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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 22, 2015 - July 14, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
5-methylheptan-3-one
EC Number:
208-793-7
EC Name:
5-methylheptan-3-one
Cas Number:
541-85-5
Molecular formula:
C8H16O
IUPAC Name:
5-methylheptan-3-one
Specific details on test material used for the study:
Name: Ethylamylketon
Chemical name: 5-methylheptan-3-one
Batch no.: Ref. No. 15531051
Appearance: colourless, liquid
Composition: 5-Methylheptan-3-one: 96.42%
3,4-Dimethyl-hexan-2 one: 1.16%
CAS No.: 541-85-5
EINECS-No.: 208-793-7
Purity: 96.42%
Production date: July 15, 2015
Expiry date: July 15, 2016
Storage Room temperature (20 ± 5°C); keep containers tightly closed in a well-ventilated place
Stability Stable at room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Animals
Species / Strain: Hsd. Han: WIST Rats
Source: TOXI-COOP ZRT. 1103 Budapest, Cserkesz u. 90.
Hygienic level: SPF at arrival and kept in good conventional environment during the study
Number of animals:130 females, 80 males
Age of females at arrival:7.5-8.5 weeks
Body weight of females at arrival:140-165 g
Age of males at arrival: 8-9 weeks
Body weight of males at arrival: 230-260 g
Age at start of mating: females 8.5-9.5 weeks, males 12-13 weeks
Acclimatization time: 6 days for females, 26 days for males

Identification of Animals
The individual identification was performed during the pre-experimental period. The individual identification of the adult males and females was performed in the pre-experimental phase by ear punching. Males were identified from 1 to 80 and females were marked from 101 to 230.
The sperm positive females were randomized and the animals’ cages were identified by identity cards, with information about study number, strain, sex, dose group, cage number, individual animal numbers, date of mating and scheduled date of Caesarean section.
Litters: At Caesarean section, the litters were identified with the litter numbers. The flasks used for fixation and all sheets used for recording were marked with these numbers and not with the dose groups up to the end of fetal examinations (“blind” examination of fetuses).
Fetuses:In the course of the Caesarean section, after removal from the uterus the fetuses were randomly allocated and identified thereafter.
For visceral examination the fetuses were identified by finger-cutting, for skeletal examination by means of water-proof plastic ribbon tied around their neck.

Housing Conditions
Animal health:Only healthy animals were used for the study. Health status was certified by the breeder.
Animal room:16/ C
Housing: pre-mating period: 1-3 females /cage,2 males/cage
during mating hours: 1 male with 1- 3 females
during pregnancy: 1-3 sperm positive females /cage
Cage type:Type II polypropylene/polycarbonate with stainless steel covers equipped by self-feeding baskets
Bedding: Certified laboratory wood bedding (Lignocel¿ Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG; D-73494 Rosenberg Holzmühle 1 Germany). The cages and bedding were changed twice a week.
Room sanitation: At the end of each working day floors were swept and then mopped with an acceptable disinfectant. Water bottles were cleaned on a rota basis as required during the course of the study.

Environmental Conditions
Illumination: Artificial light, from 6 a.m. to 6 p.m.
Temperature: 21-22 °C
Relative humidity: 33 - 47 %
Ventilation: above 10 air exchanges/hour by central air-conditioning system.
Environmental conditions were maintained by an air-condition system. Temperature and relative humidity were verified and recorded daily during the study.

