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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Acetoacetamide
EC Number:
227-774-4
EC Name:
Acetoacetamide
Cas Number:
5977-14-0
Molecular formula:
C4H7NO2
IUPAC Name:
acetoacetamide
Test material form:
solid: crystalline
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.

Test animals

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

Administration / exposure

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]
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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.

Effect levels (maternal animals)

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

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

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.

Effect levels (fetuses)

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

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables


 

Applicant's summary and conclusion

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.