Acetoacetamide

Endpoint summary

Description of key information

No adverse effects were found on male and female clinical observations, functional observations, body weight, food consumption, haematology and coagulation, clinical biochemistry, urinalysis and gross pathological findings at necropsy. There were also no test item-related effects on estrous cyclicity, nipple retention, anogenital distance, precoital interval and duration of gestation, reproductive indices, pup thyroid weight and pup thyroxine hormone, pup external findings on PND 0 and at death. No test item-related effects on number of corpora lutea, implantation sites, % pre and post implantation loss, litter data, litter weights, reproductive indices and number of live pups on PND 0, 4 and 13 in HD group were observed when compared with controls.

Adverse effects of Acetoacetmide were noted at a dose level of 100 mg/kg body weight/day for general systemic toxicity (in terms of histopathological findings in thyroid and liver in parental males and females at 300 and 1000 mg/kg). Thus, the NOAEL for general systemic toxicity could be established at 100 mg/kg body weight/day.

No adverse effects of Acetoacetmide were noted at a dose level of 1000 mg/kg body weight/day for reproductive and developmental toxicity. Thus, the NOAEL for reproductive and developmental toxicity screening could be established at 1000 mg/kg body weight/day.

 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

12 June 2017 - 8 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

This study was designed as a dose range finding study and in this respect followed the procedures as indicated in the following internationally accepted guidelines and recommendations:

OECD Guidelines for Testing of Chemicals, Section 4, No. 407, “Repeated Dose 28-Day
Oral Toxicity Study in Rodents”, adopted 03 October, 2008

Qualifier:

no guideline available

Principles of method if other than guideline:

This study was designed as a dose range finding study and in this respect did not conform to any specific test guideline. It followed the procedures as indicated in the
following internationally accepted recommendations:
Commission Regulation (EC) No. 440/2008, L 142, Annex Part B, May 30, 2008

GLP compliance:

no

Remarks:

study conducted in a GLP accredited facility

Limit test:

no

Specific details on test material used for the study:

Batch No.: 12535
Physical State: crystalline solid
Colour: white to yellowish
Molecular Weight: 101.11 g/mol
pH Value: 7.7
Purity: > 98 %
Expiry Date: 13 May 2018
Storage Conditions: room temperature, protected from light

Species:

rat

Strain:

Wistar

Remarks:

Wistar rats, Crl: WI(Han)

Details on species / strain selection:

This test was performed on the rat (Wistar rats, Crl: WI(Han)), it is commonly used for such studies as is the preferred rodent species.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Housing and Feeding Conditions

Full barrier in an air-conditioned room
Temperature: 22 ± 3 °C
Relative humidity: 55 ± 10 %
Artificial light, sequence being 12 hours light, 12 hours dark
Air change: 10 x / hour
Free access to Altromin 1324 maintenance diet for rats and mice
Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
The animals were group housed (3 animals / sex / cage) in IVC cages (type III,
polysulphone cages) on Altromin saw fibre bedding
Adequate acclimatization period (at least five days)

Number and Sex of Animals

24 animals (12 males and 12 females) were used for the study (3 male and 3 female animals per group).

Preparation of the Animals

Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Only healthy animals were used for the study. Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively. Each animal was marked for individual identification with an ear tattoo and/or tail number.

Route of administration:

oral: gavage

Details on route of administration:

The test item formulation and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight

Vehicle:

water

Details on oral exposure:

Daily for 14 days. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Analytical verification of doses or concentrations:

yes

Remarks:

assessed as part of a separate GLP analytical study Study No. 173604

Details on analytical verification of doses or concentrations:

Dose formulation analysis for verification of concentration and homogeneity of prepared formulations was not performed in this dose range finding study.
Prior to the start of the subsequent OECD 422 study stability and homogeneity of the test item in the vehicle will be investigated.

Duration of treatment / exposure:

14 days, daily oral dosing.

