4-hydroxy-4-methylpentan-2-one

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

There are three fertility studies performed with 4-hydroxy-4-methylpentan-2-one:

1- An OECD 422 combined reprotoxicity study (MHW 1997)

2- An OECD 421 fertility study preliminary to OECD 443 (Sisti , 2020)

3- An extended one generation study with F2 extension (Sisti, 2020 -OECD 443)

OECD 422 study (MHW 1997)

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.

OECD 421 study (Sisti, 2020)

This GLP study was performed according to OECD 421 as a preliminary study to the extended one generation study.

Based on the results, the parental NOAEL (No Observed Adverse Effect Level) was considered to be below to 750 mg/kg/day for males and females since some effects during the in-life phase in females (transient decrease in body weight gain and decrease in food consumption) and adverse effects on kidneys in males examined microscopically were evident at 750mg/kg/day.

The NOAEL for the toxicity on fertility could be considered to be 750mg/kg/day.

The NOAEL for the developmental of pups could be considered to be 250 mg/kg/day on the basis of the effects on body weights of pups and in relation to the progressive decrease in pups’ survival observed during the lactation period.

3-Extended one generation study with F2 extension (OECD 443, Sisti 2020):

The pre- and postnatal effects of Diacetone alcohol on development, as well as a thorough

evaluation of systemic toxicity in pregnant and lactating females and young and adult

offspring were investigated.

On the basis of the results of this study, the dosage of 200 mg/kg/day was considered

theNOAEL for toxicity in males (based on adverse kidneys effects at 600 mg/kg/day).

The dosage of 600 mg/kg/day was considered the NOAEL both for toxicity and reproductive

performance in females (the maternal toxicity observed in parental generation was not

confirmed in Cohort 1B females).

Considering that the increase in post natal loss seen in lactating parental females, at dose

level of 600 mg/kg/day, was also evident in Cohort 1B at birth and with slight higher incidence

on Day 4 post partum, when compared to the control group, it cannot be excluded the

relation with the test item at this dose level and then as conservative way the NOAEL for

developmental toxicity of pups could be at 200 mg/kg/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

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 for cross-reference:

reference to same study

Qualifier:

according to guideline

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 or test system 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

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)

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

other: kidney and liver effects

Organ:

adrenal glands

kidney

liver

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.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of experimental phase Allocation of animals to groups 23 April 2019 - First day of mating 14 May 2019 - End of experimental phase Last day of necropsy for females 24 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 july 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

The purpose of this study was to provide information on toxic effects on rats of both sexes
after repeated dosing with the test item Diacetone alcohol, as well as any effects of the
test item on male and female reproductive performance, such as gonadal function, mating
behaviour, conception, development of conceptuses, parturition and lactation of the
offspring.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley SD rat was the species and strain of choice because it is accepted by
many regulatory authorities and there are ample experience and background data on this
species and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

A total of 105 Sprague Dawley SD rats (45 males and 60 virgin females) were ordered from
Charles River Italia S.p.A., Calco (Lecco), Italy. The animals were approximately 7 to 8 weeks
old and weighing 200-225 g for males and approximately 196 to 204 g for females at receipt.
After arrival, theweight range for each sex was determined and the animalswere temporarily
identified within the cage by means of a coloured mark on the tail. A health check was
performed by a veterinarian.
An acclimatisation period of 26 days was allowed before the start of treatment, during which
time the health status of the animals was assessed by thorough observations and oestrous
cycle was monitored.
The animalswere housed in a limited access rodent facility. Animal roomcontrolswere set to
maintain temperature and relative humidity at 22°C±2°C and 55%±15% respectively; actual
conditions were monitored, recorded and the records retained. No relevant deviations
from these ranges were recorded during the study. There were approximately 15 to 20 air
changes per hour and the rooms were lit by artificial light for 12 hours each day.
From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulfone
solid bottomed cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate,
Varese). Nesting material was provided inside suitable bedding bags and changed at least
twice a week.
During mating, animals were housed one male to one female in clear polysulfone cages
measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor (Tecniplast Gazzada
S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected
and changed daily.
After mating, the males were re-caged as they were before mating.
The females were transferred to individual solid bottomed cages for the gestation period,
birth and lactation (measuring 42.5×26.6×18.5 cm). Nesting material was provided inside
suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was
provided as necessary and changed at least 2 times a week.
Drinking water was supplied ad libitum to each cage via water bottles.
A commercially available laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4,
20019, SettimoMilanese (MI), Italy) was offered ad libitum throughout the study. There was
no information available to indicate that any non-nutrient substance likely to influence
the effect of the test item was present in the drinking water or the diet. Records of analyses
of water and diet are kept on file at the test facility.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The test item was administered by oral route. The oral route was selected as it is a possible route of exposure of the test item in man.
Dose levels of 50, 250 and 750mg/kg/daywere selected by the Sponsor based on information from previous studies.

