Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Slight changes in reproductive performance and pup viability at high gavage 4-hydroxy-4-methylpentan-2-one doses (1000 mg/kg bw/d) may have been secondary to maternal toxicity (reduced weight gain, increased relative kidney, liver, and adrenal weights with histopathological lesions) in a screening reproductive toxicity study with 4-hydroxymethyl-4-methyl-2-pentanone (NHW, 1997).

OECD 422 study

The reproductive toxicity of 4-hydroxymethyl-4-methyl-2-pentanone was evaluated in a combined oral repeated-dose toxicity and reproductive/developmental toxicity screening test in rats (MHW, 1997). This study was conducted in accordance with OECD test guideline 422 (GLP status unknown). 4-hydroxy-4-methylpentan-2-one was administered at dose levels of 0 (water vehicle), 30, 100, 300, or 1000 mg/kg bw/day. Findings in parental animals included decreased locomotion and decreased response to stimulation at 300 and 1000 mg/kg bw/day males. Results of hematology and blood chemistry revealed increases in platelet count, glutamic oxalacetic transaminase, choline esterase, total protein, total cholesterol, total bilirubin, blood urea nitrogen, creatinine, and calcium, as well as a decrease in glucose, at 1000 mg/kg bw/day. Increased kidney weights were noted at 300 and 1000 mg/kg bw/day in males, and increased liver and adrenal weights were noted in the 1000 mg/kg bw/day males. Histological evaluation of kidney tissues confirmed the presence of hyaline droplets in the proximal tubular epithelium in males at 100 mg/kg bw/day or more, and basophilic tubules in the 300 and 1000 mg/kg bw/day males. Hepatocellular hypertrophy was noted in the liver in the 1000 mg/kg bw/day males, and vacuolizaiton of the cells of the zona fasciculata were noted in the adrenals of the 300 and 1000 mg/kg bw/day males. In females, one animal in the 1000 mg/kg bw/day group was euthanized during delivery. Reduced premating body weight gain was noted in the high-dose females. Histopathological changes to the liver and adrenals also were noted, as well as an increase in liver weight. Dilatation of the distal tubules and fatty degeneration of the proximal tubule epithelium in the kidneys was noted in the 300 and 1000 mg/kg bw/day females. Reproductive effects included decreased fertilization rate, number of implantations, and implantation rate at the 1000 mg/kg bw/day dose level. Offspring effects included reduced overall birth rate, delivery rate, number of live pups, live birth weight, number of live pups at day 4 of lactation, and survival at day 4 of lactation at 1000 mg/kg bw/day. In one 1000 mg/kg bw/day litter, no pups survived due to death or cannibalism. Based on these findings, the NOAEL for reproductive function in males and females, as well as for development of offspring, is considered to be 300 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study without detailed documentation
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
animals exposed for 10 days less than recommended. No date reported
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: SD(Crj:CD(SD)) SPF
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan Co (955 Kanbayashi, Yatabe-machi, Niihari-gun, Ibaragi-ken)
- Age at study initiation: 8 week old male rats and 7 week old female rats
- Weight at study initiation: 358 g for males and 211 g for females
- Housing: animals were raised in barrier system cages arranged in 5 stainless steel racks
- Diet (e.g. ad libitum): Nihon Nosan Kogyo Co., solid food lab MR, stock, available ad libitum
- Water (e.g. ad libitum): Kanagawa prefecture water supply available ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 40-60
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
VEHICLE
- Vehicle: Japanese Pharmacopeia purified water
- Lot/batch no. (if required): 180889, 180964
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until fertilization (4 days in all cases)
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Since the solution was confirmed to be stable for at least 7 days, it was prepared weekly and used within 7 days. The intial and final preparations were analyzed, and confirmation was made that specific concentrations were prepared. Analysis of the raw material of the test substance was conducted.
Duration of treatment / exposure:
44 days for males and 41-45 days for females
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
30, 100, 300, or 1000 mg/kg body weight
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
Dose levels were selected on the basis of results from a 14-day dose-range finding study.
Positive control:
None used
Parental animals: Observations and examinations:
Observations on mortality rates, external appearance and behavior were made on a daily basis throughout the administration period.

Individual body weight measurements were conducted on the date administration started (immediately before administration started) and at 7 day intervals, as well as on the final date of administration and the kill date. After pregnancy, the females were measured at day 0, 7, 14 and 20 of gestation and day 0 and 4 of lactation. Food consumption was coordinated with the body weight measurement date, and the amount of food consumed during a 24 hour period until the following day was measured. The final measurement for food consumption was conducted on day 43 of administration in males and day 3 of lactation in females. During the mating period, food consumption was not measured.

The time from the start of cohabitation to successful copulate, the copulation rate [(number of animals successfully mating/number of animals cohabitating) x 100], the fertilization rate [(number of fertilized females/number of animals successfully mating) x 100], the delivery rate [(number of females delivering pups/number of pregnant females) x 100] and the gestation period for those with confirmed delivery (period from day 0 of gestation to the date delivery was confirmed) was conducted from the results of the mating and delivery observation.

Urinary Examination in Males: Fresh urine was collected on day 38 and day 41 of administration, and pH, occult blood, protein, glucose, ketone bodies, bilirubin, and urobilinogen were regularly examined. Recovered rat urine from the metabolic cages (2~3 hours) was subject to external observations, measurements on specific gravity and urinary sediment examinations..

Collection of blood samples was conducted immediately prior to dissection on the day after the administration period ended (45 days after the start of administration). No food was given to the animals after 5:00pm on the day prior to blood sampling. Blood samples were collected from the abdominal aorta under ether anesthesia, and the following items were examined; Red blood cells (RBC), Hemoglobin (Hb), Hematocrit (Ht), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), White blood cells (WBC), Platelets (Plat), Reticulocyte count (RET), Prothrombin time (PT), and Activated partial thromboplastin time (APTT)

Blood smeared samples were created for calculating the white blood cell count percentage but since changes in the white blood cells were not confirmed, observations were not conducted. Serum was separated from one part of the collected blood sample and the following items measured; Total protein (T.P.), Albumin (Alb.), A/G ratio (A/G), Glucose (Glu.), Triglycerides (T.G.), Total cholesterol (T-Cho.), Total bilirubin (T-Bil.), Blood urea nitrogen (BUN), Creatinine (Crea.), GOT, GPT, ¿-GTP, LDH, ALP, Cholinesterase (ChE), Calcium (Ca), Inorganic Phosphorus (P), Sodium (Na), Potassium (K), Chlorine (Cl)


Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Histopathological examinations were performed on the testes, epididymides, prostate and seminal vesicle. No other sperm parameters were examined.
Litter observations:
After confirming the completion of delivery, the number of pups in the abdomen was counted (total of surviving and stillbirths), and the delivery rate [(total number of pups delivered/number of implantations) x 100] was calculated. Also, the anogenital distance was calculated by gender for each group.

Observations were made on the new pups for external abnormalities, including inside the oral cavity. Additionally, the overall condition and mortalities were verified on a daily basis, and the birth rate [number of surviving pups confirmed at birth/total number of pups delivered] x 100] and the pup survival rate [number of surviving pups at day 4 of lactation/number of surviving pups confirmed at birth] x 100] was determined.

Overall body weights were measured by gender on day 0 and day 4 of lactation for the surviving pups, and the mean weight per animal was calculated.


Postmortem examinations (parental animals):
The timing for euthanasia was soon after discovery for animals near death, after blood samples were taken in males scheduled for euthanasia, after observations were completed on day 4 of lactation for females scheduled for euthanasia, four days after the expected delivery date for females who were not confirmed to be pregnant after the expected delivery date had passed, and the date when mortalities were confirmed in females where all of the pups were dead after delivery. All were subject to exsanguination under ether anesthesia and the following items were examined. Necropsy: Visual inspection of all internal organs. The number of corpus luteums in the ovaries and the number of implantations in the uterus were studied in females, and the implantation rate [(number of implantations/number of corpus luteums) x 100] calculated. Measurement of the weight of organs: the weights (absolute weight) of the brain, heart, liver, kidney, spleen, adrenals, thymus for both males and females were measured, along with the testes and epididymides for males and the weight ratios (relative weights) calculated. Both the left and right sides of the kidneys, adrenals and epididymides were weighed together. Histopathological examinations were performed on the brain, pituitary gland, eyeballs, thyroid gland (including the glandura parathyroid), thymus, heart, lungs, kidney, spleen, stomach, small intestine (duodenum, jejune, ileum), large intestine (appendix, colon, rectum), pancreas, bladder, bone marrow and areas with visual abnormalities for all cases in the control group and the 1000 mg/kg group as well as females in other groups who were not pregnant.
Additionally, histopathological examinations were performed on the testes, epididymides, prostate and seminal vesicle in the males, and on the ovaries, uterus, vagina and mammary glands in the females.

