Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

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.

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.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
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)
Deviations:
yes
Remarks:
see Overall remarks
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
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
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test System
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous
Age at the start of the treatment period: approx. 14-15 weeks old
Body weight at the allocation of the animals to the experimental groups:
males: 327 – 368 g (mean: 350.88 g, ± 20 % = 280.70 – 421.05 g)
females: 204 - 251 g (mean: 226.60 g, ± 20 % = 181.21 – 271.92 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes.
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.

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)
- 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.
- Nesting material was provided latest on GD 18 for all mated females
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Preparation of the Animals
Prior to the start of the treatment period, a detailed clinical observation outside the home cage was made. None of the animals showed pathological signs before the first administration.
Before initiation of dosing, all females were screened for two weeks for regular estrous cyclicity and females (10 females/ group) with regular estrous cycle (4-5 day cycle) were used in the study.
Before the first administration, all animals to be 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. Randomisation was performed with validated IDBS Workbook 10.1.2 software.
Each animal was marked with its identification number by individual ear tattoo and/or tail marking.





Route of administration:
oral: gavage
Vehicle:
water
Remarks:
aqua ad iniectabilia (sterile water)
Details on exposure:
The vehicle was selected in consultation with the sponsor based on the test item’s characteristics and testing guideline.
The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulation was vortexed and/or stirred until visual homogeneity was achieved.
Based on the results of stability testing (Eurofins Munich Study No. 173603), the test item formulations were prepared at least once every 12 days (within stability time frame as given by Eurofins Munich Study No. 173603). The prepared formulation was stored protected from light and at room temperature. Formulates were kept under magnetic stirring during the daily administration. The vehicle was also used as control item.


According to the results of a previous dose range finding study (BSL Munich Study No. 173597, non GLP) and in consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose).

Control: 0 mg/kg/d
Low Dose: 100 mg/kg/d
Medium Dose: 300 mg/kg/d
High Dose: 1000 mg/kg/d


The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering or a maximum dose of 1000 mg/kg bw/day. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the high dose group.

The test item 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. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.









Details on mating procedure:
Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the mating. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
The cages were arranged in such a way that possible effects due to cage placement were minimised.




Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study (Eurofins Munich Study No. 173603).
Study pre start stability analysis was included on the samples from high dose and low dose group and the investigation was made for 0 h, 6 h (RT), 12 day (RT), 12 day (2-8 °C) and 12 day -15 to -35 °C.
Prestart homogeneity investigation included the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
Since the test item was shown to be homogenous according to Eurofins Study No. 173603 (after at least 30 min without stirring), samples were not collected during the study for the investigation of homogeneity and only samples were taken for substance concentration in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) from all groups (16 samples).
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 173604) and until then stored under appropriate conditions based on available stability data. The B-samples were retained at -15 to -35 °C at BSL Munich (test facility) and discarded after completion of the final study report.
The phase plan was amended to the study plan. The results are reported in the appendix of the final report.



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. Then in females, treatment was done during the gestation period and up to post-natal day 12. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.
Frequency of treatment:
daily
Details on study schedule:
Arrival of the Test Item: 18 May 2017
Study Initiation Date: 11 August 2017
Amendment to Study Plan: 28 August 2017
Delivery of Animals: 08 August 2017
Acclimatisation Period: 08 August 2017 - 14 August 2017
Experimental Starting Date: 14 August 2017
Treatment Period: 28 August 2017 – 20 October 2017
Necropsies: 25 September 2017- 26 September 2017 (male),
16 October 2017 - 20 October 2017 (female)
Experimental Completion Date: 20 October 2017
Completion Date of Delegated Phase (Histopathology): 01 June 2018
Completion Date of Delegated Phase (Formulation Analysis): 21 December 2017
Study Completion Date: date of the study director’s signature






Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
100 animals (40 males and 60 females) were included in the study. All females were screened for regular estrous cycles for 14 days before the treatment initiation and only 40 females (10 females/group) showing regular estrous cycles were continued in the study. Remaining not selected 20 females were discarded without any observations or used for other appropriate studies/procedures.
Control animals:
yes, concurrent vehicle
Positive control:
No
Parental animals: Observations and examinations:
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.