Details on mating procedure:

Pairing was monogamous (one male to one female).
A vaginal smear was taken from all females from the day after the start of pairing until
positive identification of copulation (spermidentification, vaginal plug in situ or copulation
plugs found in the cage tray). The female was paired with the same male until positive
identification occurred or 14 days had elapsed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed in a separate study (RTC Study no. A3523) in order to validate the
analytical method and the preparation procedure. The analytical method was validated in RTC Study no. A3523 in the range from 5 to 200 mg/mL. Linearity, accuracy and precision
were within the limits stated in ERBC SOPs for solutions (r> 0.98; accuracy 90-110%;
precision CV<5%).
Stability: in RTC Study no. A3523, a 28 hour stability and an 8 day stability at room temperature
were verified in the range from 5 to 200 mg/mL.
Assessment of preparation procedure: the proposed preparation procedure for the test
item was checked in the range from 5 to 200 mg/mL by chemical analysis (concentration)
in RTC Study no. A3523 to confirmthat the method was suitable. Final results for all levels
were within the acceptability limits stated in ERBC SOPs for concentration (90-110%).
Samples of the preparations prepared during the current study (the first and the last week
of treatment) were analysed to check the concentration. Results of the analyses were within
the acceptability limits stated in ERBC SOPs for concentration of solutions (90-110%).
Chemical analysis was carried out by the Analytical Chemistry Department using a gas
chromatography (GC) with flame-ionisation detection (FID). The software used for this
activity was Chromeleon version 7.2 (Thermo Scientific).

Duration of treatment / exposure:

Males: animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to
pairing, through the pairing period and thereafter until the day before necropsy performed
on Days 30 and 31 of the study. Animals were dosed for a total of 29 or 30 days.

Females: animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to
pairing and thereafter during pairing, post coitum and post partum periods until Day 13
post partum or the day before sacrifice.

Frequency of treatment:

animals were dosed once a day, 7 days a week
The dose volume was 4 mL/kg body weight

Dose / conc.:

50 mg/kg bw/day

Remarks:

Group 2

Dose / conc.:

250 mg/kg bw/day

Remarks:

Group 3

Dose / conc.:

750 mg/kg bw/day

Remarks:

Group 4

No. of animals per sex per dose:

Each group comprised 10 male and 10 female rats

Control animals:

yes, concurrent vehicle

Details on study design:

This study design was performed according to OECD Guideline no. 421 and in compliance with GLP. All doses (0, 50, 250 and 750 mg/kg/day) were administered orally, by gavage. The control group received corn oil. The dose volume was 4 mL/kg body weight.

Positive control:

none

Parental animals: Observations and examinations:

Mortality
Throughout the study, all animals were checked early in each working day in the morning
and in the afternoon. At weekends and Public Holidays a similar procedure was followed
except that the final check was carried out at approximately mid-day. This allowed post
mortem examinations to be carried out during the working period of that day.
Clinical signs
Once before commencement of treatment and at least once daily during the study, each
animal was observed and any clinical signs recorded.
Observations were performed at the same time interval each day, the interval was selected
taking into consideration the presence of post-dose reactions.
All observations were recorded for individual animals.