For females in the 30, 100 and 300 mg/kg groups who had been pregnant, examinations were performed on the liver and adrenals on both males and females with confirmed changes thought to impact toxicity, as well as the kidneys and areas with visual abnormalities.
Postmortem examinations (offspring):
Whenever a fatality was noted, the survivors were euthanized using ether chloroform on the necropsy day (day 4 of lactation) for the new females, and the major organs in the chest were visually examined.
Statistics:
Statistical significance (danger rate of 5% or less) with the control group on the mean values and frequencies was conducted using the following methods. Parametric data such as body weight, food consumption, hematological and blood chemical data, organ weight, number of corpus luteum, number of implantations, gestation period, and number of pups delivered were subject to Bartlett’s distribution test. If the distribution was normal, an distribution analysis for the normal positions was conducted, and if a significant variance was confirmed from those results, a comparative test was performed on each group compared with the control group using either the Dunnett method or the Scheffe method (if the size of the groups differed). If the distribution was not normal and for non-parametric data such as implantation rate, delivery rate, pregnancy rate, rate of surviving pups, and qualitative data for urine examinations, Kruskal-Wallis analysis of variance was performed, and if a significant variance was confirmed for those results, a comparative test was performed on each group compared with the control group using either the Dunnett method or the Scheffe method (if the size of the groups differed). Categorical data such as the survival rate for newborns, copulation rate, fertilization rate, birth rate, gender ratio of newborns, changes in the overall condition and manifestation of pathological abnormalities were subject to ¿2 tests. Pathological abnormalities were also noted in the control group, and the data for findings where the impact of the test substance exhibited a difference in the distribution for the degree of changes was separated into two categories and tested.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Decreased spontaneous locomotion and less response to stimulation by making noise or by contact were noted in the early stages of the administration period in males at 300 and 1000 mg/kg/d
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increases in the weights of the kidney of males at 300 and 1000 mg/kg/d, and at 1000 mg/kg/d increases in the weights of the liver in males and females and adrenals in males
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Enlarged livers at 1000 mg/kg/d
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes in the liver (males and females at 1000 mg/kg/d), kidney (males at 100, 300 and 1000 mg/kg/d) and adrenals (males at 1000 mg/kg/d)
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Tendency for a decrease in fertilization rates, number of implantations and implantation rate at 1000 mg/kg/d
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Decreased spontaneous locomotion (7 animals in the 300 mg/kg group and 10 in the 1000 mg/kg group) and less response to stimulation by making noise by knocking on the cage or by touching the animals (5 in the 300 mg/kg group, 10 in the 1000 mg/kg group) was noted. Changes in the 300 mg/kg group were mild, and only noted on day 1 or days 1~2 of administration.
In the 1000 mg/kg group, almost no spontaneous locomotion was noted after administration of day 1 of administration, and a lethargic state was noted with a clear decrease in reactivity to stimulation. There was a trend towards recovery of these changes in the evening, and they had recovered by the time of administration on the following day. Changes in conjunction with the repeat administration were mild, and within 7 days, were not noted. Changes in other males included one in the 1000 mg/kg group with loss of fur and scabbing. In the females, reduced spontaneous locomotion (6 in the 300 mg/kg group, 10 in the 1000 mg/kg group) and reduced reactivity (5 in the 300 mg/kg group, 10 in the 1000 mg/kg group) was noted to the same extent as the males. Furthermore, reduced spontaneous locomotion and a lowered body temperature was noted one of the euthanized females in the 1000 mg/kg group on the euthanization date.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Changes to body weight and food consumption were not seen.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Relative to reproductive function in parent animals, there was a tendency for a decrease in fertilization rates, number of implantations and implantation rate.
(1) Copulation Rate and Fertilization Rate
Copulation occurred for all animals in the control group and each of the test substance administration groups within four days of the start of the copulation period. No differences were noted in the number of days required for copulation. The fertilization rate was 90% in the control group, 100% in the 30 mg/kg group, 80% in the 100 mg/kg group, 90% in the 300 mg/kg group and 60% in the 1000 mg/kg group, and while not a statistically significant change, the 1000 mg/kg group exhibited a reducing trend.

(2) Number of Corpus Luteum, Number of Implantations and Implantation Rate
In the control group, there were 17.1 corpus luteum, 16.6 implantations and a 97.3% implantation rate while in the 300 mg/kg and less groups, there were 18.3~18.6 corpus luteum, 17.4~18.1 implantations and 95.4~98.2% implantation rates, but a significant difference was not noted. In the 1000 mg/kg group, the 18.2 corpus luteum, 14.2 implantations and 79.0% implantation rate was not statistically significant but a decreasing trend was noted for the number of implantations and implantation rate.

(3) Delivery Rate and Gestation Period
The delivery rate for the control group and all of the groups administered the test substance was 100%. The gestation period for the control group was 22.4 days, and was in the range of 22.4~22.9 days in all of the groups administered the test substance, and significant changes were not noted.

(4) Delivery and Lactation Conditions
In the euthanized dam in the 1000 mg/kg group, vaginal hemorrhaging was noted during the evening of the expected delivery date (day 22 of gestation), and on the following day, the head of one animal dropped to the vaginal opening but the fetus was weak and was unable to be delivered. During necropsy, in addition to the one stillborn in the vagina, confirmation was made of 17 stillborns still in the uterus. One in a different 1000 mg/kg group was confirmed to deliver on the evening of the expected delivery date but only one was confirmed upon observation the following morning, and the rest had been cannibalized. This one live pup perished by the following day.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The testes and epididymides weights were not affected by the treatment.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Of the males causing successful pregnancies, one out of six in the 1000 mg/kg group exhibited enlarged livers. Of the females experiencing smooth delivery and lactation, three out of four in the 1000 mg/kg group exhibited enlarged livers. For those with confirmed pregnancies in each of the groups, hypertrophic adrenals were noted in one out of four of the females in the 1000 mg/kg group.

For the females in the 1000 mg/kg group where all of the pups perished after delivery, nothing abnormal was noted. One of the euthanized females in the 1000 mg/kg group exhibited an enlarged and faded liver, enlarged adrenals, scattered black dots on the mucous membranes of the glandular stomach and small intestine (primarily the ileum) and atrophy of the spleen. Changes other than these findings were noted but they were sporadic and not confirmed to be related to administration of the test substance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Changes thought to be caused by administration of the test substance were noted in the liver, kidney and adrenals (see same study in section 7.5.1.).
Histological examination of testes and epididymides was unremarkable.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No significant effects at this dose (excluding the sex- and species-specific hyaline droplet nephropathy of the males)
Clinical signs:
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
reducing trend in the 1000 mg/kg group for the overall birth rate, delivery rate, number of live pups, live birth rate, number of live pups at day 4 of lactation and survival rate at day 4 of lactation
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
The total number of pups delivered per animal in the control group was 15.9, with a delivery rate of 96.1%. The number of newborns was 15.7, the live birth rate was 98.7%, the gender ratio was 1.07, the number surviving at day 4 of lactation was 15.6, with a survival rate of 99.2%. In groups 300 mg/kg or less, the values were similar to these targets in the control group. In the 1000 mg/kg group, the total number of pups delivered was 11.6, with a delivery rate of 78.7%. The number of newborns was 10.0, the live birth rate was 88.7%, the number surviving at day 4 of lactation was 8.0, with a survival rate of 69.4%, so even though statistically significant differences with the control group could not be confirmed, there was a decreasing trend shown in all parameters.

BODY WEIGHT (OFFSPRING)
The body weight at day 0 of lactation was 6.8g for males and 6.4g for females in the control group, while the range for males was 6.8~7.1g and for females was 6.5~6.7g in each of the groups administered the test substance. The body weight at day 4 of lactation was 10.5g for males and 10.0g for females in the control group, while the range for males was 10.4~11.2g and for females was 10.1~10.6g in each of the groups administered the test substance. No significant changes in the weights were noted for day 0 and day 4 of lactation.

GROSS PATHOLOGY (OFFSPRING)
Regarding external abnormalities, there was one case in the control group with overlapping abnormalities of a torn eyelid and agnathia, and one case in the 1000 mg/kg group with a rudimentary tail but these abnormalities are not believed to be caused by administration of the test substance. No internal abnormalities were noted in any of the pups. For abnormalities in organs, thymic vestiges in the neck, umbilical artery vestiges and pelviectasis was noted in 7 (4.7%) in the control group, 5 (2.7%) in the 30 mg/kg group, 3 (2.1%) in the 100 mg/kg group, 11 (7.3%) in the 300 mg/kg group and 6 (11.7%) in the 1000 mg/kg group. While the rate of occurrence in the 1000 mg/kg group showed a higher trend than that of the control group, it was not significantly different.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
The non-observable adverse effect levels for reproductive function in males and females as well as for development of offspring are considered to be 300 mg/kg/day.
Executive summary:

The reproductive toxicity of diacetone alcohol (DAA) was evaluated in a combined oral repeated-dose toxicity and reproductive/developmental toxicity screening test in rats. This study was conducted in accordance with OECD test guideline 422 (GLP status unknown). DAA was administered at dose levels of 0 (water vehicle), 30, 100, 300, or 1000 mg/kg bw/day. Findings in parental animals included decreased locomotion and decreased response to stimulation at 300 and 1000 mg/kg bw/day males. Results of hematology and blood chemistry revealed increases in platelet count, glutamic oxalacetic transaminase, choline esterase, total protein, total cholesterol, total bilirubin, blood urea nitrogen, creatinine, and calcium, as well as a decrease in glucose, at 1000 mg/kg bw/day. Increased kidney weights were noted at 300 and 1000 mg/kg bw/day in males, and increased liver and adrenal weights were noted in the 1000 mg/kg bw/day males. Histological evaluation of kidney tissues confirmed the presence of hyaline droplets in the proximal tubular epithelium in males at 100 mg/kg bw/day or more, and basophilic tubules in the 300 and 1000 mg/kg bw/day males. Hepatocellular hypertrophy was noted in the liver in the 1000 mg/kg bw/day males, and vacuolizaiton of the cells of the zona fasciculata were noted in the adrenals of the 300 and 1000 mg/kg bw/day males.

In females, one animal in the 1000 mg/kg bw/day group was euthanized during delivery. Reduced premating body weight gain was noted in the high-dose females. Histopathological changes to the liver and adrenals also were noted, as well as an increase in liver weight. Dilatation of the distal tubules and fatty degeneration of the proximal tubule epithelium in the kidneys was noted in the 300 and 1000 mg/kg bw/day females. Reproductive effects included decreased fertilization rate, number of implantations, and implantation rate at the 1000 mg/kg bw/day dose level. Offspring effects included reduced overall birth rate, delivery rate, number of live pups, live birth weight, number of live pups at day 4 of lactation, and survival at day 4 of lactation at 1000 mg/kg bw/day. In one 1000 mg/kg bw/day litter, no pups survived due to death or cannibalism. Based on these findings, the NOAEL for parental systemic toxicity is considered to be 100 mg/kg/day and the NOAEL for reproductive function in males and females, as well as for development of offspring, is considered to be 300 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP guideline study
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

There are two main developmental studies peformed according to OECD 414: one performed in rats and one in rabbit.