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Details on study design:

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 14 days. During the treatment period, the animals were observed each day for signs of toxicity. Animals which may die or may be humanely sacrificed in a condition of impending or predictable death were subjected to necropsy. At the conclusion of the test, the surviving animals were sacrificed and subjected to necropsy.

Positive control:

No

Observations and examinations performed and frequency:

All Animals were observed for clinical signs during the entire treatment period of 14 days.

The body weight was recorded once before assignment to the experimental groups and on study days 1, 8 and 14 during the treatment period as well as on the day of necropsy.

Food consumption was measured on study days 1, 8 and 14 for each cage.

Haematological parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals - - see any other information om materials and methods.

Parameters of clinical biochemistry were examined at the end of the treatment prior to or as part of the sacrifice of the animals - see any other information om materials and methods.

Sacrifice and pathology:

On study day 15, all animals of the study were sacrificed using anesthesia (ketamine, xylazine) and were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Other examinations:

Organ weights were taken from all sacrificed animals as soon as possible. Paired organs were weighed together - see any other information om materials and methods.

Statistics:

Parameters such as body weight gain and food consumption were calculated for each animal as the difference in weight measured from one week to the next.
The relative organ weights were calculated in relation to the brain weight and in relation to the body weight (measured at necropsy) and presented as percentage.

All results were reported in tabular form (summarised in mean or summary tables and/or listed in individual data tables).

Toxicology and pathology data were captured either on paper according to appropriate SOPs or using the validated computerised system Ascentos® System (version 1.1.3,
Pathology Data Systems Ltd.).

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred in the control or any of the dose groups during the treatment period with Acetoacetamide.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in the control or any of the dose groups during the treatment period of this study. All animals were subjected to a detailed gross necropsy at the end
of the treatment period on day 15.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effects on body weight and body weight gain were observed during the treatment period of the male and female dose groups when compared to the respective controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no effects of Acetoacetamide on food consumption during the treatment period of the male and female dose groups when compared to the respective controls.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

At the end of the treatment period, no effects on the investigated haematology parameters were seen in the male and female animals of the dose groups compared to the respective control group.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Investigation of clinical biochemistry parameters at the end of the treatment period revealed no effects of Acetoacetamide in the treated male and female animals
compared to the corresponding control group.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

At the end of the treatment period, there were no effects of Acetoacetamide on organ weights of the male and female dose groups when compared to the respective controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

As no mortality occurred during the treatment period with Acetoacetamide, all males and females of the control and the dose groups were subjected to a detailed gross necropsy
at the end of the treatment period.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

On the basis of this 14-day dose range finding oral toxicity study with Acetoacetamide in male and female Wistar rats with dose levels of 100 (LD), 300 (MD), and 1000 (HD) mg/kg body weight/day, the following conclusions can be made:
The animals in this study showed no clinical signs and no mortality. Moreover, test item treated animals showed no difference in body weight, body weight gain and food
consumption compared to the control animals. Also macroscopic findings at the time of necropsy and organ weights showed no significant test item related effects throughout all dosing groups.
Under the conditions of the study performed, it is assumed, that Acetoacetamid has no toxic effects on male and female Wistar rats.
Based on the data generated from this dose range finding study, dose levels of 0 (C), 100 (LD), 300 (MD) and 1000 (HD) mg/kg body weight per day are suggested for the subsequent main study (OECD 422) with Acetoacetamide.

Executive summary:

In a 14 -day dose range finding study groups of male and female rats were dosed by gavage with dose levels of 0, 100, 300, and 1000 mg/kg bw/day; no mortality and no signs of toxicity were observed in any of the dose levels used. The dose levels suggested for a higher tier repeated dose oral toxicity study are 100, 300 and 1000 mg/kg bw/day.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-11 to 2018-06-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Name: Acetoacetamide
CAS No.: 5977-14-0
Batch No.: 12535
Physical State: crystalline solid
Colour: white to yellowish
Molecular Weight: 101.11 g/mol
pH Value: 7.7
Purity: 99.4 %
Expiry Date: 13 May 2018
Storage Conditions: room temperature, protected from light
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Species:

rat

Strain:

Wistar

Remarks:

Wistar Crl: WI(Han)

Details on species / strain selection:

A commonly used species and strain of rat employed in this type of study globally.