Body weight
Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to positive identification of mating and on
Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1, 4, 7, 13 post partum
and just before necropsy.
Individual bodyweight data recorded for females during the mating period are not tabulated
in this report but will be archived with all raw data.

Food consumption
The weight of food consumed by each cage of males and females was recorded weekly
during the pre-mating period starting from Day 1 of dosing and thereafter up to mating.
Individual food consumption for mated females was measured on Days 7, 14 and 20 post
coitum starting from Day 0 post coitum and on Days 7 and 13 post partum starting from
Day 1 post partum.

Vaginal smears and oestrus cycle
Before allocation - Stock females
All females ordered for the study were evaluated pre-exposure for oestrous cyclicity and
animals that exhibited anomalies in the oestrous cycle were not allocated to the study.
Oestrous cycle was monitored by vaginal smears for 2 weeks before allocation.
These data are not tabulated in this report, but will be archived with the raw data.
Females allocated to groups
Vaginal smears were taken in the morning starting from Day 1 of dosing up to positive
identification of copulation was made.
The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of
mating)
Before dispatch to necropsy
Vaginal smears were also taken from all females, before dispatch to necropsy and the
oestrous cycle phase recorded. No vaginal smears were taken from one female sacrificed
for humane reasons.
Clinical pathology investigations
4.4.1 Blood collection for thyroid hormone determination (T3, T4 and TSH)
Blood collection was performed for hormone determination from all animals at termination.
No samples were taken from the female (no. X1190053) sacrificed for humane reasons.
Males: blood samples (approximately 0.8 mL) for hormone determination were collected
under isoflurane anaesthesia from the retro-orbital sinus. The order of collection was
equalised between groups.
Females: as a part of the necropsy procedure, blood samples (approximately 0.8 mL) for
hormone determination were withdrawn from the abdominal vena cava under isoflurane
anaesthesia. The order of collection was equalised between groups.
Pups on Days 4 and 14 post partum: on Day 4 post partum, as part of the necropsy procedure,
blood samples (approximately 0.2 mL) were taken from 2 pups from each litter (1
male and 1 female, if possible).
On Day 14 post partum, as part of the necropsy procedure, blood samples (approximately
0.5 mL) were taken from 2 pups from each litter (1 male and 1 female, if possible).
Blood samples were withdrawn under light ether anaesthesia from the heart (intracardiac
puncture). The order of collection was equalised between groups.
Parental animals and pups: samples were transferred into tubes of appropriate capacity
containing no anticoagulant and centrifuged at room temperature.

Oestrous cyclicity (parental animals):

Pairing was monogamous (one male to one female).
A vaginal smear was taken from all females from the day after the start of pairing until
positive identification of copulation (spermidentification, vaginal plug in situ or copulation
plugs found in the cage tray). The female was paired with the same male until positive
identification occurred or 14 days had elapsed.

Litter observations:

Parturition and gestation length
A parturition check was performed from Day 20 to Day 25 post coitum.
Gestation length was calculated as the time between the day of successful mating (Day 0
post coitum) and the day of birth when the parturition was defined complete (Day 0 post
partum).

Pups identification, weight and observation
As soon as possible after parturition was considered complete (Day 0 post partum), all pups
(live and dead) were counted, sexed and live pups were identified.
Live pups were individually weighed on Days 1, 4, 7 and 13 post partum.
Pups dying during the lactation period were weighed before the dispatch to necropsy.
Observations were performed once daily for all litters from Day 0 post partum.
After culling, all pups were sacrificed with the dams on Day 14 post partum.