1 -a) Main Rat developmental study (Rousseau, 2016)
The potential toxic effects of the test item, diacetone alcohol, was evaluated on the pregnant female and on embryonic and fetal development, following daily oral administration (gavage) to pregnant female rats from implantation to the day before scheduled hysterectomy (Day 6 to Day 20post-coitum(p.c.) inclusive) (Rousseau, 2016a). This GLP study was carried out according to OECD test guideline No. 414 (22 January 2001). The dose-levels were selected in agreement with the Sponsor, on the basis of the results of the previous study.Three groups of 24 mated female Sprague-Dawley rats daily received the test item, as solution in corn oil, from Day 6 to Day 20p.c., by gavage, at dose-levels of 100, 300 or 1000 mg/kg/day. A group of 24 mated females received the vehicle only under the same experimental conditions and acted as a control group. A constant dosage-volume of 5 mL/kg/day was used.The animals were checked at least once daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals.On Day 21p.c., animals were sacrificed and submitted for a macroscopicpost-mortemexamination. A hysterectomy was performed and the numbers ofcorpora lutea, implantations, uterine scars, early and late resorptions and live and dead fetuses were recorded. Kidneys and livers were weighed. The fetuses were weighed, sexed and subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices, soft tissue and skeletal (bones and cartilage) examination. A gross evaluation of placenta was undertaken. All fetuses were then discarded.
The test item concentrations in the administered dose formulations analyzed in Weeks 1 and 2 remained within an acceptable range of variations (+3.5% to +8.7%) when compared with the nominal values (± 10% of the nominal concentrations). There were 21/24, 21/24, 22/24 and 23/24 pregnant females in controls, 100, 300 and1000 mg/kg/day groups, respectively. All pregnant females had viable fetuses.There were no unscheduled deaths.Excessive salivation (recorded as ptyalism) and tremors were observed at 1000 mg/kg/day.These findings were considered to be non-adverse effects of the test item treatment.There were no effects on body weight, body weight change or food consumption at any dose-levels when compared with controls.There were no test item treatment related maternal findings at necropsy.There were no test item treatment-related effects on gravid uterus and carcass weights and hysterectomy data.Astatisticallysignificantincrease was noted in mean relative liver and kidney weight values at 1000 mg/kg/day when compared with controls.
There were no effects on mean fetal body weight and sex ratio. There were no test item treatment-related findings at external examination, and soft tissue examinationof the fetuses. Unossified or incomplete ossification of various part of the skeleton were noted in all litters at 1000 mg/kg/day. These findings were associated with presence of cartilage and were considered to be non-adverse effects of the test item treatment.
Diacetone alcohol was daily administered as solution in corn oil at dose-levels of 100, 300 or 1000 mg/kg/day, from Day 6 to Day 20 p.c., by oral route (gavage), to mated female Sprague-Dawley rats.
On the basis of the results obtained in this study:
- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 1000 mg/kg/day,
- the No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 1000 mg/kg/day.

1 -b) Range-finding study in rats (Rousseau, 2016)
The potential toxic effects of the test item, diacetone alcohol, was evaluated on the pregnant female and on embryonic and fetal development, following daily oral administration (gavage) to pregnant female rats from implantation to the day before scheduled hysterectomy (Day 6 to Day 20 post-coitum (p.c.) inclusive), in order to select dose-levels for a further main study (Rousseau, 2016b). Three groups of eight mated female Sprague-Dawley rats daily received the test item, as solution in corn oil, from Day 6 to Day 20p.c., by gavage, at dose-levels of 100, 300 or 1000 mg/kg/day. A group of eight mated females received the vehicle only under the same experimental conditions and acted as a control group. A constant dosage-volume of 5 mL/kg/day was used. The animals were checked at least once daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 20p.c.,blood samples were taken from all animals for the determination of plasma levels of the test item. On Day 21p.c., animals were sacrificed and submitted for a macroscopicpost-mortemexamination. A hysterectomy was performed and the numbers ofcorpora lutea, implantations, uterine scars, early and late resorptions and live and dead fetuses were recorded. Kidneys and livers were weighed. The fetuses were weighed, sexed and subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices. A gross evaluation of placenta was undertaken. All fetuses were then discarded. 
There were 5/8, 8/8, 8/8 and 6/8 pregnant females in controls, 100, 300 and 1000 mg/kg/day groups, respectively. All pregnant females had viable fetuses. All animals from all test item-treated groups were exposed to the test item. There were no unscheduled deaths. hypersalivation was notedon a dose-related manner in all test item-treated groups, for 1 to 5 days. Reddish vaginal discharge was noted for 2 days in one female treated at 100 mg/kg/day. There were no effects on body weight, body weight change or food consumption at any dose-levels when compared with controls. Enlarged livers were observed from 100 mg/kg/day. There were no test item treatment-related effects on gravid uterus and carcass weights and hysterectomy data. There were no test item treatment-related effects on liver weights. A dose-related increase was noted in mean relative kidney weight values when compared with controls. There were no test item-related effects on placental, fetal body weights or sex ratio. There were no findings at external examination of fetuses. Therefore 1000 mg/kg/day could be selected as a high dose-level for the next study.

2 -a) Rabbits main developmental study (Bentz, 2019)

The potential toxic effects of diacetone alcohol on the pregnant female and, on embryonic and fetal development was evaluated following daily oral administration (gavage) to pregnant female rabbits from implantation to the day prior to the scheduled hysterectomy [Days 6 to 28 post-coitum (p.c.) inclusive] (Bentz, 2019). Three groups of 24 time-mated New Zealand White rabbits were administered the test item, daily by gavage from Days 6 to 28p.c.inclusive at 100, 300 or 800 mg/kg/day. A dose volume of 5 mL/kg/day was used. One additional group of 24 females received the vehicle alone (drinking water treated by reverse osmosis) under the same experimental conditions. Test item concentration was checked in formulations given in the first and last weeks of the study.The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 29p.c., surviving females were euthanized and submitted to a macroscopicpost-mortemexamination. Hysterectomy was performed and the numbers ofcorpora lutea, implantations, early / late resorptions and, live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and skeletal/cartilage abnormalities.

Test item concentrations in formulations remained within an acceptable range of variation (± 10% of the nominal values). There were 17/24, 18/24, 20/24 and 21/23 pregnant dams in the groups treated at 0,100, 300 or 800 mg/kg/day, respectively.At 800 mg/kg/day, one female was prematurely euthanized on Day 20 p.c.for ethical grounds (no food consumption associated with body weight loss). This death was considered to be test item-related in view of the transient body weight loss and reduced food consumption recorded among surviving females treated at this dose-level. There were no test item-related clinical signs or necropsy findings in surviving females. There were no effects on mean gravid uterus and carcass weights, net body weight change. There were no test item treatment-related effects on hysterectomy parameters and noeffects on mean percentage of male fetusesor mean fetal body weight.

At fetal examination:

At 800 mg/kg/day, five litters had one malformed fetus (omphalocele, meningoencephalocele, dilated and/or overriding aorta, and/or split frontal bone); a test item effect was considered likely at this dose. The increased incidence of pale/small spleen(variations) at this dose could also be test item-related.At 300 mg/kg/day, Dilated aorta, also noted in 2 litters at 300 mg/kg/day, was potentially test item-related. 

Under the experimental conditions and results of this study:

.            the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 300 mg/kg/day based on the effects on body weight change and food consumption having led to the premature euthanasia of one female at 800 mg/kg/day,

.            the NOAEL for embryo-fetal development was considered to be 100 mg/kg/day based on external (head and abdominal closure defects), visceral (malformed aortas) and skeletal (split frontal) malformations at 800 mg/kg/day, and on the malformed aortas at 300 mg/kg/day.

2 -b) Rabbits preliminary developmental study (Bentz, 2018)

The objective of this non-GLP preliminary study was to evaluate the potential toxic effects of the test item in the pregnant female and on embryonic and fetal development, following daily oral administration (gavage) to pregnant female rabbits during embryonic and fetal periods [from implantation to the day prior to the scheduled hysterectomy: Days 6 to 28 post-coitum (p.c.) inclusive], in order to select dose levels for a further main study.

At 100 and 300 mg/kg/day, there were no unscheduled deaths, notoxicologically significanteffects onmean body weight andmean food consumption, no test item-related effects on mean gravid uterus weight, or mean fetal body weight. At 1000 mg/kg/day, one female was prematurely euthanized on Day 22p.c. following 19% body weight loss and nearly no food and water consumption. When compared with controls, at this dose there were lower mean body weight(down to -10% on Days 15 and 19p.c.)with mean body weight lossfrom Days 6 to 12 p.c., together withlow mean food consumption (down to -57% on Days 6 to 9p.c., p<0.001). Mean gravid uterus weight was -25% lower than in controls which was considered to be linked to the lower mean number of fetuses (likely not test-item-related) and mean fetal body weight (not considered to be toxicological significant). An effect of the test item treatment on high mean pre-implantation loss in all test item-treated groups (26.6, 22 and 19%, respectively,vs.12.6% in controls) and mean post-implantation loss at 100 mg/kg/day (15%vs.10.9% in controls) was considered to be unlikely (no dose-relationship or statistical significance). Paw hyperflexion was noted in one litter at 100 mg/kg/day and one litter at 1000 mg/kg/day; an effect of the test item was considered to be unlikely (no dose-relationship, no correlating external findings, low incidences). All these findings will need to be confirmed in the main embryofetal toxicity study with a higher number of animals per group.

On the basis of the experimental conditions and results of this study, the dose of 1000 mg/kg/day was considered to have exceeded the Maximum Tolerated Dose in pregnant rabbits based on the mortality at this dose.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 October 2015 -- 10 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, le Genest-Saint-Isle, France
- Age/Weight: at the beginning of the treatment period, the animals were 10-11 weeks old and had a mean body weight of 297 g (range: 256 g to 356 g)
- Fasting period before study: no
- Housing: polycarbonate cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 4 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 16 November 2015 to 10 December 2015
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Type of formulation
(visual observation):
- solution in the vehicle

Preparation procedure:
According to the analysis and stability study describing the preparation procedure (homogeneity and stability testing) for a range of concentrations covering the lowest and highest used in this study
Frequency and storage of preparations (control and test item dose formulations):
- based on test item dose formulation stability and vehicle expiry

Delivery conditions:
- at room temperature, protected from light

VEHICLE
- Justification for use and choice of vehicle: suitable formulation in corn oil
Used for preparation of test item dose formulations and administered to control group (control dose formulation).
Corn oil was already chosen as a vehicle in previous oral pharmacokinetic study following administration with the test item.

- Concentration in vehicle: 20, 60, and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: GC-FID
Test item concentrations: remained within an acceptable range of variation compared to nominal values in Weeks 1 and 2.
Details on mating procedure:
- Proof of pregnancy: detection of a vaginal plug
Duration of treatment / exposure:
Day 6 to Day 20 post-coitum inclusive
Frequency of treatment:
Daily
Duration of test:
21 days
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, on the basis of the results of the following previous in which the test item, was administered daily by gavage to pregnant female rats from implantation to the day before scheduled hysterectomy (Day 6 to Day 20 post-coitum inclusive) at dose-levels of 100, 300 or 1000 mg/kg/day.
No mortality was observed. All females were pregnant, except three control animals and two females treated at 1000 mg/kg/day. All pregnant females had viable fetuses.
There were no effects on body weight, body weight change or food consumption at any dose-levels when compared to controls. There were no clinical signs, except ptyalism observed on a dose-related manner in all test item-treated groups.
At hysterectomy, no test item effects were noted on maternal or litter data.
Blood samples taken 6 hours after dosing on Day 20 p.c. for the determination of plasma levels of the test item demonstrated a dose-related internal exposure.
Therefore, 100, 300 and 1000 mg/kg/day were selected.

