure week
- Dose groups that were examined: 5 males and 5 females randomly selected from each group
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Bizarre behavior, Tremor

IMMUNOLOGY: No

OTHER: BLOOD COAGLUATION
- Time schedule for collection of blood: randomly selected animals from each group one day after the last treatment (i.e. on the day of necropsy)
determination of blood clotting times
- Parameters checked in table [2] were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY: Yes, the tissues indicated in Table 4 were prepared for microscopic examination and weighed, respectively. 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.
Clinical signs:
no effects observed
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:
no effects observed
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
Description (incidence and severity):
There were no test item related effects on the weights of examined organs in male or female animals at any dose level (150, 450 and 1000 mg/kg bw/day).
The weights of examined organs (absolute and relative to body and brain weights) were comparable to their control in male animals at 150, 450 and 1000 mg/kg bw/day.
Minor change in the heart weights were observed in female animals at 450 mg/kg bw/day (absolute heart weight) and at 1000 mg/kg bw/day (absolute and relative to body and brain weights) in female animals at 1000 mg/kg bw/day compared to their control. These findings were considered to be of little or no biological relevance in the lack of related histological alterations. In addition, all individual values of heart weights (absolute and relative to body and brain weights) were well within the historical control range.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Specific macroscopic alterations related to the test item were not detected in the organs or tissues at any dose levels (150, 450 or 1000 mg/kg bw/day) at the necropsy.
In the control group, one side pyelectasia (2/12 male and 1/11 dam) and 1.5 x1.5 cm Hernia diaphragmatica (1/11 dam) were observed.
At 150 mg/kg bw/day, one side pyelectasia was seen in one male animal (1/12) and there were no macroscopic findings in the organs or tissues in female animals (12/12).
Necropsy observation revealed one side pyelectasia in single male (1/12) and female (1/12) animals administered with 450 mg/kg bw/day.
At 1000 mg/kg bw/day, both sides pyelectasia (1/12 male), uneven and spotted surface of kidneys (1/12 male) and brownish red colored thymus (2/12 male) and thymic hemorrhage (2/12 female) were detected at the necropsy.
Pyelectasia and Hernia diaphragmatica are common observations in untreated experimental rats of this strain with similar age. Pyelectasia occurred independently from doses and with low incidence, therefore these findings were considered to be toxicologically not relevant. Diaphragmatic hernia was seen in control dam only.
Uneven and spotted surface of the kidneys is also a species-specific individual alteration occurring in not treated rats of this strain on the basis of literature data and necropsy observations of several years.
Brownish-red color of the thymus refers to thymic hemorrhage, which is considered to be related to the exsanguination procedure due to the circulatory disturbance.
In addition, the frequency noted in this study fits well within the historical control range
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) animals 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.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
OESTROUS CYCLE: 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: 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: 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:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
urinalysis
other: systemic toxicity
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical 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.
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
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). Non 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.

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.

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

Endpoint conclusion
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, and an additional 4 week study consistently supporting the finding that hydroxyacetone is relatively non-toxic when administered repeatedly. Hence, the database is of high quality.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose toxicity: oral_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.

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.

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

Justification for classification or non-classification

The available study provide no indication for treatment related effects including specific target organ toxicity.

In this key study, the NOAEL was found to be 1000 mg/kg due to the lack of any test-item related effects.

According to Regulation 1272/2008, substances are classified as specific target organ toxicants following repeated exposure by the use of expert judgement, on the basis of the weight of all evidence available, including the use of recommended guidance values which take into account the duration of exposure and the dose/concentration which produced the effect(s). Substances are classified in category 2 for target organ toxicity (repeat exposure) on the basis of observations from appropriate studies in experimental animals in which significant toxic effects, of relevance to human health, were produced at generally moderate exposure concentrations. Guidance values for classification into category 2 are 10 < C100 mg/kw bw/d in a subchronic study, corresponding to300 mg/kg in subacute studies. The oral administration of the test item to rats, at dose levels of 150, 450 or 1000 mg/kg bw/day was well tolerated and did not result in any adverse toxicological findings, no LOAEL up to the limit dose could be determined. Hence, no classification according to Regulation 1272/2008 is triggered.