Sex:

male/female

Details on test animals or test system and environmental conditions:

1. Full barrier in an air-conditioned room
2. Temperature: 22 ± 3 °C
3. Relative humidity: 55 ± 10 %
4. Artificial light, sequence being 12 hours light, 12 hours dark
5. Air change: 10 x / hour
6. Free access to Altromin 1324 maintenance diet for rats and mice
7. Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
8. Animals were housed in groups of 5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages during the premating period for both males and females and during post-mating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
9. Nesting material were provided latest on GD 18 for all mated females
10. Adequate acclimatisation period (at least 5 days) under laboratory conditions

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage, this is a potential route of incidential exposure in man.

Vehicle:

water

Details on oral exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration analysis of formulation samples was determined at three concentrations, 20 mg/mL, 60 mg/mL and 200 mg/mL in study weeks 1, 3, 5 and in the last week of the study.
The mean recoveries observed for the LD dose group was between 99.4 % and 105.1 % of the nominal value, between 99.3 % and 105.3 % for the MD dose group and between 98.3 % and 103.0 % of the nominal value for HD dose group. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 102.0 %, 101.5 %, and 100.2 % of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10 %.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control: application volume for all groups was 5 mL/kg body weight

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low Dose: application volume for all groups was 5 mL/kg body weight

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Medium Dose: application volume for all groups was 5 mL/kg body weight

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High Dose: application volume for all groups was 5 mL/kg body weight

No. of animals per sex per dose:

10 males and 10 females.

Control animals:

yes, concurrent vehicle

Details on study design:

The study followed the OECD 422 Guideline.

Positive control:

No

Observations and examinations performed and frequency:

Clinical Observations
General clinical observations was made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations include spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.


Functional Observations
Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the last week of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each group outside the home cage using a functional observational battery of tests. 
Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye) were recorded.

Body Weight and Food Consumption
The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13 along with the pups. All animals were weighed directly before termination.
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.


Haematology
Haematological parameters were examined in 5 randomly selected males and females (only lactating females were evaluated) from each group at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in EDTA-coated tubes. The following haematological parameters were examined: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono),
eosinophils (Eos), basophils (Baso) and large unstained cells (Luc).

Blood Coagulation
Coagulation parameters from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in citrate tubes. The following coagulation parameters were examined: prothrombin time (PT) and activated partial thromboplastin time (aPTT).

Clinical Biochemistry
Parameters of clinical biochemistry from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in serum separator tubes. The following parameters of clinical biochemistry were examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP),
albumin (Alb), urea, total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na) and potassium (K).
Blood samples were collected from 2 female pups/litter on day 4 after birth, from all dams and 2 pups/litter at termination on day 13, and from all adult males at termination. All blood samples were stored under appropriate conditions. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroxine (T4). Pup blood was pooled by litter for T4 analysis.
Two pups per litter were sacrificed on day 4 after birth and blood samples were taken for possible serum hormone assessments. Two pups per litter were female pups to reserve male pups for nipple retention evaluations, except in the event that removing these pups left no remaining females for assessment at termination. No pups were eliminated when litter size dropped below 8 pups. When there was only one pup available above a litter size of 8, only one pup was sacrificed.


Urinalysis
A urinalysis was performed with samples from 5 randomly selected males prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance was recorded. The following parameters were measured using qualitative indicators (Heiland Urine Stripes URI 10SL): specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood and leukocytes.