Oestrous cyclicity (parental animals):
Estrous cycles were monitored before treatment initiation to select for the study females with regular estrus cyclicity. Vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.
Sperm parameters (parental animals):
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. Please refer to histopathology section.
Litter observations:
The duration of gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after the delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of littering
(PND 0) and on PND 4 and PND 13. Live pups were identified by tattooing. In addition to the observations of the parent animals, any abnormal behaviour of the offspring was recorded.
The anogenital distance (AGD) of each pup was measured on PND 0 and normalised to the cube root of body weight measured on the same day. The number of nipples/areolae in male pups was counted on PND 12.




Postmortem examinations (parental animals):
Pathology
All surviving males were sacrificed any time after the completion of the mating period (after a minimum dosing period of 37-38 days) and females were sacrificed on the respective PND 13 by using anesthesia (ketamine/xylazine). All surviving pups were killed by cervical dislocation on PND 13.
Vaginal smears were examined on the day of necropsy (except for animal no. 59, 70, 73, 75) to determine the stage of estrous cycle.
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 coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions 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 [testes (paired weight), epididymides (paired weight), prostate, seminal vesicles and coagulating glands (complete weight), uterus with cervix, ovaries (paired weight), thyroid/parathyroid glands (from 1 pup/sex/litter/group and from all adult males and females) – weighed after fixation (complete weight), thymus, liver, spleen, kidneys (paired weight), brain, adrenal (paired weight), heart and pituitary gland] of 5 randomly selected male and female animals (only lactating females were evaluated) from each group were recorded as soon as possible except for the organ weights of the female LD group were only 4 randomly selected animals were weighed. Paired organs were weighed together. Organ weights of animals found dead or 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 all adult males and females were preserved. Weight of thyroid/parathyroid glands was measured after fixation.
Adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, eyes, optic nerves, Harderian gland, femur with knee joint, heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (mandibular), lymph nodes (mesenteric), lymph nodes (axillary), mammary gland area (male and female), oesophagus, ovaries, oviducts, pancreas, pituitary, prostate and seminal vesicles with coagulating glands as a whole, rectum, salivary glands (sublingual, submandibular), sciatic nerve, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar segments), spleen, sternum (with bone marrow), stomach, testes, thymus, thyroid/parathyroid glands, tongue, trachea, ureters, urinary bladder, uterus with cervix and vagina of the same selected animals from each group were preserved in 4% neutral-buffered formaldehyde except for testes and epididymides that were fixed in Modified Davidson’s Fixative for approximately 24 hours before they were transferred to 70% ethanol. All animals found dead and/or intercurrently euthanised for animal welfare reasons were subjected to a gross necropsy and the organs preserved for a histopathological examination.
Thyroid/parathyroid glands from 1 pup/sex/litter/group sacrificed on PND 13 and non-selected adult animals were preserved for potential histopathological examination.

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. . Histopathological examination of thyroid gland from pups and from the remaining non-selected adult animals was not deemed necessary as no test item related histopathological findings were observed in thyroid/parathyroid gland of selected animals and there was also no test item related effect observed on T4 hormone level in males and pups sacrificed on PND 13.
A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study or which were euthanised due to morbidity.
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 thickness of 2-4 µm and stained with hematoxylin and examined in control and HD animals.
These examinations were not extended to animals of all other dosage groups as no treatment-related changes were observed in any organ/tissue of 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 were performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation were 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). Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.
The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of compliance and quality assurance statement) to the study director.
The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon the completion of the study.





Postmortem examinations (offspring):
Dead pups and all surviving pups on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.
Statistics:
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. If required other statistical method were used. 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):
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.
Dermal irritation (if dermal study):
not examined
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:
no effects observed
Description (incidence and severity):
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.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
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.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
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:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results P0
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
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 function: sperm measures:
no effects observed
Description (incidence and severity):
see Details on results P0: Histopathology
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
see Details on results P0: Thyroid Hormone (T4) Analysis, Precoital Interval and Duration of Gestation, Pre- and Postnatal Data, Reproductive Indices
Clinical Observations
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 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 Development
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
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.

Haematology and Coagulation
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
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).

Urinanalysis
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.

Functional Observations
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.