Food and Water Supply
Animals received ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany and tap water, as for human consumption, ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used. Water quality control analysis was performed periodically.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The test item was administered orally (by gavage) from gestational day 5 to 19, daily. The treatment volume was 2 mL/kg/bw.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Five samples were taken from different places of the dosing formulations and analysed with GC-FID.
Sample preparation for the GC analysis: Samples were diluted with n-hexane in duplicate except for the control. A volume of 0.5 mL of the internal standard solution was added before the samples were filled up to 10 mL final volume.
The measured concentrations of the Ethylamylketon formulations varied between 95 and 106 percent of the nominal concentration.
Details on mating procedure:
The females were paired to males in the mornings for two to four hours (one male: one to three females) until the number of sperm positive females / group achieved at least twenty two. Vaginal smears were prepared from each female, stained with 1 % aqueous methylene blue solution and examined for presence of sperm and for estrus cycle. The day of mating was regarded as day 0 of pregnancy (vaginal plug and/or sperm in the vaginal smear). Sperm positive females were separated and caged in groups of 2 to 3 animals, individual caging was avoided.

Randomization
The sperm positive females were allocated if possible to each experimental group on each mating day in such a way that the group averages of the body weight were as similar as possible on the first day of gestation. Females paired with the same male were allocated to different groups on the same mating day.
Duration of treatment / exposure:
The test item was administered orally (by gavage) from gestational day 5 to 19, daily.
Frequency of treatment:
Daily
Duration of test:
15 days
Doses / concentrationsopen allclose all
Dose / conc.:
80 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
Clinical Observations
General clinical observations of the sperm positive females were made once a day, after treatment at approximately the same time, considering the peak period of anticipated effects after dosing.
Mortality
Observations for signs of morbidity and for mortality were made twice daily, at the beginning and before the end of the working period.
Body Weight
The body weight of the male animals was not measured.
The body weight of the female rats was measured at least once in the pre-mating period, but was not statistically evaluated. Body weight of sperm positive females was measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (accuracy of 1 g).
Corrected body weight was calculated for the 20th day of pregnancy (body weight on day 20 minus the weight of the gravid uterus).
5.6 Food Consumption
The food consumption was measured between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20 by re-weighing the non-consumed diet (accuracy: 1 g).
Examination for Sign of Implantation
On gestation days 13 and/or 14 the sperm positive females were checked for the presence of vaginal bleeding which indicated the implantation of conceptuses

Ovaries and uterine content:
All sperm positive females were sacrificed by decapitation under deep Isofluran anaesthesia on day 20 of gestation. The abdomen was opened, the uterus with cervix and left ovary were removed and weighed. The right ovary was placed into a Petri dish after removal. After removing the uterus gross pathology of dams' viscera was performed.
The number of corpora lutea in each ovary and implantation sites in each uterine horn, live fetuses, early and late embryonic death and fetal death were counted. Animals in which unambiguous implantation sites, but not fetuses, were found were considered as pregnant. Uteri that appear non-gravid were further examined to confirm the non-pregnant status.
Fetal examinations:
Fetuses were removed from the opened uterus and were sunk in a Petri-dish filled up with water. Spontaneous movement of fetuses was observed as a viability assessment. Euthanasia of the fetuses was performed by hypothermia.
The fetuses were washed with tap water and randomly laid on a filter paper with written ordinal numbering. Bleeding from the umbilical cord after it had been cut was observed as an indication of viability before euthanasia.
Each live fetus and its placenta was weighed individually (fetuses accuracy 0.01 g, placentas accuracy 0.001 g), and subjected to external examination. The gender of the fetuses was determined according to the anogenital distance. The fetuses were individually identified and about the half of each litter was subjected to visceral examination and the other half for skeletal examination. The body of those subjected to visceral examination was fixed in Sanomiya mixture. After fixation the bodies were micro dissected by means of a dissecting microscope.
The heads were examined by Wilson's free-hand razor blade method. The abdominal region of those subjected to skeletal examination was opened, the viscera and skin of fetuses were removed and the cadaver was fixed in alcian-blue-acetic acid-ethanol mixture. After fixation in isopropanol the skeletons were stained by KOH-Alizarin red-S method and the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded
Statistics:
Statistical analysis was performed with SPSS PC+ software. The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, the histogram has been checked first. If the data were almost normal, the data were transformed with a simple function to bring the distribution to normality. Where after transforming the data (x3) no significant heterogeneity was detected, ANOVA was carried out followed by Duncan's Multiple Range test to assess the significance of inter-group differences
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a non-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. Chi2 test was performed if feasible.
Dams or litters were excluded from the data evaluation in cases of:
- Sperm positive but non pregnant females (total exclusion)
- Body weight, body weight gain and food consumption of dams with complete intrauterine death of conceptuses (partial exclusion)
Although these animals were excluded from the data evaluation the final report includes all data of these animals, too.
A male/female fetus was considered as retarded in body weight, when its weight was below the average minus twofold standard deviation of the control male/female fetuses (3.01 and 2.84 g respectively).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg bw/day digging in the bedding and salivation of the animals was observed directly after application from the first to fourth treatment day up to the observations after the last treatment. Approximately from the mid or the second half of the treatment period incoordination and reduced activity of all animals, laying down of 20 of 22, dyspnoea in 9 dams was recorded. Creeping apart of the legs was observed in seven dams on the last to three days of treatment. These clinical signs started approximately 10 minutes after application and disappeared in two or three hours. Piloerection of five females appeared at the last days of the treatment and porfirine discoloration around the nose was recorded for one on the last treatment day. Three dams died on days 17 or 18 in this dose group after showing incoordination, reduced activity and laying down up to the day before (last clinical observation).
The mortality and the above mentioned clinical signs were judged to be a consequence of a toxic effect of the test item.