Sacrifice and pathology:

Pathology

All males were sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days) and females were sacrificed on the respective PND 13 by using anesthesia (ket amine/xylazine). All surviving pups were killed by cervical dislocation on PND 13.
Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle.
Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coag ulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesio ns of all adult animals were preserved in 4 % neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson's Solution for 24 hours and then transferred to 70 % ethanol.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Organ Weights

The wet weight of the organs (Table 8) of 5 randomly selected male and female animals (only lac tating females were evaluated) from each group was recorded as soon as possible. Paired organs were weighed together. Organ weights of the animal euthanised for animal welfare reasons were not recorded.
Reproductive organs (testes, epididymides, prostate with seminal vesicles and coagulating glands, uterus with cervix and ovaries) were weighed from all animals.
Thyroid/parathyroid glands from 1 pup/sex/litter/group (if possible) (sacrificed on PND 13) and from al l adult males and females were preserved. Weight of thyroid/parathyroid glands was measured after f ixation.

Histopathology

A full histopathology was carried out on the preserved organs and tissues of all animals of the control and high dose groups which were sacrificed at the end of the treatment period. Thyroid/parathyroid glands from pups and from the adult animals were examined. Thyroid/parathyroid glands from pups and from the remaining non-selected adult animals were not examined as no major histopathological findings were observed in thyroid/parathyroid gland of selected male and female adult animals and T4 hormone levels of males and day 13 pups.
A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study.

Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thi ckness of 2-4 um and stained with hematoxylin and examined in control and HD animals and in non-pregnant female animals of the LD and MD animals. Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were also examined in the mating partners of the non-pregnant female LD and MD animals.
These examinations were extended to animals of all other dosage groups for treatment-related changes in the stomach that are observed in the high dose group.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for histopathology). The study phases from test site 1 and 2 were performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012) [10]. Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP's of the test sites

Postmortem examinations (offspring)

Pathology

All surviving pups were killed by cervical dislocation on PND 13.
Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Organ Weights

Thyroid/parathyroid glands from 1 pup/sex/litter/group (if possible) (sacrificed on PND 13) and from all adult males and females were preserved. Weight of thyroid/parathyroid glands was measured after fixation.

Histopathology

Thyroid/parathyroid glands from pups and from the adult animals were examined. Thyroid/parathyroid glands from pups and from the remaining non-selected adult animals were not examined as no maj or histopathological findings were observed in thyroid/parathyroid gland of selected male and female adult animals and T4 hormone levels of males and day 13 pups.

Other examinations:

Reproductive indices: Copulation and fertility indices were calculated (see results for full details).

Offspring viability indices: Assessed throughout the post-partum period.

Statistics:

The findings of this study were evaluated in terms of the observed effects, the necropsy and the microscopic findings.

The gestation length, pre-coital interval, the number of live births and post-implantation loss, the number of pups with grossly visible abnormalities, the number of implantations, corpora lutea, AGD, litter size and litter weights were summarized in tabular form.

Parameters like body weight gain and food consumption were calculated as the difference in weight measured from one week to the next. The relative organ weights were calculated in relation to the body weight (measured at necropsy) and were presented as percentage.

All results were reported in a tabular form (summarised in mean or summary tables and/or listed in individual data tables).

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non -parametric Kruskal-Wallis Test and a post-hoc Dunn's Test, based on the results of homogeneity and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed for all animals in the male and female control and dose groups until terminal sacrifice. During the weekly detailed clinical observation, no relevant differences between the groups were found. None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in the control or any of the dose groups during the treatment period of this study. All animals survived their scheduled study period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In both males and females, there was no test item treatment related effect observed on body weight and body weight gain in the dose groups during the study period.
Statistical significance was found for a moderately lower body weight gain in male HD group between premating day 1 to postmating day 14 and premating day 1 to terminal sacrifice when compared to control group. As there was no dose dependency and all male animals in all dose groups were without clinical findings, the statistical significance is not considered to be an adverse effect due to treatment with test item. No statistical significances were seen in the female groups.
Mean body weight gain between day 0 and day 4 in the lactation phase was lower in the female HD group than in the control group. This finding is considered to be incidental and not related to adverse effects of the test item as no dose depency was seen during lactation phase.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No test item related or statistically significant effect on food consumption was observed in males and females during the whole study period.
For the males food consumption was lower during the entire premating period for all dose groups, except for the MD group in the first half of premating. A dose dependency was not observed.
Marginally reduced mean food consumption was found for females in the HD group during premating, gestation and lactation phase, but no dose dependency was found during study period.
In correlation to the body weight and body weight gain, the food consumption in both males and females tended to increase with the progress of the study in the control, the LD, the MD and the HD group.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