Organ Weights
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. 
Regarding reproductive organs there was a decrease for the mean and relative organ weight prostate with seminal vesicles and coagulating glands in the HD group (absolute 14.49 % and relative 12.32 % below control), but a slight increased mean and relative weight in the LD and MD group. In the females a slight decrease of approx. 16 % below control group was seen in the LD group for mean and relative weight of ovaries. As no dose dependency was observed for reproductive organs, the findings are considered to be of no toxicological relevance.
There were no further considerable differences in organ weight between dose groups and control group.

Pathology
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.

Histopathology
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.

Thyroid Hormone (T4) Analysis
No treatment-related changes were observed for T4 in adult males of the LD and MD groups, when compared to the controls. In the adult male HD group, T4 was markedly decreased with statistical significance. T4 analysis in adult male animals at terminal sacrifice showed a tendency to decreased mean values of the dose groups compared to control group without dose dependency for male pups. A marked decrease of T4 is recorded for the male animal HD group (39.88 nmoL/L) when compared to C group (64.57 nmoL/L).

Estrous Cyclicity
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.
Deviations from the physiological 4 or 5 day cycle in the rat were observed in animals of the control, LD and HD group. Cycles with less than 4 days were seen in 4 animals in the control, 2 animals in the LD and 3 animals in the HD group. Additionally, for one control animal the cycle was more than 5 days. These deviations are not considerd to be related to treatment with test item as deviations occurred mainly in the control groups and occasionally in the dose groups.

Precoital Interval and Duration of Gestation
There were no test item-related or statistically significant effects observed on the duration of precoital interval and the duration of gestation in the dose groups when compared to the control group.

Pre- and Postnatal Data
There were no test item treatment related effects observed on the number of corpora lutea, implantation sites, live pups on PND 0, 4 and 13, percent preimplantation loss and post implantation loss in treatment groups when compared with the control group.

Reproductive Indices
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. However, a reduced viability index (No. of live offspring at day 4 / No. of live offspring at birth X 100) of 88.9 % during PND 4-13 was observed for one animal in the HD group One pup of dam No. 75 (HD group) was missing between PND 4 and PND 13 and resulted in a slightly lower mean viability index (Viability Index (%) = (No. of live offspring at day 4 / No. of live offspring at birth) X 100) of 98.89 % for the HD group. This is considered to be incidential and not test item related.







Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results F1: Pup External Findings
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
see Details on results F1
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see Details on results F1
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results F1
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
see Details on results F1
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
see Details on results F1: Thyroid Hormone (T4) Analysis, Thyroid Weight on PND 13
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Litter Data
There were no test item treatment related or statistically significant effects observed in treatment groups on litter data parameters like group mean total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, still birth, runt on PND 0 as well as number of live pups, male pups, number of female pups and sex ratio on PND 4 and PND 13 when compared with the controls.

Litter Weight Data
There was no test item related effect on pup mean weight, total litter weight, female litter weight on PND 0, PND 4 and PND 13 observed in treatment groups when compared with the controls. There was no statistically significant change in dose groups compared to corresponding controls.

Pup Survival Data
No test item related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in treatment groups was observed when compared to the control group. A marginally higher mean total mortality of pups between PND 4 and PND 13 was observed in the HD group (1.11%) compared to the control (0.00%). This increase is due to one dam No. 75 which lost one pup during this time period. This single mortality was considered as incidental and not related to the treatment with the test item.

Anogenital Distance and Nipple Retention
In males and females, no statistically significant effect on pup weight and absolute and relative anogenital distance in treatment groups was observed when compared to the controls.
No statistically significant effect was observed on nipple retention in the pups of any of the groups when compared with the controls. Group mean number of nipple retention in HD was slightly higher without achieving statistical significance when compared with the control group and is therefore considered to be not related to treatment with test item.

Thyroid Hormone (T4) Analysis and Pup Thyroid Weight on PND 13
No treatment-related changes were observed for mean male and female pup thyroid/parathyroid weight on PND 13, for thyroxine (T4) in PND 13 pups, or T4 in adult males of the LD and MD groups, when compared to the controls. In the adult male HD group, T4 was markedly decreased with statistical significance
However, there is an increase of mean thyroid/parathyroid weight on PND 13 for male pups (HD: 0.0064 g, MD: 0.0067 g) when compared to control (C: 0.0057 g), which is only slightly seen in female pups (HD and LD: 0.0064 g, MD: 0.0063 g) when compared to control (C: 0.0060 g). No dose dependency or statistical significance was seen for male and female pups and therefore, no adverse effect is considered.
T4 analysis on PND 13 for male pups and adult male animals at terminal sacrifice showed a tendency to decreased mean values of the dose groups compared to control group without dose dependency for male pups. A marked decrease of T4 is recorded for the male animal HD group (39.88 nmoL/L) when compared to C group (64.57 nmoL/L).

