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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study without detailed documentation
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
animals exposed for 10 days less than recommended. No date reported
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 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 animals)

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
Description (incidence and severity):
Increases in the weights of the kidney of males at 300 and 1000 mg/kg/d, and at 1000 mg/kg/d increases in the weights of the liver in males and females and adrenals in males
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Enlarged livers at 1000 mg/kg/d
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes in the liver (males and females at 1000 mg/kg/d), kidney (males at 100, 300 and 1000 mg/kg/d) and adrenals (males at 1000 mg/kg/d)
Other effects:
not examined

Reproductive function / 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)

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.