- Rationale for animal assignment: computerized stratification procedure
Maternal examinations:
MORBIDITY AND MORTALITY:
- Time schedule: at least twice a day during the treatment period.

CLINICAL OBSERVATIONS:
- Time schedule: once a day during the treatment period.

BODY WEIGHT:
- Time schedule: on Days 2, 4, 6, 9, 12, 15, 18 and 21 p.c..

FOOD CONSUMPTION:
- Time schedule: on Days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c..

POST-MORTEM MACROSCOPIC EXAMINATION:
- Sacrifice on Day 21 p.c..
- Examined: principal thoracic and abdominal organs and the weight of the gravid uterus.
Ovaries and uterine content:
The ovaries and uterine content were examined after termination, including::
- number of corpora lutea,
- number and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of uterine scars,
- number and distribution of implantation sites.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Other : number dead and live, body weight, sex
Statistics:
Data were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous) or by Fisher exact probability test (proportions).

Indices:
% Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
% Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites
Clinical signs:
no effects observed
Description (incidence and severity):
Details on maternal toxic effects:
Excessive salivation (recorded as ptyalism) was observed at 1000 mg/kg/day. Tremors were noted in one female treated at 1000 mg/kg/day on Days 17 and 18 p.c. These findings were considered to be non-adverse effects of the test item treatment.

Other clinical signs (such as reddish vaginal discharge, cutaneous lesions and chromorhinorrhea) were observed in some control and/or test item animals with absence of any dose-relationship.
Nodosity on mammary gland was also noted from Day 19 p.c. in one female treated at 1000 mg/kg/day. These findings observed in rats of this strain when housed in laboratory conditions, and were not attributed to the test item treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight gain were not affected by the test item treatment when compared with control values.
At 1000 mg/kg/day, significantly higher mean body weight gain (+25 g vs. +20 g in controls, p<0.05) was recorded between Days 9 and 12 p.c. when compared with controls. This difference was mainly due to one isolated animal and thus considered to be of no toxicological importance.
Net body weight change (body weight change over the treatment period adjusted for the gravid uterus weight) was increased at 100 and 300 mg/kg/day when compared with controls, but no dose-relationship was observed.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test item effects on food consumption when compared to controls.
At 1000 mg/kg/day, significantly higher mean food consumption (25 g vs. 22 g in controls, p<0.01) was recorded between Days 9 and 12 p.c. when compared with controls. This difference was mainly due to one isolated animal and thus considered to be of no toxicological importance.
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:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant increase was noted in mean relative liver (19.06 vs. 16.5 g, p<0.01) and kidney (2.30 vs. 2.11 g, p<0.01) weight values at 1000 mg/kg/day when compared with controls.
Mean gravid uterus weights and carcass weights of test item-treated animals were similar to control values.
Gross pathological findings:
no effects observed
Description (incidence and severity):
A mass on mammary gland was observed in one female at 1000 mg/kg/day and a scab on skin was noted in one female given 100 mg/kg/day. These macroscopic lesions were observed in isolated animals, without any dose relationship, and are commonly observed in rats of this strain when housed in laborator y conditions. They are thus considered not to be test item treatment-related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified
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
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
The few external malformations recorded without any dose-relationship in low- and mid-dose levels group s are common observations in this species and strain. There were no external malformations in fetuses from the high-dose group.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no test item treatment-related cartilage abnormalities.
At 1000 mg/kg/day, 23/23 litters had fetuses with unossified or incomplete ossification of various part of the skeleton. These findings were associated with presence of cartilage and were considered to be test item treatment-related but not adverse.
There were no test-item treatment related skeletal malformation.
At 1000 mg/kg/day, on litter had a fetus, with knobby ribs. This malformation was associated with other thoracic skeletal variations (thickened and wavy ribs). Taking into account the absence of associated or similar findings in other groups and the isolated occurrence of this finding, a test item treatment-related effect was considered unlikely.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no test item treatment-related soft tissue malformations. The soft tissue malformations recorded without any dose relationship in control, low- and mid-dose-levels groups were observed in isolated fetuses. There were no soft tissue malformations in fetuses from the high-dose group.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The few external variations recorded in mid- and high-dose-levels groups are common observations in this species and strain. They are thus considered not to be test item treatment-related.
There were no test item treatment-related soft tissue variations.
The soft tissue variations recorded without any dose-relationship in control, low- and mid-dose-levels groups are common observations in this species and strain. There were no soft tissue variations in fetuses from the high-dose group.
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

Clinical signs observed in animals during the study are summarized in the following table:

 

Dose-level (mg/kg/day)

0

100

300

1000

Tremors

0

0

0

1

Reddish vaginal discharge

6

3

4

1

Chromorhinorrhea

2

0

0

0

Ptyalism

0

0

0

6

Cutaneous lesion on neck ventral area

0

2

1

0

Nodosity on mammary gland

0

0

0

1

Number of affected animals

8/24

5/24

5/24

8/24

 

Mean body weights and mean body weight changes are summarized in the following table:

 

Dose-level (mg/kg/day)

0

100

300

1000

 

Body weight (g)

 

 

 

 

 

Day 6p.c.

300

298

298

300

 

 Difference from controls (%)

 (-1)

(-1)

(0)

Day 21p.c.

448

443

446

445

 

Difference from controls (%)

 

(-1)

(0)

(-1)

 

Body weight change (g)

 

 

 

 

 

Days 9 - 12p.c.

+20

+19

+24

+25*

 

Difference from controls (%)

 

(-5)

(+20)

(+25)

 

Days 6 - 21p.c.

+147

+145

+149

+145

 

Difference from controls (%)

 

(-1)

(+1)

(-1)

 

*: p<0.05.

 

Mean food consumptions (g/animal) are summarized in the following table:

 

Dose-level (mg/kg/day)

0

100

300

1000

. Days 6 - 9p.c.

19

19

19

18

. Days 9 - 12p.c.

22

22

23

25**

. Days 12 - 15p.c.

25

24

24

25

. Days 15 - 18p.c.

29

28

28

29

. Days 18 - 21p.c.

28

29

29

29

**: p<0.01.

 

Mean gravid uterus weights, net weight changes and carcass weights in pregnant females are summarized in the following table:

 

Dose-level (mg/kg/day)

0

100

300

1000

Gravid uterus weight (g)

106.7

96.2

100.4

104.0

Difference from controls (%)

 

(-10)

(-6)

(-3)

Carcass weight (g)

340.9

347.0

345.9

341.3

Difference from controls (%)

 

(+2)

(+1)

(+0)

Net body weight change from Day 6p.c.(g)

40.6

49.1

48.3

41.2

Difference from controls (%)

 

(+21)

(+19)

(+1)

Liver and kidney weight values are summarized in the following table:

 

Dose-level (mg/kg/day)

0

100

300

1000

Finalnetbody weight (g)

334.7

341.2

342.0

338.8

Liver:

         Mean weight

16.50

16.93

17.86

19.06**

         Mean % net body weight

4.90

4.94

5.21

5.62**

Kidneys:

         Mean weight

2.11

2.15

2.20

2.30**

         Mean % net body weight

0.64

0.63

0.64

0.68*

*: p<0.05, **: p<0.01.

 

Pregnancy parameters are summarized in the following table:

 

Dose-level (mg/kg/day)

0

100

300

1000

Number of females with live fetuses at termination

21

21

22

23

Mean number ofcorpora luteaper animal

14.8

14.9

14.7

15.3

Mean number of implantations per animal

14.1

13.1

13.5

13.9

Mean pre-implantation loss (%)

4.1

10.8

8.5

8.1

Mean number of fetuses per animal

13.1

12.0

12.6

12.9

Dead fetuses (%)

0.0

0.0

0.0

0.0

Mean number of implantation scars

0.0

0.0

0.1

0.0

Mean number of early resorptions

1.0

0.9

0.8

0.8

Mean number of late resorptions

0.0

0.2

0.0

0.2

Mean post-implantation loss (%)

6.9

10.2

8.9

7.4

 

Mean fetal body weights and percentages of male fetuses (sex-ratio) are summarized in the following table:

 

Dose-level (mg/kg/day)

0

100

300

1000

Mean fetal body weight (g)

5.83

5.69

5.72

5.70

Difference from controls (%)

 

(-2)

(-2)

(-2)

Male fetuses (g)

5.98

5.85

5.85

5.84

Difference from controls (%)

 

(-2)

(-2)

(-2)

Female fetuses (g)

5.70

5.58

5.57

5.58

Difference from controls (%)

 

(-2)

(-2)

(-2)

 

Mean percentage of male fetuses (%)

 

49.7

44.4

46.7

47.5

Fetal and litter incidences of external variations are summarized in the following table:

 

Fetal (Litter) incidences (%) of external variations:

 

Dose-level (mg/kg/day)

0

100

300

1000

Number of litters

21

21

22

23

Number of fetuses

275

252

277

297

Litters affected, n (%)

0 (0)

0 (0)

1 (4.5)

2 (8.7)

Fetus affected, n (%)

0 (0)

0 (0)

3 (1.1)

2 (0.7)

Main findings

 

 

 

 

Local edema, F (L)

0 (0)

0 (0)

0 (0)

1 (1)

Protruding tongue, F (L)

0 (0)

0 (0)

3 (1.1)

2 (0.7)

 

Fetal and litter incidences of external malformations are summarized in the following table:

 

Fetal (Litter) incidences (%) of external malformations

 

Dose-level (mg/kg/day)

0

100

300

1000

Number of litters

21

21

22

23

Number of fetuses

275

252

277

297

Litters affected, n (%)

0 (0)

1 (4.8)

1 (4.5)

0 (0)

Fetus affected, n (%)

0 (0)

1 (0.4)

3 (1.1)

0 (0)

Main findings

 

 

 

 

Cleft palate, F (L)

0 (0)

0 (0)

3 (1)

0 (0)

Mandibular micrognathia, F (L)

0 (0)

0 (0)

3 (1)

0 (0)

Omphalocele, F (L)

0 (0)

1 (1)

0 (0)

0 (0)

F: fetal incidence, L: litter incidence.