Slight salivation of 14 of 21 as well as digging in the bedding of all females (from the third treatment day the earliest up to the last treatment) were recorded also in the 300 mg/kg bw/day dose group. Salivation and digging in the bedding observed in the 300 mg/kg bw/day groups were considered to be related to treatment with the test item.

There was no mortality in the 300 and 80 mg/kg bw/day groups as well as no clinical signs were recorded in the 80 mg/kg bw/day dose group.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
3 sperm positive females died before scheduled termination in the 750 mg/kg bw/day dose group. Misgavage was not excluded as the reason for death of one of these dams.The mortality was judged to be a consequence of a toxic effect of the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significantly lower mean body weight from gestation day 11 to 20 and reduced body weight gain from gestation day 5 to 14, weight loss from gestation day 14 to 20, lower corrected body weight and a negative corrected body weight gain (all p<0.01) was indicated in the 750 mg/kg bw/day dose group compared to controls. These reductions were attributed to the treatment and considered to be adverse.

The body weight parameters were similar in the 300 and 80 mg/kg bw/days groups compared to the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significantly (p<0.01) reduced mean food consumption of the dams was indicated during the whole treatment period in the 750 mg/kg bw/day dose group compared to controls which was attributed to the treatment and was judged as adverse.

There were no relevant differences among the 300, 80 mg/kg bw/day and the control group values.

A slight but statistically significant (p<0.05) increase was indicated in the food consumption
between days 3 and 5 in the 750 mg/kg bw/day group which was judged to be irrelevant considering that this was the pre-treatment period
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment related macroscopic alterations were recorded only for the 750 mg/kg bw/day group animals (13 of 22). 12 dams had necropsy findings associated with the stomach (pinprick sized and/or dark, clotted bloody points, clotted bloody surface, not flexible mucosa of glandular stomach, slightly mucous surface, distended to markedly distended stomach filled up with food/stomach content or empty stomach). The stomach of all animals in this group was preserved for possible histopathology and one control organ was retained for comparison.

There were macroscopic findings recorded also for the females which died before scheduled necropsy (No. 222 had dark red lungs and shoamy secretum in the trachea, No. 228 dark red lungs, distended stomach filled up with food, several pinprick sized clotted blood coloured dark points in the glandular stomach and No. 129 empty stomach, clotted blood in the internal surface and clotted blood coloured point in the glandular stomach). In case of dam No. 222 a technical error was neither proved nor excluded considering the shoamy fluid in the trachea as a possible sign for misgavage. In addition bloody vaginal orifice and one fetus which sticked in the vagina was recorded for dam No. 116 at 750 mg/kg bw/day which was associated with the complete postimplantation loss in this dam
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
There were no test item related changes indicated in the mean number of corpora lutea, implantations, viable fetuses and their sex distribution, percentage of pre- and postimplantation loss.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Intrauterine parameters
There were no test item related changes indicated in the mean number of corpora lutea, implantations, viable fetuses and their sex distribution, percentage of pre- and postimplantation loss.