The test item had no toxicologically relevant effect on haematology and blood coagulation parameters analysed at the end of the treatment period of this study.
Statistical significance was only achieved for an increase of the mean reticulocyte value in the male HD group when compared to control group. Reticulocytes in all other male dose groups and female groups (only lactating females) were not affected. Furthermore, RBC values are slightly decreased in the male HD group not reaching statistical significance. Under consideration of the lack of dose dependency for reticulocytes in the male groups and absence of findings in the female dose groups (only lactating females), the statistical significance is not considered to be of toxicological or biological relevance.
No test item related effect was observed on coagulation parameters in males and females when compared with the respective controls. No statistical significance was seen for all coagulation parameters in the male and female dose groups.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The test item had no toxicologically relevant effect on parameters of clinical biochemistry analysed at the end of the treatment period of this study.
Statistical significance was found for a slightly decreased TP mean value of the male HD group when compared to control group. For male LD and MD groups the mean value was slightly decreased without reaching statistical significance. No clear dose dependency was seen for this parameter. No decrease or dose dependency was seen for TP in the female dose groups (only lactating females) when compared to control group. Based on these findings, the statistical significance for TP is not considered to be biological or toxicological relevant.
Furthermore, no statitistical significance was found for all parameters in the female dose groups (only lactating females).

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

For some animals in the LD, MD and HD group elevated levels of glucose were measured with the highest value for animal no. 28 (> 1000 mg/dl). Under consideration of the glucose values in serum this finding is not test item-related. Serum mean glucose levels in all dose groups are higher when compared to control group but are seen without dose dependency and no statistical significance.
Elevated levels of erythrocytes and urobilinogen were found in the urine of few male animals in the LD, MD and C group, elevated levels of leucocytes and bilirubin were seen in all male dose groups and the male C group. Protein levels were seen in all male dose groups including the C group. Therefore, this effect on urine parameters was not considered to be test item related. Positive nitrite value in one male control animal is considered to be incidental.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