Pup External Findings
No test item related gross external abnormalities of toxicological relevance on PND 0-12 and at death were observed in the pups of any of the groups.
Few specific findings on PND 0 like dark snout (pup no. 9 from dam 57 of MD group and pup no. 1 from dam 73 of HD group), dry skin (pup no. 3 from dam 60 of MD group and pup no. 1-10 from dam 68 of HD group) and wound at the right flank (pup no. 5 from dam 68 of HD group) were observed. Additionally, findings like dark head, neck, back (pup no. 13 from dam no. 47) and dark tail tip (pup no. 6 from dam no. 48) were seen in the control group.
Pup no. 1 from control dam no. 44 was found dead on PND 0 and pup no. 6 from dam no. 75 of HD group was not found on PND 9 and is considered to be cannibalised.
External findings like absent hair coat on head (pup no. 1-9 and 12-13 from dam no. 50 of LD group) and on back (pup no. 1-9 from dam no. 79 from the HD group) were recorded at death.
These external findings were considered to be spontaneous in nature and not related to treatment with the test item.



Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
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.
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 embryofetal development after repeated dose administration in Wistar rats.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of63 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 aqua ad injectabilia (sterile water), the vehicle used in this study. The 4 groups comprised 10 male and 10 femaleWistarrats.Before dosing all females were screened for two weeks for regular estrous cyclicity and animals (10 females/ group) with regular estorus cycle (4-5 day cycle) were used in the study.

The following doses were evaluated:

Control (C):                 0           mg/kg body weight/day

Low Dose (LD):          100    mg/kg body weight/day

Medium Dose (MD):   300  mg/kg body weight/day

High Dose (HD):         1000  mg/kg body weight/day

The test item formulation was prepared at least at the latest once every 12 days (within stability time frame as given by Eurofins Munich Study No. 173603). The test item was dissolved in aqua ad injectabilia and administered daily during14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 12 in females. Males were dosedfor 28 days. Dose volumes were adjusted individually based on weekly body weight measurements. Theadministrationvolumewas 5 mL/kg body weight.

During the period of administration, the animals were observed each day for signs of toxicity. At the conclusion of the test animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

Haematological and clinical biochemistry evaluations were performedon blood samples collected at terminal sacrifice fromfive randomly selected males and females from each group. Urinalysis wasperformedon samples collected at terminal sacrifice fromfive randomly selected males from each group.

Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behavior observations were performed in the week before the treatment from all animals and in the last week of treatment infive randomly selected males and femalesof each group.

After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition, on day 4 and day 13 post-partum. The anogenital distance (AGD) of each pup was measured on PND 0.

The number of nipples/areolae in male pups was counted on PND 12. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4).

The males were sacrificed after completion of the mating period on treatment day 29and the females along with their pups were sacrificed on post natal day 13.

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

Pups sacrificed on post-natalday 4 or 13 or those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the preserved tissues (adrenal glands, testes, epididymides, ovaries, oviducts, uterus with cervix, vagina, prostate and seminal vesicle with coagulating gland) was performed on high dose and control animals. Histopathological examinations were extended to animals of all other dosage groups as treatment-related changes were observed in the high dose group. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation ofadditional hematoxylin-PAS (Periodic Acid Schiff)stained slides.All gross lesions macroscopically identified were examined microscopically in all animals.

Summary Results

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. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%. 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.

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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Satisfactory.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

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.

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.


Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 August 2017 to 17 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD 422 Guideline
Version / remarks:
Combined Repeated Dose Oral Toxicity Study with the Reproduction / Developmental Toxicity Screening Study
Deviations:
yes
Remarks:
see Overall remarks
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) rat
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
Vehicle:
water
Details on 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 %.
Details on mating procedure:
Mating was performed using a ratio of 1:1 (male to female) (if possible). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the mating. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrous cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
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
Duration of test:
See above - Details on exposure [study followed the OECD 422 Guideline]
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.
Maternal examinations:
Clinical Observations
General clinical observations were 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 included 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.