Sperm positive but non pregnant females:

-No corpora lutea and no implantation:
Control group: No. 221
80 mg/kg bw/day dose group: Nos. 154, 123, 143, 104, 138
300 mg/kg bw/day dose group: Nos. 174, 176, 197
750 mg/kg bw/day dose group: Nos. 161

- Corpora lutea present, no implantation:
Control group: No. 141
80 mg/kg bw/day dose group: No. 170
750 mg/kg bw/day dose group: No. 207

Total postimplantation loss (partial exclusion):
750 mg/kg bw/day dose group: No. 116


The mean number of corpora lutea and implantations was statistically significantly higher (p<0.05 and p<0.01 respectively) in the 750 mg/kg bw/day group which was considered as unrelated to the treatment. The whole number of post-implantation loss was statistically significantly higher in the 750 mg/kg bw/day group according to the Chi square test but not if the mean percent was evaluated. Moreover the value of 13.7% was within the control background data range (5.0-14.1%). Hence this slight increase was considered to be more likely unrelated to the treatment. There were no significant differences in the mean number of viable fetuses and their sex distribution and no significant increase of early- and late embryonic death was observed in the test item treated groups.

Moreover, the mean percentage and number of early embryonic death as well as number of preimplantation loss and total intrauterine mortality was statistically significantly lower in the 80 mg/kg bw/day group.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
The whole number of post-implantation loss was statistically significantly higher in the 750 mg/kg bw/day group according to the Chi square test but not if the mean percent was evaluated.
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no significant differences in the mean number of viable fetuses and their sex distribution and no significant increase of early- and late embryonic death was observed in the test item treated groups.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
The mean number of corpora lutea and implantations was statistically significantly higher (p<0.05 and p<0.01 respectively) in the 750 mg/kg bw/day group which was considered as unrelated to the treatment.
Details on maternal toxic effects:
Severe clinical signs, markedly reduced food consumption and body weight gain, weight loss (after correction) as well as macroscopic changes of the stomach of the dams were observed in the 750 mg/kg bw/day dose group.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
gross pathology
mortality
Dose descriptor:
NOEL
Effect level:
80 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Fetal- and placental weight was statistically significantly lower and the relative placental weight was statistically significantly higher (all p<0.01) in the 750 mg/kg bw/day dose group which was considered to have a relationship with the treatment of the dams with the test item.
There was no statistically significant difference in the body weight of the fetuses and in the relative placental weight in the 300 and 80 mg/kg bw/day groups. The placental weight was slightly but statistically significantly lower in the 80 mg/kg bw/day group. Considering that there was no statistical significance indicated in the relative placental weight in this group and that the value was within the historical control range (654.9-747.5) and as moreover no statistical significance was indicated in the 300 mg/kg bw/day group, this change was judged to be not adverse.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Body weight retardation (males below 3.01 g for and females below 2.84 g) was evaluated as an external variation. Nearly all fetuses (196 of 207) were considered to be body weight retarded (males below 3.01 g and females below 2.84 g) in the 750 mg/kg bw/day dose group which was attributed to the treatment and was considered to be a consequence of maternal toxicity.
Variations
There was no dose response indicated in the incidence of body weight retardation in the 300 and 80 mg/kg bw/day groups.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no significant differences in the mean number of viable fetuses and their sex distribution and no significant increase of early- and late embryonic death was observed in the test item treated groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no significant differences in the mean number of viable fetuses and their sex distribution and no significant increase of early- and late embryonic death was observed in the test item treated groups.
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no external malformations recorded in the experimental groups.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
There were four malformations found in the 750 mg/kg bw/day group, one each in the control and 300 mg/kg bw/day and none in the 80 mg/kg bw/day group.