In males, slightly and statistically significantly higher supported rearing count in LD group before initiation of treatment was observed when compared to control. As this difference was seen before start of treatment and all other parameters of functional observation were unremarkable, it is considered to be incidental and biologically not relevant.
In females, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared with the controls.
There were no biologically relevant differences observed in body temperature between the groups and no ophthalmoscopic findings occured in any of the observed male and female animals.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The mean absolute and relative organ weights of thyroid / parathyroid increased dose-dependently with statistical significance for the male and female HD group (mean organ weight for males: LD 16.11 %, MD 24.28 %, HD 33.91 % compared to control, mean organ weight for females: LD -7.14 %, MD 10.34 %, HD 24.85 % compared to control) and slight statistical significance for relative weight in the male MD group (relative organ weight: MD 0.0087 % compared to control group 0.0070 %). This finding correlates to a minimal to slight follicular hypertrophy in most animals of the male and female HD group and six males in the MD group.
For the male HD group the mean weight of spleen is 14.72 % below controls. Whereas in all male groups the mean and relative organ weight was below control, the mean and relative organ weight for spleen weight in the female groups was above control (up to 11.66 % for MD group). Therefore no toxicological effect was seen on spleen weight.
In the male and female groups the values for thymus showed marked individual variability. The mean and relative mean weights for the male LD group were up to 14.88% (relative weight) above the control group. In the female groups, the mean weights were only for the MD group above control (35.86 % for mean absolute weight). In the absence of dose dependency no toxicological effect can be concluded for thymus weight.
No dose dependent decrease or increase was seen for kidney in the male or female groups. For the male groups an increase was only found in the MD group (relative weight 13.83 % and absolute weight 9.71 % above control group), in the LD and HD groups a decrease in organ weight was found (up to 13.53 % below control). The highest increase was seen for the relative weight in the female LD group (11.95 % above control). No dose dependency was found for female dose groups. Therefore, no toxicological effect on kidney was found.
In the male groups a marked decrease for the mean and relative organ weight of adrenal glands was found in the HD group (absolute weight 24.82 % and relative weight 19.91 % below control), but no dose dependency was seen. For the females an increase without dose dependency of the relative and mean organ weight was seen in all groups. Under consideration of the absence of dose dependency no toxicological effect can be concluded.
Mean and relative organ weight for heart was markedly increased in the male MD group when compared to control group (absolute 18.64% and relative 23.31%) and is considered to be incidental, as no such effect was found in the female dose groups and no dose dependency occurred.
A slight decrease for the mean and relative organ weight liver was seen in the male groups, with a decrease of absolute weigth up to 12.34 % in the HD group below control group. This was not found in the female groups, where all mean values with exception of the mean value in the MD group were increased compared to control group. Under consideration of organ weight determination no clear correlation to the histopathological observation of a minimal hepatocellular hypertrophy noted in male animals of the HD group can be found.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Male animal no. 35 in the HD group showed a small right coagulating gland at necropsy. Histopathologically, this finding was not attributed to treatment with test item. No histological correlation was seen.
There were no further findings recorded at necropsy for all other animals in all groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the stomach minimal to moderate multifocal mixed inflammatory cell infiltrates in the submucosa of the glandular stomach were seen and in the limiting ridge. Whereas in control animals slight mixed inflammatory cell infiltrates were observed in the limiting ridge of one animal only. The accumulation of inflammatory cells in the submucosa of the glandular stomach was observed in animals from the high dose group only and was most likely induced by the local irritating effect of the test item.

In kidneys from male rats hyaline droplets were seen and represent an accumulation of secondary lysosomes within the cytoplasm and contain alpha-2u-globulin that reversibly binds to the inducing xenobiotics and/or metabolites. Hyaline droplets deposition is a male rat specific event, and it is of no toxicological relevance in humans.

The test item did not produce any histological evidence of toxicity in the reproductive organs and tis sues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, oviducts, uterus and cervix, and vagina. Furthermore, there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In most animals per sex at 1000 mg/kg bw, but also in six males at 300 mg/kg/ bw, a minimal to slight follicular hypertrophy was seen for thyroid glands. Additionally, increased minimal hepatocellular hypertrophy was noted in males at 1000 mg/kg bw. No test item-related increase of this finding was observed in females. The findings observed for thyroid glands and liver at histopathological evaluation were considered to be test item-related.
There were no abnormalities in the testes, epididymides and accessory sex glands that could be related to treatment in the control and dose animals.
There were no further findings that distinguished controls from test item-treated animals for all other organs.

Other effects:

no effects observed

Description (incidence and severity):

Clinical Biochemistry:
The test item had no toxicologically relevant effect on parameters of clinical biochemistry analysed at the end of the treatment period of this study.
Statistical significance was found for a slightly decreased TP mean value of the male HD group when compared to control group. For male LD and MD groups the mean value was slightly decreased without reaching statistical significance. No clear dose dependency was seen for this parameter. No decrease or dose dependency was seen for TP in the female dose groups (only lactating females) when compared to control group. Based on these findings, the statistical significance for TP is not considered to be biological or toxicological relevant.
Furthermore, no statitistical significance was found for all parameters in the female dose groups (only lactating females).