Culling and pups selection for blood collection (serum hormone) at necropsy
On Day 4 post partum, the size of each litter was adjusted by eliminating extra pups by random
selection to yield, as nearly as possible, four pups per sex per litter. Partial adjustment
(for example, 5 males and 3 females) was acceptable.
On Day 4 post partum, at least one culled male and one culled female were selected for
hormone determination.

Anogenital distance (AGD)
The AGD of each pup was measured on Day 1 post partum. The AGD was normalized to
the cube root of body weight collected on Day 1 post partum.

Nipple count
The presence of nipples/areolae in male pups was checked on Day 13 post partum, on the
day before dispatch to necropsy.

Clinical pathology investigations
4.4.1 Blood collection for thyroid hormone determination (T3, T4 and TSH)
Blood collection was performed for hormone determination from all animals at termination.
No samples were taken from the female (no. X1190053) sacrificed for humane reasons.
Males: blood samples (approximately 0.8 mL) for hormone determination were collected
under isoflurane anaesthesia from the retro-orbital sinus. The order of collection was
equalised between groups.
Females: as a part of the necropsy procedure, blood samples (approximately 0.8 mL) for
hormone determination were withdrawn from the abdominal vena cava under isoflurane
anaesthesia. The order of collection was equalised between groups.
Pups on Days 4 and 14 post partum: on Day 4 post partum, as part of the necropsy procedure,
blood samples (approximately 0.2 mL) were taken from 2 pups from each litter (1
male and 1 female, if possible).
On Day 14 post partum, as part of the necropsy procedure, blood samples (approximately
0.5 mL) were taken from 2 pups from each litter (1 male and 1 female, if possible).
Blood samples were withdrawn under light ether anaesthesia from the heart (intracardiac
puncture). The order of collection was equalised between groups.
Parental animals and pups: samples were transferred into tubes of appropriate capacity
containing no anticoagulant and centrifuged at room temperature.

Bioanalysis - Thyroid hormone determination (T3, T4 and TSH)
Samples were assayed to determine the serum levels of Total triiodothyronine (total T3),
Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by a multiplex assay,
using LuminexMagpix system and the MILLIPLEX MAP Rat ThyroidMagnetic Bead Panel
kit (MerkMillipore, cat. no. RTHYMAG-30K).
The determination was restricted as detailed below:
1. Samples from all parental males from all groups
2. Samples from pups on Day 14 post partum
The results of these analyses are presented as individual data, mean and standard deviation.

Postmortem examinations (parental animals):

Necropsy
The clinical history of parental males and females was studied and a detailed post mortem
examination was conducted (including examination of the external surface and orifices).
Changes were noted, the requisite organs weighed and the required tissue samples preserved
in fixative and processed for histopathological examination.
Females
All females were examined also for the following:
– external and internal abnormalities;
– number of visible implantation sites (pregnant animals);
– number of corpora lutea (pregnant animals).
Uteri of apparently non-pregnant female or uteri of female with no visible implantations
were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

Organ weights
Parental animals
From all animals completing the scheduled test period, the organs indicated in the Annex of the study protocol were dissected free of fat and weighed.

Tissues fixed and preserved
Samples of all the tissues listed in Annex of the study protocol were fixed and preserved in 10% neutral
buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides
which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol).

Histopathological examination
The tissues required for histopathological examination are listed in Annex.
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5
micrometer thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained
with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium
(staging of spermatogenic cycle) was also performed. The evaluation took into account
the tubular stages of the spermatogenic cycle, in order to identify treatment-related effects,
such as: missing germcell layers or types, retained spermatids, multinucleated or apoptotic
germcells and sloughing of spermatogenic cells into the lumen. Identification of the stages
of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952 and
referred to the comprehensive reviews on the subject: Russell, 1990; Creasy, 1997; Creasy,
2002. The PAS- H stained sections were used to identify the spermatogenic stages. For ease
and clarity of reporting, only tissues with findings are reported in the incidence tables.
The examination was restricted in the first instance as detailed below:
i Tissues specified in section 4.5.7 from all males and females in the control and high
dose groups killed at term.
ii Tissues specified in section 4.5.7 from the female (no. X1190053) killed during the
treatment period.
iii All abnormalities in all groups.
As treatment-related findings were found in the liver of high dose animals of both sexes
and in kidneys of high dose treated males, the histopathological evaluation was extended
to the liver of animals of both sexes and to kidneys for males to the corresponding animals
of low and intermediate doses.
The ratios of organ weight to body weight were calculated for each animal.