One fetus had a hemicentric ossification of a thoracic centrum with a bipartite cartilage in the 750 mg/kg bw/day dose group. A similar finding was observed in the control group where the cartilage of a thoracic centrum was dumb-bell shaped (additionally bipartite ossification of this thoracic centrum). Considering the low incidence and the occurrence of a similar change in the control group as well as that hemicentric vertebra occurs sporadically also in control animals according to the background data of Toxi-Coop Zrt. (Appendix XXIV/A), this finding was judged to be unrelated to the treatment.

A rib malformation as displaced articulation to the vertebra and bifurcate shape was found in the 300 mg/kg bw/day dose group. Taking into account that this was a single case and not in the high dose, this was judged to be unrelated to the treatment.

Three fetuses in two litters had malformations of the pectoral girdle (bent scapula), the forelimb (bent radius or/and humerus and ulna) and the hindlimb bones (bent femur in three and slightly bent fibula as well as thickened tibia in one fetus) in the 750 mg/kg bw/day dose group. Malformations of these bones are part of the background data of Toxi-Coop Zrt. These abnormalities found with a moderate incidence were judged to be related to the severe maternal toxicity.

Significantly increased the incidence of the skeletal variations in the 750 mg/kg bw/day dose group due to the retarded or markedly incomplete ossification of the skull bones (including bipartite supra occipital and unossified hyoid), less than 3 ossified sternebra, incomplete or unossified centra of cervical, thoracic, lumbar or sacral vertebrae as well as lumbar or sacral arches, unossified or incompletely ossified pubic and less than 3/3.5 ossified metacarpal/metatarsal. These variations were related to a delay in ossification in association with the low fetal weight.

In summary, increased incidence of skeletal variations (due to delayed ossification) was indicated corresponded with the lower fetal weight in the 750 mg/kg bw/day group likely due to maternal toxicity.

There were no significant differences in the incidence of other type of skeletal variations among the dose groups. There was no increase indicated in the variations observed in the 300 and 80 mg/kg bw/day groups.


Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item effect indicated regarding malformed fetuses. Markedly dilated lateral brain ventricles were observed in one fetus at 80 and situs inversus totalis in one at the 300 mg/kg bw/day group. Both of these malformations were judged to be unrelated to the treatment considering the low incidence, the lack of dose response and that these findings occur sporadically also in control animals according to the background pregnancy and fetal data of Toxi-Coop Zrt. Visceral variations as hydroureter and malpositioned testis were recorded with low incidence and unrelated to the treatment.

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
fetal/pup body weight changes
changes in litter size and weights
skeletal malformations
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
fetal/pup body weight changes
changes in litter size and weights
skeletal malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: forelimb
skeletal: hindlimb

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
750 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
In the conducted prenatal developmental toxicity study of Ethylamylketon, a NOAEL of 300 mg/kg bw/day (maternal toxicity & developmental toxicity) was determined. Based on these results, Ethylamylketon is not classified as toxic to reproduction.
Executive summary:

Groups of 24 sperm-positive female Hsd. Han: WIST Rats were treated with Ethylamylketon by oral administration at three dose levels of 80, 300 and 750 mg/kg bw/day and one control group of 23 sperm positive females from day 5 up to and including day 19 post coitum daily. The control animals were given the vehicle (corn oil) alone. The treatment volume was 2 mL/kg/bw.

Formulation analytics (checking of homogeneity and achieved concentrations of the test item in the dosage forms) was performed two times during the treatment period using a validated GC-FID method. The measured concentrations of the Ethylamylketon formulations varied between 95 and 106 percent of the nominal concentration.