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

System:

endocrine system

Organ:

liver

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Reproductive function / performance (P0)

Reproductive function: estrous cycle: no effects observed

Test item had no biologically significant effect on the estrous cycle analysed during 2 weeks premating period after the first administration in treatment groups when compared to the controls. There were no significant differences in the length or sequence of cycle stages between the treatment groups and the control group and no statistical significances were calculated.

Reproductive performance: no effects observed

There were no test item-related effects on the reproductive indices (copulation, viability, fertility and delivery indices) in the dose groups when compared to the control group.

Conclusions:

On the basis of this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with Acetoacetamide in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:
No mortality occurred in this study.
No adverse effects were found on male and female clinical observations, functional observations, body weight, food consumption, haematology and coagulation, clinical biochemistry, urinalysis and gross pathological findings at necropsy.
There were also no test item-related effects on estrous cyclicity, nipple retention, anogenital distance, precoital interval and duration of gestation, reproductive indices, pup thyroid weight and pup thyroxine hormone, pup external findings on PND 0 and at death. No test item-related effects on number of corpora lutea, implantation sites, % pre and post implantation loss, litter data, litter weights, reproductive indices and number of live pups on PND 0, 4 and 13 in HD group were observed when compared with controls.
For organ weight thyroid / parathyroid a test item-related dose dependent increase with statistical significance (absolute and relative for male and female HD group and relative for male MD group) was seen. Absolute and relative organ weight for liver was decreased in all male groups and male thyroxine hormone was statistically significant decreased in the male HD group compared to control group. Test item-related minimal to slight follicular hypertrophy for thyroid glands in male and female HD group and male MD group was found at histopathological evaluation. Additionally, test item-related hepatocellular hypertrophy in male HD group was recorded.
Adverse effects of Acetoacetmide were noted at a dose level of 100 mg/kg body weight/day for general systemic toxicity (in terms of histopathological finings in thyroid and liver in parental males and females at 300 and 1000 mg/kg). Thus, the NOAEL for general systemic toxicity could be established at 100 mg/kg body weight/day.
No adverse effects of Acetoacetmide were noted at a dose level of 1000 mg/kg body weight/day for reproductive and developmental toxicity. Thus, the NOAEL for reproductive and developmental toxicity screening could be established at 1000 mg/kg body weight/day.

Executive summary:

The aim of this study was to assess the possible effects of Acetoacetamide on male and female fertility and embryo-fetal development after repeated dose administration in Wistar rats in an OECD 422 study design. The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. Animals of an additional control group were handled identically as the dose groups but received sterile water, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats.

No adverse effects were found on male and female clinical observations, functional observations, body weight, food consumption, haematology and coagulation, clinical biochemistry, urinalysis and gross pathological findings at necropsy. There were also no test item-related effects on estrous cyclicity, nipple retention, anogenital distance, precoital interval and duration of gestation, reproductive indices, pup thyroid weight and pup thyroxine hormone, pup external findings on PND 0 and at death. No test item-related effects on number of corpora lutea, implantation sites, % pre and post implantation loss, litter data, litter weights, reproductive indices and number of live pups on PND 0, 4 and 13 in HD group were observed when compared with controls.

Adverse effects of Acetoacetmide were noted at a dose level of 100 mg/kg body weight/day for general systemic toxicity (in terms of histopathological finings in thyroid and liver in parental males and females at 300 and 1000 mg/kg). Thus, the NOAEL for general systemic toxicity could be established at 100 mg/kg body weight/day.

No adverse effects of Acetoacetmide were noted at a dose level of 1000 mg/kg body weight/day for reproductive and developmental toxicity. Thus, the NOAEL for reproductive and developmental toxicity screening could be established at 1000 mg/kg body weight/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Satisfactory

System:

endocrine system

Organ:

liver

thyroid gland

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

Justification for classification or non-classification

The test substance does not warrent classification under (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.