Postmortem examinations (offspring):

Pups
All pups found dead in the cage were examined for external and internal abnormalities.
All culled pups sacrificed on Day 4 post partum were subjected to an external examination.

Pups at Day 14 post partum
All live pups sacrificed on Day 14 post partum were examined for external abnormalities.
Internal examination
Gonads were inspected from all pups in order to confirm the sex previously determined by
external examination.
All pups with abnormalities were retained in a 10% neutral buffered formalin.

Nipple count retention on Day 14 post partum
No nipples were found in pups when checked on Day 13 post partum and then no ventral
regions were retained on the day of necropsy (Day 14 post partum) .

Pups at Day 14 post partum
Thyroids were weighed from one male and one female pups selected for blood collection
for hormone determination and preserved in 10% neutral buffered formalin.
The thyroid weights were determined after fixation.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables, the significance
of the differences amongst group means was assessed by Dunnett’s test or a
modified t test, depending on the homogeneity of data. Statistical analysis of histopatholgical
findings was carried out by means of the non-parametric Kolmogorov Smirnov test.
The non-parametric Kruskal-Wallis analysis of variance (non-continuous variables) was
used for the other parameters. Intergroup differences between the control and treated
groups were assessed by the non-parametric version of theWilliams test. The criterion for
statistical significance was p < 0.05. The mean values, standard deviations and statistical
analysis were calculated from actual values in the computer without rounding off.

Reproductive indices:

Group mean values, where possible, were calculated for all parameters. Data from non
pregnant female were excluded fromgroup mean calculations as considered
appropriate by the Study Director.
The following reproductive indices were calculated:

Males
Copulatory Index (%) = No. of males with confirmed mating/No. of males cohabitatedx 100
Fertility Index (%) = No. of males which induced pregnancy/No. of males cohabitated x 100

Females

Copulatory Index (%) =No. of females with confirmed mating/No. of females cohabitated x 100
Fertility Index (%) =No. of pregnant females/No. of females cohabitated x 100

Offspring viability indices:

Pre- implantation loss was calculated as a percentage from the formula:

(No. of corpora lutea – no. of implantation) / No. of corpora lutea x 100

Pre-natal loss was calculated as a percentage from the formula:

(No. of visible implantations – Live litter size at birth)/ No. of visible implantations x 100

Post natal loss at Day 0 post partum was calculated as a percentage from the formula:

Total litter size – Live litter size/Total Litter size X 100

Post natal loss at Day 4 post partum (before culling) was calculated as a percentage from the formula:

(Live litter size at birth - live litter size at Day 4 (before culling)/Live litter size at birth X 100

Post natal loss at Day 13 post partum (after culling) was calculated as a percentage from the formula:

(Live litter size on Day 4 (after culling) – Live litter size on Day 13 )/Live litter size on Day 4 (after culling) X 100


Anogenital distance in pups was presented as normalized to the cube root of body weight collected on Day 1 post partum.
Sex ratios was calculated at birth, on Day 4 and on Day 14 post partum and will be presented as the percentage of males per litter.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No significant clinical signs were observed throughout the study in all treated animals
of both sexes.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant difference in body weights and body weight gain were
recorded in treated males compared to the control group, throughout the study. Transient
decrease in body weight gain on Day 7 post partum, as resulting in some fluctuation in the
body weight during the first part of the lactation period, was noted on Day 7 post partum
in all treated females with no clear dose-relationship. Considering that this variation was
noted on occasion it cannot be concluded clearly any toxicologically test item-related effect.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

On Days 7 and 13 post partum, treatment-related effect on food consumption, which was
statistically significantly decreased in females receiving 750 mg/kg/day, was recorded (-18%
p<0.05). At all dose levels no effects on food consumption were observed in treated males
during the study.

