4-hydroxy-4-methylpentan-2-one

Toxicity to reproduction

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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:

no

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 oral exposure:

VEHICLE
- Vehicle: Japanese Pharmacopeia purified water
- Lot/batch no. (if required): 180889, 180964

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 animals/sex/dose

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

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on kill date
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on kill date
- Animals fasted: Yes
- How many animals: all

URINALYSIS: Yes
- Time schedule for collection of urine: collected on day 38 and day 41 of administration
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

Reproductive examinations

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:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no deaths among the males. In the females, one animal in the 1000 mg/kg group was weakened during delivery and so was euthanized once reaching a near death state. Other than this one case, no deaths were noted.

BODY WEIGHT AND WEIGHT GAIN
There were no significant changes noted in the weights or weight increases in males. In females, a significant reduction in the amount of weight gain (-31%) during the premating period was noted in the 1000 mg/kg group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Neither males nor females in the groups administered the test substance exhibited significant changes in food consumption.

HAEMATOLOGY
There was a significant increase (+16%) in the number of platelets noted in the 1000 mg/kg group male rats.

CLINICAL CHEMISTRY
In the 1000 mg/kg group in males (females not done), significant increases (50% ) in GOT, choline esterase (x1.8), total protein (10%), total cholesterol (76%), total bilirubin (33%), blood urea nitrogen (19%), creatinine (17%) and calcium (6%) were noted, as well as a significant reduction in glucose (23%). Furthermore, while not a significant difference, an increasing trend was noted in the 300 mg/kg group for choline esterase and total protein, an increasing trend was noted in the 1000 mg/kg group for ¿-GTP and albumin, along with a decreasing trend for the A/G ratio and triglycerides. Significantly low values were exhibited for alkaline phosphatase when compared to the control group but these were not dose dependent changes.

URINALYSIS
There were no significant changes noted for any of the examination items in the groups administered the test substance.

ORGAN WEIGHTS
Significant increases in the absolute (15%) and relative (13%) weights of the kidneys were noted in males in the 300 mg/kg group, and the same increasing trend was noted in the 1000 mg/kg group. Additionally, in the 1000 mg/kg group, significant increases in the absolute (21%) and relative (24%) weights of the adrenals were noted, along with a significant increase in the relative (17%) weight of the liver, and there was an increasing trend exhibited for the absolute weights of the liver. In the females, significant increases in the absolute (26%) and relative (25%) weights of the liver were noted in the 1000 mg/kg group.

GROSS PATHOLOGY, HISTOPATHOLOGY: NON-NEOPLASTIC
Changes thought to be caused by administration of the test substance were noted in the liver, kidney and adrenals.
Five out of ten males in the 1000 mg/kg group exhibited central lobular hepatocellular hypertrophy. Increased deposition of hyaline droplets in the proximal tubular epithelium of the kidney was noted in 8/10 in the 100 mg/kg group, 10/10 in the 300 mg/kg group and 9/10 in the 1000 mg/kg group, exhibiting a strong dose dependency. Additionally, there were 6 animals with increased basophilic tubules in the kidneys in the 300 and 1000 mg/kg group and dilation of the distal tubules noted in 4/10 animals in the 1000 mg/kg group. Vacuolization of the cells of the zona fasciculata in the adrenals was noted in one in the 300 mg/kg group and 5 in the 1000 mg/kg group. Central lobular hepatocellular hypertrophy was noted in 5/10 males in the 1000 mg/kg group.

In the 1000 mg/kg group, central lobular hepatocellular hypertrophy was noted in 6/10 females, vacuolization of the cells of the zona fasciculata in the adrenals in 3/10 females and dilation of the distal tubules of kidneys in 3/10 females.

However, in males and females unsuccessful at impregnation and in females where all of the pups died, there were no changes noted in the pituitary gland and reproductive organs that were thought to be caused by administration of the test substance. In one euthanized female in the 1000 mg/kg group, the fatty degeneration of the proximal tubule epithelium and dilation of distal tubules in the kidneys noted was stronger than the changes in the survivors. Furthermore, central lobular hepatocellular hypertrophic necrosis was noted, along with necrosis of the mucous membranes of the glandular stomach and ileum, hypertrophy of the cells of the zona fasciculata in the adrenals and atrophy of the spleen and thymus. In addition to the findings given above, changes in each of the organs were noted but they were sporadic and not confirmed to be related to administration of the test substance.

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)

Key result

Dose descriptor:

NOEL

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect

Critical effects observed:

not specified

Conclusions:

Based on the above mentioned results, the non-observable adverse effect levels for repeat dose toxicity of diacetone alcohol in rats are considered to be 100 mg/kg/day in males and females (excluding the sex- and species-specific hyaline droplet nephropathy of the males).

Executive summary:

A repeated dose study according to OECD TG 422 was conducted with 4-hydroxy-4-methyl-2-pentanone (= diacetone alcohol, or HMP). In this study, 10 rats/sex/group were dosed by gavage with 0, 30, 100, 300, or 1000 mg/kg/day of 4-hydroxy-4-methyl-2-pentanonein distilled water for approximately 45 days. Decreased locomotor activity and stimulation response were observed at 300 and 1000 mg/kg/day for both sexes. Altered blood parameters, dilatation of the distal tubules of the kidneys, hepatocellular hypertrophy, and vacuolization of the zona fasciculata of the adrenals were observed in males from the 1000 mg/kg/day group. Females at 300 mg/kg/day showed dilatation of the distal tubules and fatty degeneration of the proximal tubular epithelium in the kidneys. At 1000 mg/kg/day, female body weight gain was reduced, liver weight was increased, hepatocellular hypertrophy was noted, and kidney lesions similar to those at 300 mg/kg/day were also recorded. In addition, increased incidence and/or severity of hyaline droplets in the tubular epithelium was noted in males at 100 mg/kg/day and greater. The kidney effects observed are suggestive of chronic progressive nephropathy (CPN) and a2µ-globulin nephropathy, a condition not relevant for human risk assessment (U. S. EPA. (1991) Alpha2µ-globulin: Association with chemically induced renal toxicity and neoplasia in the male rat. Risk Assessment Forum. EPA/625/3-91/019F). Therefore, the NOAEL for this study was 100 mg/kg/day for males and females (excluding the sex- and species-specific hyaline droplet nephropathy observed in the males).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

4-hydroxy-4-methylpentan-2-one

EC Number:

204-626-7

EC Name:

4-hydroxy-4-methylpentan-2-one

Cas Number:

123-42-2

Molecular formula:

C6H12O2

IUPAC Name:

4-hydroxy-4-methylpentan-2-one

Details on test material:

- Name of test material (as cited in study report): diacetone alcohol CAS# 123-42-2
- Physical state: liquid
- Analytical purity: 99.8%
- Stability under test conditions: stable
- Storage condition of test material: sealed in a container, in a cool, dark location

Test animals

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

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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

Details on results (P0)

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.

Effect levels (P0)

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)

Target system / organ toxicity (P0)

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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

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

Applicant's summary and conclusion

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.