 

Fetal and litter incidences of soft tissues variations are summarized in the following table:

 

Fetal (Litter) incidences (%) of soft tissue variations

 

Dose-level (mg/kg/day)

0

100

300

1000

Number of litters

21

21

21

23

Number of fetuses

132

121

131

144

Litters affected, n (%)

3 (14.3)

4 (19.0)

5 (23.8)

0 (0.0)

Fetus affected, n (%)

8 (6.1)

5 (4.1)

5 (3.8)

0 (0.0)

Main findings

 

 

 

 

Vessels: Short innominate artery, F (L)

3 (1)

3 (2)

2 (2)

0 (0)

Vessels: Absent innominate artery, F (L)

4 (2)

2 (2)

3 (3)

0 (0)

Dilated ureter, F (L)

1 (1)

0 (0)

0 (0)

0 (0)

F: fetal incidence, L: litter incidence.

Fetal and litter incidences of soft tissues variations are summarized in the following table:

 

Fetal (Litter) incidences (%) of soft tissue malformations

 

Dose-level (mg/kg/day)

0

100

300

1000

Number of litters

21

21

21

23

Number of fetuses

132

121

131

144

Litters affected, n (%)

1 (4.8)

1 (4.8)

1 (4.8)

0 (0.0)

Fetus affected, n (%)

1 (0.8)

1 (0.8)

1 (0.8)

0 (0.0)

Main findings

 

 

 

 

Absent kidney, F (L)

1 (1)

1 (1)

0 (0)

0 (0)

Vessels: absent aortic arch, F (L)

0 (0)

0 (0)

1 (1)

0 (0)

Absent ureter, F (L)

0 (0)

1 (1)

0 (0)

0 (0)

F: fetal incidence, L: litter incidence.

 

Dose-related fetal skeletal variations are summarized in the following table:

 

Fetal (Litter) incidences (%) of dose-related skeletal variations

 

Dose-level (mg/kg/day)

0

100

300

1000

HCD

Number of litters

21

21

22

23

182

Number of fetuses

143

131

146

153

1249

Hyoid : unossified

0 (0)

0 (0)

0 (0)

1.3 (8.7)

0 (0)

Thoracic vertebra: incomplete ossification of centrum

6.3 (28.6)

6.1 (38.1)

7.5 (31.8)

9.2 (39.1)

18.4 (57.1)

Lumbar vertebra: bipartite ossification of centrum

0 (0)

0 (0)

0 (0)

0.7 (4.3)

0 (0)

Caudal vertebra: unossified centrum

0 (0)

0 (0)

0.7 (4.5)

3.3 (8.7)

0.4 (2.7)

Caudal vertebra: incomplete ossification of arch

0 (0)

0 (0)

0 (0)

0.7 (4.3)

1.2 (6.0)

Extra stenebral ossification site

0 (0)

0 (0)

0 (0)

0.7 (4.3)

0.2 (1.6)

Wavy ribs

0 (0)

0 (0)

0 (0)

0.7 (4.3)

0.9 (5.5)

Thickened ribs

0 (0)

0 (0)

0 (0)

1.3 (4.3)

0.6 (3.3)

Unossified 1st metacarpals

0 (0)

0 (0)

0 (0)

0.7 (4.3)

0.2 (1.1)

Incomplete ossification of metacarpals

10.5 (28.6)

7.6 (28.6)

8.9 (31.8)

11.1 (30.4)

0.2 (1.6)

Forepaw: unossified proximal phalanx

49.7 (95.2)

41.2 (95.2)

56.2 (86.4)

68.0 (100.0)

5.3 (21.4)

F: fetal incidence, L: litter incidence.

HCD: Historical Control Data (Sprague-Dawley rat, Janvier Le Genest - France, March 2013 to June 2014).

In bold and underlined: higher fetal and/or litter incidencesvs.upper limits of the HCD and contemporaneous controls.

 

Conclusions:
The test item, was daily administered as a solution in corn oil at dose-levels of 100, 300 or 1000 mg/kg/day, from Day 6 to Day 20 p.c., by oral route (gavage), to mated female Sprague-Dawley rats.
On the basis of the results obtained in this study:
- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 1000 mg/kg/day,
- the No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 1000 mg/kg/day.
Executive summary:

The potential toxic effects of the test item, diacetone alcohol, was evaluated on the pregnant female and on embryonic and fetal development, following daily oral administration (gavage) to pregnant female rats from implantation to the day before scheduled hysterectomy (Day 6 to Day 20 post-coitum (p.c.) inclusive). This GLP study was carried out according to OECD test guideline No. 414 (22 January 2001). The dose-levels were selected in agreement with the Sponsor, on the basis of the results of the previous study. Three groups of 24 mated female Sprague-Dawley rats daily received the test item, as solution in corn oil, from Day 6 to Day 20 p.c., by gavage, at dose-levels of 100, 300 or 1000 mg/kg/day. A group of 24 mated females received the vehicle only under the same experimental conditions and acted as a control group. A constant dosage-volume of 5 mL/kg/day was used. The animals were checked at least once daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 21 p.c., animals were sacrificed and submitted for a macroscopic post-mortem examination. A hysterectomy was performed and the numbers of corpora lutea, implantations, uterine scars, early and late resorptions and live and dead fetuses were recorded. Kidneys and livers were weighed. The fetuses were weighed, sexed and subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices, soft tissue and skeletal (bones and cartilage) examination. A gross evaluation of placenta was undertaken. All fetuses were then discarded.

The test item concentrations in the administered dose formulations analyzed in Weeks 1 and 2 remained within an acceptable range of variations (+3.5% to +8.7%) when compared with the nominal values (± 10% of the nominal concentrations). There were 21/24, 21/24, 22/24 and 23/24 pregnant females in controls, 100, 300 and1000 mg/kg/day groups, respectively. All pregnant females had viable fetuses. There were no unscheduled deaths. Excessive salivation (recorded as ptyalism) and tremors were observed at 1000 mg/kg/day. These findings were considered to be non-adverse effects of the test item treatment. There were no effects on body weight, body weight change or food consumption at any dose-levels when compared with controls. There were no test item treatment related maternal findings at necropsy. There were no test item treatment-related effects on gravid uterus and carcass weights and hysterectomy data. A statistically significant increase was noted in mean relative liver and kidney weight values at 1000 mg/kg/day when compared with controls.

There were no effects on mean fetal body weight and sex ratio. There were no test item treatment-related findings at external examination, and soft tissue examination of the fetuses. Unossified or incomplete ossification of various part of the skeleton were noted in all litters at 1000 mg/kg/day. These findings were associated with presence of cartilage and were considered to be non-adverse effects of the test item treatment.

Diacetone alcohol was daily administered as solution in corn oil at dose-levels of 100, 300 or 1000 mg/kg/day, from Day 6 to Day 20 p.c., by oral route (gavage), to mated female Sprague-Dawley rats.

On the basis of the results obtained in this study:

- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 1000 mg/kg/day,

- the No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 1000 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 October 2018 - 23 November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
yes
Remarks:
some animals had 4 days of acclimation only instead of 5 days as recommended per guideline. This deviation was deemed necessary for organizational purposes and was considered to not have an impact on the validity of the study
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Centre LAGO (Vonnas, France)
- Age/Mean body weight at study initiation: at the beginning of the treatment period, the females were approximately 18-20 weeks old and had a mean body weight of 3580 g (range: 2955 g to 4205 g). The females were sexually mature and primigravid.
- Fasting period before study: no
- Housing: individually
- Diet: “type 110C”, batch No. 18101 (SAFE, Augy, France)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of 4 or 5 days before the beginning of treatment period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 5 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 8h dark/16h light

IN-LIFE DATES: 22 October 2018 to 23 November 2018
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Drinking water treated by reverse osmosis
Details on exposure:
PREPARATION OF DOSING FORMULATIONS:
Solution in the vehicle
Concentration in vehicle: 20, 60 and 160 mg/mL.
Amout of vehicle: 5 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: Gas Chromatography with FID detection (GC-FID)
Test item concentrations: were within an acceptable range of variation compared to nominal values (± 10%) in the first and the last weeks of study treatment
Stability: The dose formulations containing the test item and prepared as a solution at 5 mg/mL and 200 mg/mL in drinking water treated by reverse osmosis were found to be stable after 11 days of storage at room temperature and protected from light.
Details on mating procedure:
- Purchased time-pregnant
- Proof of pregnancy: vaginal plug (at the breeder's facility); referred to as Day 0 post coitum
Duration of treatment / exposure:
Days 6 to 28 post coitum (p.c.) inclusive
Frequency of treatment:
Daily
Duration of test:
until Day 29 p.c.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected in agreement with the Sponsor, on the basis of the results of a preliminary embryofetal toxicity study where pregnant females were treated by gavage at 100, 300 and 1000 mg/kg/day from Days 6 to 28 p.c. with the test item.

At 1000 mg/kg/day, 1 female was prematurely euthanized on Day 22 p.c. for ethical reasons (loss of 19% of body weight from Day 6 p.c., almost no food and water consumption).
There were no relevant clinical signs in the other females from this group. These latter females lost weight from Days 6 to 12 p.c. generally and then regained weight. Their food consumption was reduced from Days 6 to 15 p.c. (mainly in the first days of treatment). The only necropsy finding was whitish area on the liver in one surviving female; a relationship to the test item could not be ruled out. At hysterectomy, mean fetal body weight was 7% lower than in controls.
There were no toxicologically relevant effects at 100 and 300 mg/kg/day.

Following the death at 1000 mg/kg/day in the preliminary study, 800 mg/kg/day was selected as the high dose level and was expected to produce mild maternal toxicity. The low-dose and mid-dose were selected using a ratio representing approximately a 3-fold interval (i.e. 100 and 300 mg/kg/day).

- Rationale for animal assignment: stratification procedure
Maternal examinations:
MORTALITY/MORBIDITY:
- Time schedule: once a day before the treatment period and at least twice a day during treatment period.

CLINICAL OBSERVATIONS:
- Time schedule: at least once a day, 1 to 3 hours after dosing on the day of dosing, for the recording of clinical signs (including evidence of abortion).