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, PND 9 and PND 13 along with 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.

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.

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.

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.

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 (11.20). Blood from the abdominal aorta of the animals was collected in serum separator tubes. From 2 preferably 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, blood samples were collected from the defined site in serum separator tubes. 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 thyroid hormones (T4). Pup blood was pooled by litter for thyroid hormone analysis. Further assessment of T4 in blood samples from the dams and day 4 pups was not deemed necessary based on the fact that 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
Two pups per litter were sacrificed on day 4 after birth and blood samples were taken for possible serum hormone assessments. No pups were eliminated as litter size droped below 8 pups. As 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 and females (only lactating females were evaluated) prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance were recorded.

Estrous cyclicity
Estrous cycles were monitored before treatment initiation to select for the study females with regular estrus cyclicity. Vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.


Ovaries and uterine content:
Examined at terminal necropsy.
Fetal examinations:
The females were allowed to litter. The litter observations were:

Litter observations
The duration of gestation was recorded and is calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after the delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of littering (PND 0) and on PND 4 and PND 13. Live pups were identified by tattooing. In addition to the observations of the parent animals, any abnormal behaviour of the offspring were recorded.
The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = A GD/Cube root of pup weight). The number of nipples/areolae in male pups was counted on PND 12.
Statistics:
The findings of this study were evaluated in terms of the observed effects, the necropsy and the mic roscopic 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).
Indices:
Assessed at necropsy and throughout the post-partum period (see results section).
Historical control data:
The laboratories historical control data was used where appropriate.
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.
Dermal irritation (if dermal study):
not examined
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):
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):
no effects observed
Description (incidence and severity):
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:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
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.
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):
no effects observed
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.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Under consideration of the absence of dose dependency no toxicological effect can be concluded.
Gross pathological findings:
effects observed, non-treatment-related
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, 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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no test item treatment related effects observed on the number of corpora lutea, implantation sites, live pups on PND 0, 4 and 13, percent preimplantation loss and post implantation loss in treatment groups when compared with the control group.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no test item treatment related or statistically significant effects observed in treatment groups on litter data parameters like group mean total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, still birth, runt on PND 0 as well as number of live pups, male pups, number of female pups and sex ratio on PND 4 and PND 13 when compared with the controls.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
No test item related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in treatment groups was observed when compared to the control group. A marginally higher mean total mortality of pups between PND 4 and PND 13 was observed in the HD group (1.11%) compared to the control (0.00%). This increase is due to one dam No. 75 which lost one pup during this time period. This single mortality was considered as incidental and not related to the treatment with the test item.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
There were no test item-related or statistically significant effects observed on the duration of precoital interval and the duration of gestation in the dose groups when compared to the control group.
Changes in number of pregnant:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were observed for mean male and female pup thyroid/parathyroid weight on PND 13, for thyroxine (T4) in PND 13 pups, or T4 in adult males of the LD and MD groups, when compared to the controls.
In males and females, no statistically significant effect on pup weight and absolute and relative anogenital distance in treatment groups was observed when compared to the controls.
No statistically significant effect was observed on nipple retention in the pups of any of the groups when compared with the controls.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
In males and females, no statistically significant effect on pup weight and absolute and relative anogenital distance in treatment groups was observed when compared to the controls.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No test item related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in treatment groups was observed when compared to the control group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Based on litter data.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Based on litter data.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
There were no test item treatment related effects observed on the number of corpora lutea, implantation sites, live pups on PND 0, 4 and 13, percent preimplantation loss and post implantation loss in treatment groups when compared with the control group.
External malformations:
no effects observed
Description (incidence and severity):
Based on litter data.
Skeletal malformations:
not examined
Visceral malformations:
no effects observed
Description (incidence and severity):
Based on litter data.
Other effects:
no effects observed
Description (incidence and severity):
Based on litter data.
Details on embryotoxic / teratogenic effects:
No embryo-fetal toxicity was evident.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no


 

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.
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 embryofetal 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 femaleWistarrats.

The following doses were evaluated: 0 (control), 100, 300 and 1000 mg/kg bw/day (low dose, medium dose and high dose).

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.

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Satisfactory.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
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.

Additional information