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:

no effects observed

Description (incidence and severity):

Thyroid hormone

No alterations of toxicological significance were noted in the thyroid
hormones performed in parental animals or in pups on Day 14 post partum when compared
to the control group.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At 750 mg/kg/day treatment-related clinical signs were recorded in pups during the lactation
period. These included an increase incidence of lost pups (found dead or missing pups)
found in the litters maternally exposed at 750 mg/kg/day. No negative effects in anogenital
distance were seen in treated male or female pups, when compared to the control group.
No nipples were observed in male pups on Day 13 post partum, the day before necropsy. No
relevant necropsy findings were noted in control and treated pups sacrificed on Days 4 and
14 post partum. No differences were noted in thyroid weight between control and treated
pups. No changes were observed on terminal body, absolute and relative organ weight of
control and parental animals that completed the treatment. At macroscopic observations
all changes were considered spontaneous and incidental, having a comparable incidence
in control and treated groups. At microscopic observations treatment-related findings,
were observed in the liver of males dosed at 250 and in males and females dosed at 750
mg/kg/day and in the kidneys of males dosed at 250 and 750 mg/kg/day. In the absence of
degenerative changes in the liver, the hepatocytic hypertrophy observed was considered as
an adaptive rather than an adverse effect. Concerning the kidney, since the hyaline droplets
accumulation noted in males receiving 750 mg/kg/day was associated, in general, with
mild nephropathy (mainly represented multifocal tubular basophilia and/or hyaline cast
of renal tubules) these alterations were considered adverse. No treatment related effects
was noted in the spermatogenic cycle.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The total number of oestrous cycles observed in all females before
pairing, the pre-coital intervals, the copulatory index and fertility index did not show any
differences between control and treated groups. Gestation length was similar between
treated and control groups. Corpora lutea and number of implantation sites of treated females
were comparable to the control values.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

Copulatory and fertility indices both for males and females were similar between control
and treated groups.

Key result

Dose descriptor:

NOAEL

Effect level:

> 750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect in parental animals

Remarks on result:

other: No effets at the high dose tested

Key result

Critical effects observed:

no

Key result

Remarks on result:

other: not examined 421

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs of pups

Found dead and/or missing pups were recorded in all treated and control groups. However
the total number of lost pups (found dead plus missing pups) were particularly evident
in females receiving 750 mg/kg/day when compared to the control group, representing a
treatment related effect at this dose level.

Anogenital distance and nipple count
No differences were noted in the mean values of the AGD (anogenital distance normalised
for the cube root of the body weight performed on Day 1 post partum) of treated pups of
both sexes, compared to the control group.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Mortality of pups
Found dead and/or missing pups were recorded in all treated and control groups. However
the total number of lost pups (found dead plus missing pups) were particularly evident
in females receiving 750 mg/kg/day when compared to the control group, representing a
treatment related effect at this dose level.

Fate of females
There were no test item-related effects on pregnancy status.
One control female (no. X1190005) was found not pregnant at necropsy. Three females
(one control, one receiving 50 mg/kg/day and one receiving 750 mg/kg/day) had unilateral
implantation (nos. X1190017, X1190039, X1190063) in the right uterine horn and were not
pregnant in the left one.
The number of females with live pups on Day 14 post partum was: 9 in the control, 10 in low
dose (50 mg/kg/day), 9 in mid-dose (250 mg/kg/day) and 10 in high dose (750 mg/kg/day)
groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Live litter size was gradually reduced (mean value), during the lactation period, in females
receiving 750 mg/kg/day, with statistical significance on Day 13 post partum. The decrease
was especially evident in female nos. X1190061, X1190063, X1190071, X1190073
and X1190075. In the same period, an increased incidence in post-natal loss (expressed as
percentage) was also recorded compared to the control group.
Starting from Day 1 post partum until termination, litter weight and mean pup weight were
decreased when compared to the control group and they were statistically significant on
Days 4 (litter weight) and 13 post partum (litter and mean pup weights).
These combined effects suggesting a possible adverse effect of the test item on the developing
offspring