BODY WEIGHT:
- Time schedule: on Days 2, 4, 5, 6, 9, 12, 15, 19, 24 and 29 p.c. and prior to premature euthanasia.

FOOD CONSUMPTION:
- Time schedule: on the following intervals: Days 2-4, 4-5, 5-6, 6-9, 9-12, 12-15, 15-19, 19-24 and 24-29 p.c.

POST-MORTEM EXAMINATION:
- Sacrifice on Day 29 p.c.
- Examined: principal thoracic and abdominal organs
Ovaries and uterine content:
The ovaries and uterine content were examined after termination, including:
- Gravid uterus weight,
- Number of corpora lutea,
- Number of implantation sites,
- Number of early and late resorptions,
- Number of dead and live fetuses
- Gross evaluation of placentas
Fetal examinations:
- External examinations: Yes: all fetuses per litter
- Soft tissue examinations: Yes: all fetuses per litter
- Skeletal examinations: Yes: all fetuses per litter
- Head examinations: Yes: all fetuses per litter
- Other : fetal weight, fetal sex


Statistics:
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).
Indices:
% Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
% Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites
Historical control data:
Cf attached documents
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related clinical signs in surviving females.
A few findings were recorded without any dose-level relationship (swollen mouth area, cutaneous lesion(s), blood in the bedding, short/broken teeth, bluish appearance on buccal area and/or difficulty during gavage administration). They generally are common observations in rabbits of this physiological condition and/or strain and were noted with isolated incidence.



Mortality:
mortality observed, treatment-related
Description (incidence):
There were no unscheduled deaths at 0, 100 or 300 mg/kg/day.

At 800 mg/kg/day, one female was prematurely euthanized on Day 20 p.c. for ethical reasons. Emaciated appearance and absence of feces were observed from Day 15 p.c. The female lost 18% of body weight between Days 6 and 19 p.c. Progressive decrease in food consumption was recorded from the start of the dosing period and this female did not eat from Day 12 p.c. At necropsy, there were dilatation of the gall bladder and whitish colored areas of the stomach mucosa with dry content. A test item-related effect on this animal condition and premature death was considered likely since it occurred at the high-dose at which body weight loss and reduction of food consumption were recorded among survivors.

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See Table 1.
At 100 and 300 mg/kg/day and when compared with controls, there were no test item-related effects on mean body weight and mean body weight change.
At 800 mg/kg/day and when compared with controls, minimal mean body weight loss was noted at the beginning of the treatment period (-73 g on Days 6-9 p.c., p<0.001). Thereafter, mean body weight gains returned towards control values in surviving pregnant females.



Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See Table 2.
At 100 and 300 mg/kg/day and when compared with controls, there were no test item-related effects on mean food consumption.
At 800 mg/kg/day and when compared with controls, mean food consumption was lower at the beginning of the treatment period (down to -40% on Days 6-9 p.c., p<0.001). On Days 24-29 p.c., mean food consumption returned towards control values in surviving pregnant females.





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, non-treatment-related
Description (incidence and severity):
When compared with controls, there were no test item treatment-related findings in animals sacrificed as scheduled, on Day 29 p.c.
The few findings recorded are commonly observed findings in this species and strain.



Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Gravid uterus and carcass wrights and net body weight change from Day 6 p.c.:
See Table 3.
There were no test item-related effects on mean gravid uterus and carcass weights, and net body weight change.



Number of abortions:
no effects observed
Description (incidence and severity):
No dams with abortions.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
See Table 4.
There were no effects on hysterectomy parameters (mean number of corpora lutea, implantation sites, live fetuses, pre- and post-implantation losses).

Number of dams with pre-implantation loss = 11/17, 14/18, 10/20, 15/21, at 0, 100, 300, 800 mg/kg/day, respectively
Number of dams with post-implantation loss = 7/17, 9/18, 11/20, 9/21 at 0, 100, 300, 800 mg/kg/day, respectively
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No dams with total resorption.
Early or late resorptions:
no effects observed
Description (incidence and severity):
See Table 4.
There were no effects on hysterectomy parameters (mean number of corpora lutea, implantation sites, live fetuses, pre- and post-implantation losses).

Number of dams with resorptions = 7/17, 9/18, 11/20, 9/21 at 0, 100, 300, 800 mg/kg/day, respectively
Dead fetuses:
no effects observed
Description (incidence and severity):
See Table 4.
No dead fetuses.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
At hysterectomy on Day 29 p.c., there were 17/24, 18/24, 20/24 and 21/23 pregnant dams in the groups treated at 0, 100, 300 or 800 mg/kg/day, respectively.
There were 7, 6, 4 and 2 non-pregnant females in the groups treated at 0, 100, 300 or 800 mg/kg/day, respectively.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
mortality
body weight and weight gain
food consumption and compound intake
Abnormalities:
not examined
Description (incidence and severity):
no microscopy
Fetal body weight changes:
no effects observed
Description (incidence and severity):
See Table 5.
There were no effects on mean fetal body weight.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
See Table 4.
There were no effects on hysterectomy parameters (mean number of corpora lutea, implantation sites, live fetuses, pre- and post-implantation losses).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no effects on mean percentage of male fetuses (sex ratio).
Changes in postnatal survival:
not examined
External malformations:
effects observed, treatment-related
Description (incidence and severity):
See Table 6.
At 100 and 300 mg/kg/day, there were no test item treatment-related malformations at external examination of the fetuses. At 100 mg/kg/day there was one fetus with multiple malformations (teras), and another with face malformations. In the absence of any plausible developmental connection with other malformed fetuses and/or the absence of dose-relationship, these malformed fetuses were considered incidental.

At 800 mg/kg/day, four litters had malformed fetuses: one fetus with omphalocele, one fetus No. 11 with meningoencephalocele, one fetus No. 1 with omphalocele and one fetus No. 3 with constricted tail), and one fetus No. 6 with narrowed tail. Tail shape abnormalities (bent, constricted and narrowed tails) are common findings (bent tail was also observed in one control fetus), not linked to the other malformations seen in this group and any test item relationship was considered unlikely. However, a relationship between the test item and closure defects (omphalocele and meningoencephalocele) cannot be excluded (three litters affected with findings not observed during the June 2015 to December 2017 period).

Number of fetuses with external malformations = 1 (0.6%), 2 (1.2.%), 0 (0.0.%), 5 (2.5%), at 0, 100, 300 or 800 mg/kg/day, respectively.
Number of litters with external malformations = 1 (5.9%), 2 (11.1%), 0 (0.0.%), 4 (19.0%), at 0, 100, 300 or 800 mg/kg/day, respectively.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
See table 11.
When compared with controls and/or Historical Control data, there was no dose-level relationship or significant differences in the litter and/or fetal incidences of skeletal malformations at 100 and 300 mg/kg/day. At 100 mg/kg/day, most of the skeletal malformations were in the teras.

At 800 mg/kg/day, three litters had a malformed fetus:
- two fetuses had fused caudal vertebrae. The litter and fetal incidences remained close to the upper limit of the Historical Control Data. Therefore, a test item treatment relationship was considered to be unlikely,
- one fetus had a split frontal in addition to fused sternebrae. While isolated, split frontal is not a common malformation in this species and strain (Morita et al., 1987; at Citoxlab: seen only in a study of 2014 in a few control litters) and is also a closure defect. Therefore, a test item treatment relationship cannot be excluded.

Number of fetuses with skeletal malformations = 12 (7.5%), 5 (2.9%), 5 (2.7%), 10 (5.0%), at 0, 100, 300 or 800 mg/kg/day, respectively.
Number of litters with skeletal malformations = 8 (47.1%), 5 (27.8%), 4 (20.0%), 7 (33.3%), at 0, 100, 300 or 800 mg/kg/day, respectively.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
See Table 8.
In the control group, one female had one fetus with a ventricular septal defect which is a common finding in this species and strain (Morita et al.,1987).

At 100 mg/kg/day, one dam had one malformed fetus with malpositioned kidneys and adrenals, and multiple heart great vessels malformations (dilated aorta/pulmonary trunk and retroesophageal aortic arch). This fetus also had several external and skeletal malformations associated with a low body weight (teras already mentioned previously).

At 300 mg/kg/day, two dams had one fetus with dilated aorta.

At 800 mg/kg/day, three dams had one malformed fetus with an overriding and dilated aorta, one fetus with a ventricular septum defect, a dilated pulmonary trunk and a dilated aorta, and one fetus with a misshapen cerebrum.

Most of these findings are not dose-related or observed in controls and/or reported in Historical Control Data. However, there is tendency towards an increase in the number of fetuses with malformed aorta:
- 300 mg/kg/day: two fetuses (dilated aorta),
- 800 mg/kg/day: one fetus (overriding and dilated aorta) and one fetus (dilated aorta).
At 100 mg/kg/day, a test item effect was considered to be unlikely since the aorta malformations were only seen in the teras.

Number of fetuses with visceral malformations = 1 (0.6%), 1 (0.6%), 2 (1.1%), 3 (1.5%), at 0, 100, 300 or 800 mg/kg/day, respectively.
Number of litters with visceral malformations = 1 (5.9%), 1 (5.6%), 2 (10.0%), 3 (14.3%),at 0, 100, 300 or 800 mg/kg/day, respectively.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
External variations:
There were no test-item treatment-related variations at external examination of the fetuses.

Two variations (misshapen palate rugae and paw hyperflexion) were recorded without any dose-level relationship. They are common observations in this species and strain.

Number of fetuses with external variations = 1 (0.6%), 4 (2.3%), 0 (0.0.%), 0 (0.0%), at 0, 100, 300 or 800 mg/kg/day, respectively.
Number of litters with external variations = 1 (5.9%), 2 (11.1%), 0 (0.0.%), 0 (0.0%),at 0, 100, 300 or 800 mg/kg/day, respectively.

Visceral variations:
See Table 7.
At 100 and 300 mg/kg/day, there were no test item treatment-related variations at soft tissue examination of the fetuses.

At 800 mg/kg/day and when compared with controls, there was statistically significant increases in the numbers of fetuses with a small and/or pale spleen. The litter incidences of this these two findings remained below or close to the upper limit of the Historical Control Data. However, these findings were observed in the high-dose group only and a test-item relationship cannot be excluded.

Number of fetuses with visceral variations = 45 (28.3%), 52 (30.4%), 42 (22.6%), 61 (30.2%), at 0, 100, 300 or 800 mg/kg/day, respectively.
Number of litters with visceral variations = 15 (88.2%), 17 (94.4%), 15 (75.0%), 17 (81.0%), at 0, 100, 300 or 800 mg/kg/day, respectively.