During the study animals were checked for mortality and clinical signs. Body weight and food consumption of the dams were also recorded. The day of detection of sperm in the vaginal smear of females was regarded as day 0 of gestation. A Caesarean section and gross pathology were performed on gestational day 20. Organs of the dams were examined macroscopically.

The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and external abnormalities. The placentas were weighed and examined externally. The body of about half of each litter was subjected to visceral examination by means of a dissecting microscope after fixation in Sanomiya mixture. The heads were examined by Wilson's free-hand razor blade method. After double staining, the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

Results

In total, the number of evaluated litters was 78 (21 both in the control and 300 mg/kg bw/day, and 18 both in the 80 and 750 mg/kg bw/day dose groups).

Clinical Symptoms, Mortality, Necropsy

Marked clinical signs were observed in the 750 mg/kg bw/day dose group and three dams died on days 17 or 18. Misgavage was not excluded as the reason for death of one of these dams. Macroscopic alterations in the stomach were recorded for the 750 mg/kg bw/day group animals.

Salivation and digging in the bedding observed in the 300 mg/kg bw/day groups were considered to be related to treatment with the test item.

Body weight, food consumption

Lower body weight and body weight gain (including weight loss if the corrected body weight was calculated) was indicated in the 750 mg/kg bw/day dose group associated with markedly reduced food consumption in the whole treatment period and up to day 20 in this group. These reductions were considered to be due to the treatment with the test item.

Intrauterine parameters

There were no test item related changes indicated in the mean number of corpora lutea, implantations, viable fetuses and their sex distribution, percentage of pre- and postimplantation loss.

Fetal- and placental weight

Lower fetal- and placental weight and higher relative placental weight were indicated in the 750 mg/kg bw/day dose group which were considered to have a relationship with the treatment of the dams with the test item, likely due to maternal toxicity.

External examination of fetuses

No malformations were found.

Nearly all fetuses were considered to be body weight retarded (variation) in the 750 mg/kg bw/day dose group which was attributed to the treatment and might be caused by the maternal toxicity.

No placental abnormalities were found.

Visceral examination of fetuses

No test item effect was indicated.

Skeletal examination of fetuses

Skeletal malformations such as bent bones of the fore-and hindlimbs in three fetuses of two litters at 750 mg/kg bw/day were judged to have a relationship with the treatment and to be likely a consequence of the marked maternal toxicity.

Increased incidence of skeletal variations (due to delayed ossification) was indicated corresponded with the lower fetal weight in the 750 mg/kg bw/day group likely due to maternal toxicity.

Conclusion

Based upon these data, treatment of pregnant Hsd. Han:WIST Rats from gestational day 5 to 19 by oral administration of Ethylamylketon, caused mortality, severe clinical signs, markedly reduced food consumption and body weight gain, weight loss (after correction) as well as macroscopic changes of the stomach of the dams in the 750 mg/kg bw/day dose group. Ethylamylketon was judged not to increase the intrauterine mortality. 750 mg/kg bw/day caused lower fetal-and placental weight, increased relative placental weight and increased incidence of skeletal variations as well as malformations such as bent scapula and/or bones of the fore-and hind limbs in three fetuses of two litters that were considered as most probably a consequence of the marked maternal toxicity.

Ethlyamylketon at 300 mg/kg bw/day caused slight salivation, digging in the bedding. These findings were considered as non-adverse maternal effects in the absence of any effects on food consumption or body weight. In addition no effects on the intrauterine development of conceptuses were recorded.

Ethylamylketon caused no maternal and fetal effects at 80 mg/kg bw/day

 

Based on these observations the No Observed Adverse Effect Level (NOAEL) and No Observed Effect Level (NOEL) were determined as follows:

NOAEL maternal toxicity: 300 mg/kg bw/day

NOAEL developmental toxicity: 300 mg/kg bw/day

NOEL maternal toxicity: 80 mg/kg bw/day

NOEL developmental toxicity: 300 mg/kg bw/day