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Thyroid hormones
Pups - Day 14 post partum: thyroxine was decreased in some female pups maternally
exposed at 250 and 750 mg/kg/day. Mean group values were 14% and 15% below controls,
respectively.
Due to the minimal severity (data were within the range of historical data) and the absence
of other related findings, this change was not considered to be of toxicological significance.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

At macroscopic observations, autolysed abdominal organs were observed in the majority
of decedent pups of the treated groups. These findings did not permit to determine the
possible cause of death of the pups. In addition, no milk in stomach was evident in one
found dead pup (female) of female no. X1190077 of Group 4 (750 mg/kg/day).
Pups sacrificed on Days 4 and 14 post partum
No findings were recorded in pups sacrificed on Day 4 with the exception of three pups:
- Group 1 - female no. X1190001: one female pup with scabs on muzzle;
- Group 4 - female no. X1190075: one female pup with no milk in stomach; female no.
X1190077: one female pup with tip of tail with dark colour.
No relevant necropsy findings were noted in control and treated pups sacrificed on Day 14
post partum.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: decrease in mean pups weight and pups survival during lactation

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

600 mg/kg bw/day (nominal)

System:

other:

Organ:

other: Increase decedent pups

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Remarks on result:

other: not examined 421

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

750 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The parental NOAEL (No Observed Adverse Effect Level) was considered to be below to 750 mg/kg/day for males and females since some effects during the in-life phase in females (transient decrease in body weight gain and decrease in food consumption) and adverse effects on kidneys in males examined microscopically were evident at 750mg/kg/day.
The NOAEL for the toxicity on fertility could be considered to be 750mg/kg/day.
The NOAEL for the developmental of pups could be considered to be 250 mg/kg/day on the basis of the effects on body weights of pups and in relation to the progressive decrease in pups’ survival observed during the lactation period.

Executive summary:

This study design was performed according to OECD Guideline no. 421 and in compliance with GLP. All doses (0, 50, 250 and 750 mg/kg/day) were administered orally, by gavage. The control group received corn oil. The dose volume was 4 mL/kg body weight.

Males were treated daily for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 29 or 30 days. Females were treated for 2 weeks prior to pairing, during pairing up to Day 13 post coitum. The non pregnant female and the female sacrificed for humane reasons were dosed up to the day before necropsy.

There were no test item related mortality and no test item related effects on the pregnancy status. No significant clinical signs were observed throughout the study in all treated animals of both sexes. No relevant difference in body weights and body weight gain were recorded in treated males compared to the control group, throughout the study. Transient decrease in body weight gain on Day 7 post partum, as resulting in some fluctuation in the body weight during the first part of the lactation period, was noted on Day 7 post partum in all treated females with no clear dose-relationship. Considering that this variation was noted on occasion it cannot be concluded clearly any toxicologically test item-related effect. On Days 7 and 13 post partum, treatment-related effect on food consumption, which was statistically significantly decreased in females receiving 750 mg/kg/day, was recorded (-18% p<0.05). At all dose levels no effects on food consumption were observed in treated males during the study. No alterations of toxicological significance were noted in the thyroid hormones performed in parental animals or in pups onDay 14 post partum when compared to the control group. The total number of oestrous cycles observed in all females before pairing, the pre-coital intervals, the copulatory index and fertility index did not show any differences between control and treated groups. Gestation length was similar between treated and control groups. Corpora lutea and number of implantation sites of treated females were comparable to the control values. A treatment-related decrease on live litter size at birth was noted in females receiving 750 mg/kg/day when compared to the control value.