Cartilages:
See Table 9.
At 100 mg/kg/day and when compared with controls, there are no significant differences.

From 300 mg/kg/day onwards and when compared with controls or Historical Control Data, there are increases incidences of non-ossified metacarpal bones and proximal phalanxes (only the respective cartilages were present). The incidences being above the upper limit of the Historical Control, these findings were considered to be test item treatment-related.

Number of fetuses with skeletal cartilage findings = 87 (54.7%), 75 (43.9%), 76 (40.9%), 89 (44.1%),at 0, 100, 300 or 800 mg/kg/day, respectively.
Number of litters with skeletal cartilage findings = 17 (100%), 17 (94.4%), 17 (85%), 20 (95.2%), at 0, 100, 300 or 800 mg/kg/day, respectively.

Skeletal variations:
See Table 10.
At 100 mg/kg/day and when compared with controls, there are no test item-related effects on the litter and fetal incidences of skeletal variations.

From 300 mg/kg/day onwards, there is a tendency towards a dose-dependent increase in the incidences of skeletal variations (interparietal, ribs and metacarpal bones). The incidence of unossification or incomplete ossification of the metacarpal bones correlates with the cartilage findings (see previous paragraph). However, all incidences remained lower, equal or close to the upper limit of the Historical Control Data. Therefore, in the absence of any effect on pup body weight, a test item treatment relationship was considered unlikely.

Number of fetuses with skeletal variations = 135 (84.9%), 147 (86.0%), 165 (88.7%), 180 (89.1%),at 0, 100, 300 or 800 mg/kg/day, respectively.
Number of litters with skeletal variations = 17 (100%), 18 (100%), 20 (100%), 21 (100%), at 0, 100, 300 or 800 mg/kg/day, respectively.
Details on embryotoxic / teratogenic effects:
Malformation evaluation:
See Table 12.
There was a dose-related incidence of litters with relevant malformed fetuses from 300 mg/kg/day.
With 5 affected fetuses from 5 different litters and these malformations being not seen in historical control data or with a lower incidence in historical control data, an effect of the test item treatment was considered likely at 800 mg/kg/day. It was suspected at 300 mg/kg/day.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
skeletal malformations
visceral malformations
Abnormalities:
effects observed, treatment-related
Localisation:
external: cranium
external: umbilicus
skeletal: skull
visceral/soft tissue: cardiovascular
Description (incidence and severity):
800 mg/kg/day: external (head and abdominal closure defects: omphalocele, meningoencephalocele), visceral (malformed aortas: dilated, overriding) and/or skeletal (split frontal) malformations: 5 litters with 1 malformed fetus (1 to 2 of these malformations per fetus)

300 mg/kg/day: 2 fetus from 2 different litters with dilated aortas
Developmental effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1

Body weight and body weight change (g)

 

Dose level (mg/kg/day)

0

100

300

800

Mean body weight

 

 

 

 

Day 6 p.c.

3561

3658

3597

3579a

 

 

(+3)

(+1)

(+1)

Day 9 p.c.

3618

3696

3629

3505a

 

 

(+2)

(0)

(-3)

Day 12 p.c.

3689

3775

3690

3556a

 

 

(+2)

(0)

(-4)

Day 15 p.c.

3780

3867

3779

3635a

 

 

(+2)

(0)

(-4)

Day 19 p.c.

3845

3928

3860

3714a

 

 

(+2)

(0)

(-3)

Day 24 p.c.

3951

4028

3969

3859

 

 

(+2)

(0)

(-2)

Day 29 p.c.

3991

4083

4030

3940

 

 

(+2)

(+1)

(-1)

Mean body weight change

 

 

 

 

Days 6 - 9 p.c.

+57

+37

+33

-73#a

Days 9 - 12 p.c.

+72

+79

+60

+50a

Days 12 - 15 p.c.

+91

+93

+89

+79a

Days 15 - 19 p.c.

+65

+61

+81

+79a

Days 19 - 24 p.c.

+106

+100

+109

+97

Days 24 - 29 p.c.

+39

+55

+61

+81

Days 6 - 29 p.c.

+430

+425

+434

+348

Days 9 - 29 p.c.

+373

+388

+401

+414

(): in brackets, percentage difference (%) vs. controls.

Statistical significance vs. controls:#: p<0.001; a:including the prematurely euthanized female.

Table 2

Mean food consumption (g/animal/day)

 

Dose level (mg/kg/day)

0

100

300

800

Days 6 - 9 p.c.

184

183

177

111#a

 

 

(-1)

(-4)

(-40)

Days 9 - 12 p.c.

189

203

195

143**a

 

 

(+7)

(+3)

(-24)

Days 12 - 15 p.c.

163

172

176

133a

 

 

(+6)

(+8)

(-18)

Days 15 - 19 p.c.

194

198

187

158*a

 

 

(+2)

(-4)

(-19)

Days 19 - 24 p.c.

173

173

178

154

 

 

(0)

(+3)

(-11)

Days 24 - 29 p.c.

106

122

114

129

 

 

(+15)

(+8)

(+22)

( ) in brackets, percentage difference (%) vs. controls.

Statistical significance vs. controls *: p<0.05, **: p<0.01, #: p<0.001;a: including the prematurely euthanized female.

Table 3

Mean gravid uterus and carcass weights and net body weight change (g)

 

Dose level (mg/kg/day)

0

100

300

800

Mean gravid uterus weight

509.6

 

552.7

(+8%)

522.0

(+2%)

515.9

(+1%)

Mean carcass weight

3481

 

3531

(+1%)

3508

(+1%)

3424

(-2%)

Mean net weight change from Day 6 p.c.

-79.6

-127.5

-88.5

-168.0

() in brackets, percentage (%) differences vs. controls.

Table 4

Hysterectomy data

 

Dose level (mg/kg/day)

0

100

300

800

Number of females with live fetuses on Day 29p.c.

17

18

20

21

Mean number ofcorpora lutea per animal

12.2

12.0

11.6

11.6

Mean number of implantation sites per animal

10.4

10.2

10.3

10.3

Mean pre-implantation loss (%) per animal

14.9

14.8

10.3

11.0

Mean number of live fetuses per animal

9.4

9.5

9.3

9.6

Dead fetuses (%)

0.0

0.0

0.0

0.0

Mean number of implantation scars per animal

0.0

0.0

0.0

0.0

Mean number of early resorptions per animal

0.7

0.2

0.7

0.4

Mean number of late resorptions per animal

0.3

0.5

0.3

0.2

Mean post-implantation loss (%) per animal

9.3

6.1

10.8

6.6

 

Table 5

Mean fetal body weights

 

Dose level (mg/kg/day)

0

100

300

800

Mean fetal body weight

37.6

38.9

38.5

36.0

(males+ females)

 

(+3)

(+2)

(-4)

Mean fetal body weight (males)                                                                                                                           38.2                        39.5                         38.8                          36.6   

Mean fetal body weight (females)                                                                                                                         36.3                        37.6                        37.9                         35.6

(): in brackets, percentage difference (%) vs. controls.

 

Table 6

Litter (L) and Fetal (F) incidences of external malformations

 

Dose level (mg/kg/day)

0

100

300

800

HCD

Dams with live fetuses, n

17

18

20

21

159 + 42

Live fetuses evaluated, n

159

171

186

202

1558 + 391

. cleft face, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

. open eyes, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

. absent claw, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

. amelia, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

. phocomelia, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

. omphalocele, L(F) %

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

9.5 (1.0)

4.5 (0.5) (a)

. thoracogastroschisis , L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

. bent tail, L(F) %

5.9 (0.6)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

5.6 (0.5) (a)

. constricted tail, L(F) %

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

4.8 (0.5)

0.0 (0.0)

. narrowed tail, L(F) %

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

4.8 (0.5)

0.0 (0.0)

. meningoencephalocele, L(F) %

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

4.8 (0.5)

0.0 (0.0)

. acephalostomia, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

. iniencephaly, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

HCD: Historical Control Data (NZW Rabbits, Centre LAGO France, June 2015 to September 2016 and October 2016 to December 2017, n = 8 + 2 studies).

(a): maximum value.

Table 7

Litter (L) and Fetal (F) incidences of soft tissue variations

 

Dose level (mg/kg/day)

0

100

300

800

HCD

Dams with live fetuses, n

17

18

20

21

159 + 42

Live fetuses evaluated, n

159

171

186

202

1558 + 391

. lung(s): abnormal lobation, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

. spleen: paleness, L(F) %

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

14.3 (4.0*)

11.1 (1.2) (a)

. spleen: small, L(F) %

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

9.5 (5.4**)

8.3 (0.9) (a)

. gall bladder: enlarged, L(F) %

17.6 (2.5)

11.1 (1.8)

0.0 (0.0)

9.5 (2.5)

20.0 (3.5) (a)

. gall bladder: colored nodule, L(F) %

0.0 (0.0)

16.7 (1.8)

0.0 (0.0)

4.8 (0.5)

13.6 (2.1) (a)

. gall bladder: small, L(F) %

0.0 (0.0)

16.7 (1.8)

10.0 (1.1)

9.5 (1.5)

5.6 (0.6) (a)

. gall bladder: bilobed , L(F) %

5.9 (1.3)

0.0 (0.0)

5.0 (0.5)

0.0 (0.0)

15.0 (2.0) (a)

. liver: colored nodule, L(F) %

0.0 (0.0)

22.2 (2.3)

10.0 (1.1)

0.0 (0.0)

4.5 (0.5) (a)

. liver: colored focus, L(F) %

5.9 (0.6)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

12.5 (2.2) (a)

. liver: cyst, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

. ovary: serous cyst, L(F) %

11.8 (1.9)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

5.6 (0.6) (a)

. vessels: absent innominate artery, L(F) %

52.9 (8.2)

44.4 (7.6)

35.0 (9.1)

52.4 (11.4)

72.0 (17.9 (a)

. vessels: short innominate artery, L(F) %

47.1 (10.7)

61.1 (11.1)

40.0 (7.0)

57.1 (9.4)

94.4 (26.8) (a)

. vessels: narrowed ductus arteriosus, L(F) %

0.0 (0.0)

0.0 (0.0)

10.0 (1.1)

0.0 (0.0)

0.0 (0.0)

. vessels: absent brachiocephalic trunk, L(F) %

41.2 (5.0)

16.7 (2.3)

20.0 (3.2)

14.3 (2.0)

29.2 (4.0) (a)

HCD: Historical Control Data (NZW Rabbits, Centre LAGO France, June 2015 to September 2016 and October 2016 to December 2017, n = 8 + 2 studies).

(a): maximum value.

Statistical significance vs. controls: *: p<0.05; **: p<0.01 (in terms of number of observations).

Table 8

Litter (L) and Fetal (F) incidences of soft tissue malformations

 

Dose level (mg/kg/day)

0

100

300

800

HCD

Dams with live fetuses, n

17

18

20

21

159 + 42

Live fetuses evaluated, n

159

171

186

202

1558 + 391

. brain: misshapen cerebrum, L(F) %

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

4.8 (0.5)

0.0 (0.0)

. heart: ventricular septum defect, L(F) %

5.9 (0.6)

0.0 (0.0)

0.0 (0.0)

4.8 (0.5)

0.0 (0.0)

. kidney: malpositioned, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

. adrenal gland(s): malpositioned, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

. vessels: dilated aorta, L(F) %

0.0 (0.0)

5.6 (0.6)

10.0 (1.1)

9.5 (1.0)

0.0 (0.0)

. vessels: overriding aorta, L(F) %

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

4.8 (0.5)

0.0 (0.0)

. vessels: dilated pulmonary trunk, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

4.8 (0.5)

5.3 (0.5) (a)

. vessels: retroesophageal aortic arch, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

HCD: Historical Control Data (NZW Rabbits, Centre LAGO France, June 2015 to September 2016 and October 2016 to December 2017, n = 8 + 2 studies).

(a): maximum value.

Table 9

Litter (L) and Fetal (F) incidences of cartilage findings

 

Dose level (mg/kg/day)

0

100

300

800

HCD

Dams with live fetuses, n

17

18

20

21

159 + 42

Live fetuses evaluated, n

159

171

186

202

1558 + 391

. metacarpal bone: cartilage present, L(F) %

17.6 (4.4)

16.7 (1.8)

45.0 (5.9)

52.4* (14.4**)

36.4 (5.7) (a)

. forepaw phalanx: proximal cartilage present, L(F) %

0.0 (0.0)

0.0 (0.0)

15.0 (2.2)

19.0 (3.0*)

9.1 (1.0) (a)

HCD: Historical Control Data (NZW Rabbits, Centre LAGO France, October 2016 to December 2017, n = 2 studies).

(a): maximum value.

Statistical significance vs. controls: *: p<0.05; **: p<0.01 (in terms of number of observations).

Table 10

Litter (L) and Fetal (F) incidences of significant skeletal variations

 

Dose level (mg/kg/day)

0

100

300

800

HCD

Dams with live fetuses, n

17

18

20

21

159 + 42

Live fetuses evaluated, n

159

171

186

202

1558 + 391

. interparietal: split, L(F) %

0.0 (0.0)

0.0 (0.0)

5.0 (1.1)

23.8 (3.0*)

20.0 (2.5) (a)

. ribs: supernumerary 13th, L(F) %

94.1 (56.6)

88.9 (59.1)

100.0 (68.8*)

100.0 (73.3**)

100.0 (81.2) (a)

. metacarpal: unossified 1st, L(F) %

5.9 (1.9)

5.6 (0.6)

25.0 (3.2)

47.6** (7.9*)

33.3 (7.4) (a)

. metacarpal: incomplete ossification 1st, L(F) %

11.8 (2.5)

11.1 (1.2)

20.0 (2.7)

33.3 (8.4*)

61.1 (8.4) (a)

HCD: Historical Control Data (NZW Rabbits, Centre LAGO France, June 2015 to September 2016 and October 2016 to December 2017, n = 8 + 2 studies).

(a): maximum value.

Statistical significance vs. controls: *: p<0.05; **: p<0.01 (in terms of number of observations).

 

Table 11

Litter (L) and Fetal (F) incidences of skeletal malformations

 

Dose level (mg/kg/day)

0

100

300

800

HCD

Dams with live fetuses, n

17

18

20

21

159 + 42

Live fetuses evaluated, n

159

171

186

202

1558 + 391

. interparietal: absent, L(F) %

11.8 (2.5)

5.6 (0.6)

0.0 (0.0)

9.5 (1.0)

31.6 (3.4) (a)

. frontal: split, L(F) %

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

4.8 (0.5)

0.0 (0.0)

. skullcap: hole, L(F) %

5.9 (0.6)

5.6 (0.6)

5.0 (0.5)

0.0 (0.0)

4.5 (0.5) (a)

. maxilla: absent, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

. head: multiple malformations, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

. hyoid: absent, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

. odontoide: incomplete ossification, L(F) %

0.0 (0.0)

0.0 (0.0)

5.0 (0.5)

0.0 (0.0)

0.0 (0.0)

. thoracic vertebra(e): fused centrum, L(F) %

5.9 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

5.6 (0.6) (a)

. lumbar vertebra(e): absent, L(F) %

5.9 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

. caudal vertebra(e): fused, L(F) %

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

9.5 (1.0)

8.3 (0.9) (a)

. scapula: absent, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

. clavicle: absent, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

. sternebra(e): fused, L(F) %

23.5 (3.1)

5.6 (0.6)

15.0 (1.6)

19.0 (3.0)

27.3 (3.6) (a)

. ribs: fused, L(F) %

5.9 (0.6)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

8.3 (0.9) (a)

. ribs: branched, L(F) %

5.9 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

2.7 (0.3) (a)

. humerus: absent, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

. radius: absent, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

. ulna: absent, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

. metacarpal(s): absent, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

. forepaw phalanxes: absent, L(F) %

0.0 (0.0)

5.6 (0.6)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

HCD: Historical Control Data (NZW Rabbits, Centre LAGO France, June 2015 to September 2016 and October 2016 to December 2017, n = 8 + 2 studies).

(a): maximum value.

Table 12

Relevant malformed fetuses

 

Dose level (mg/kg/day)

0

100

300

800

 

 

 

Fetus N31309-01

(especially dilated aorta)

Fetus N31335-10

(especially dilated and overriding aorta)

 

 

 

 

 

 

 

 

Fetus N31327-09

(dilated aorta)

Fetus N31337-2

(especially omphalocele, dilated aorta)

 

Fetus N31338-11

(especially meningoencephalocele)

 

 

 

 

 

Fetus N31339-01 (omphalocele)

 

Fetus N31342-11

(especially split frontal)

Number of litters/total

0/17

0/18

2/20

5/21

Number of fetuses/total

0/159

0/171

2/186

5/202

 

Conclusions:
The test item was administered daily by gavage from Days 6 to 28 p.c. inclusive at 100, 300 or 800 mg/kg/day. A dose volume of 5 mL/kg/day was used. One additional group of 24 females received the vehicle alone (drinking water treated by reverse osmosis) under the same experimental conditions.
 
Under the experimental conditions and results of this study:
.            the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 300 mg/kg/day based on the effects on body weight change and food consumption having led to the premature euthanasia of one female at 800 mg/kg/day,
.            the NOAEL for embryo-fetal development was considered to be 100 mg/kg/day based on external (head and abdominal closure defects), visceral (malformed aortas) and skeletal (split frontal) malformations at 800 mg/kg/day, and on the malformed aortas at 300 mg/kg/day.
 
Executive summary:

The objective of this GLP study was to evaluate the potential toxic effects of the test item, on the pregnant female and, on embryonic and fetal development following daily oral administration (gavage) to pregnant female rabbits from implantation to the day prior to the scheduled hysterectomy [Days 6 to 28 post-coitum (p.c.) inclusive].

Methods

Three groups of 24 time-mated New Zealand White rabbits were administered the test item, daily by gavage from Days 6 to 28 p.c. inclusive at 100, 300 or 800 mg/kg/day. A dose volume of 5 mL/kg/day was used. One additional group of 24 females received the vehicle alone (drinking water treated by reverse osmosis) under the same experimental conditions.

Test item concentration was checked in formulations given in the first and last weeks of the study. The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 29 p.c., surviving females were euthanized and submitted to a macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantations, early / late resorptions and, live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and skeletal/cartilage abnormalities.

 

Results

 

Test item concentrations in formulations remained within an acceptable range of variation (± 10% of the nominal values).

 

There were 17/24, 18/24, 20/24 and 21/23 pregnant dams in the groups treated at 0,100, 300 or 800 mg/kg/day, respectively.

At 800 mg/kg/day, one female was prematurely euthanized on Day 20 p.c. for ethical grounds (no food consumption associated with body weight loss). This death was considered to be test item-related in view of the transient body weight loss and reduced food consumption recorded among surviving females treated at this dose-level. There were no test item-related clinical signs or necropsy findings in surviving females.

 

There were no effects on mean gravid uterus and carcass weights, net body weight change.

 

There were no test item treatment-related effects on hysterectomy parameters and no effects on mean percentage of male fetuses or mean fetal body weight.

 

At fetal examination:

  1. At 800 mg/kg/day, five litters had one malformed fetus (omphalocele, meningoencephalocele, dilated and/or overriding aorta, and/or split frontal bone); a test item effect was considered likely at this dose. The increased incidence of pale/small spleen (variations) at this dose could also be test item-related.
  2. At 300 mg/kg/day , Dilated aorta, also noted in 2 litters at 300 mg/kg/day, was potentially test item-related.

 

Conclusion

 

The test item, was administered daily by gavage from Days 6 to 28 p.c. inclusive at 100, 300 or 800 mg/kg/day. A dose volume of 5 mL/kg/day was used. One additional group of 24 females received the vehicle alone (drinking water treated by reverse osmosis) under the same experimental conditions.

 

Under the experimental conditions and results of this study:

.            the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 300 mg/kg/day based on the effects on body weight change and food consumption having led to the premature euthanasia of one female at 800 mg/kg/day,

.            the NOAEL for embryo-fetal development was considered to be 100 mg/kg/day based on external (head and abdominal closure defects), visceral (malformed aortas) and skeletal (split frontal) malformations at 800 mg/kg/day, and on the malformed aortas at 300 mg/kg/day.

 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
GLP guideline study
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

According to the information available in the rabbits study (Benz, 2019) and the treatment-related fetal malformations observed at 300 and 1000 mg/kg/day, the classification as Reprotoxic Cateqory 2 (H361 d) is warranted.