In particular, a decrease in pre-natal loss (live litter size at birth versus the mean number of implantation sites) was evident. This was considered an adverse effects. This treatment related effect on reduction in litter size continued during the lactation period at the dose level of 750 mg/kg/day and it became statistically significant decreased on Day 13 post partum. As a consequence, an increase in mean post-natal loss (expressed as percentage) data was also recorded. This was considered an adverse effects. Decreases in litter weight and mean pup weight were evident in females dosed at 750 mg/kg/day compared to the control group, starting from Day 1 post partum until termination. They were statistically significant on Days 4 (litter weight) and 13 post partum (litter and mean pup weights). Starting from birth until Day 14 post partum, a decrease in the number of male pups (-17% to -22%) and consequently in the total number of pups (-7% to -21%) was noted in females receiving 750 mg/kg/day. This latest change became significant, at statistical analysis, on Day 14 post partum. These effects was considered treatment-related, but not adverse.

At 750mg/kg/day treatment-related clinical signs were recorded in pups during the lactation period. These included an increase incidence of lost pups (found dead or missing pups) found in the litters maternally exposed at 750 mg/kg/day. No negative effects in anogenital distance were seen in treated male or female pups, when compared to the control group.

No nipples were observed in male pups on Day 13 post partum, the day before necropsy. No relevant necropsy findings were noted in control and treated pups sacrificed on Days 4 and 14 post partum. No differences were noted in thyroid weight between control and treated pups. No changes were observed on terminal body, absolute and relative organ weight of control and parental animals that completed the treatment. At macroscopic observations all changes were considered spontaneous and incidental, having a comparable incidence in control and treated groups. At microscopic observations treatment-related findings, were observed in the liver of males dosed at 250 and in males and females dosed at 750 mg/kg/day and in the kidneys of males dosed at 250 and 750 mg/kg/day. In the absence of degenerative changes in the liver, the hepatocytic hypertrophy observed was considered as an adaptive rather than an adverse effect. Concerning the kidney, since the hyaline droplets accumulation noted in males receiving 750 mg/kg/day was associated, in general, with mild nephropathy (mainly represented multifocal tubular basophilia and/or hyaline cast of renal tubules) these alterations were considered adverse. No treatment related effects was noted in the spermatogenic cycle.

Conclusion

Based on the results of the present study, the parental NOAEL (No Observed Adverse Effect Level) was considered to be below to 750 mg/kg/day for males and females since some effects during the in-life phase in females (transient decrease in body weight gain and decrease in food consumption) and adverse effects on kidneys in males examined microscopically were evident at 750mg/kg/day.

The NOAEL for the toxicity on fertility could be considered to be 750 mg/kg/day.

The NOAEL for the developmental of pups could be considered to be 250 mg/kg/day on the basis of the effects on body weights of pups and in relation to the progressive decrease in pups’ survival observed during the lactation period.

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

Reference

Reference 1

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

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system 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

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

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

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

Abnormalities:

no effects observed

Fetal body weight changes:

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.

Reference 2

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

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. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system 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:

body weight and weight gain

food consumption and compound intake

mortality

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. At 100 mg/kg/day there was one fetus (N31299-01) with multiple malformations (teras), and another (N31286-06) 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.

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	3579^a
		(+3)	(+1)	(+1)
Day 9 p.c.	3618	3696	3629	3505^a
		(+2)	(0)	(-3)
Day 12 p.c.	3689	3775	3690	3556^a
		(+2)	(0)	(-4)
Day 15 p.c.	3780	3867	3779	3635^a
		(+2)	(0)	(-4)
Day 19 p.c.	3845	3928	3860	3714^a
		(+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	+50^a
Days 12 - 15 p.c.	+91	+93	+89	+79^a
Days 15 - 19 p.c.	+65	+61	+81	+79^a
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	133^a
		(+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 Category 2 (H361 d) is warranted.