3-methylbutanone

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2/2012 - 5/2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted to OECD protocols in a GLP-compliant lab

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-methylbutanone

EC Number:

209-264-3

EC Name:

3-methylbutanone

Cas Number:

563-80-4

Molecular formula:

C5H10O

IUPAC Name:

3-methylbutan-2-one

Constituent 2

Reference substance name:

Methyl Isopropyl Ketone

IUPAC Name:

Methyl Isopropyl Ketone

Constituent 3

Reference substance name:

2-Butanone, 3-methyl

IUPAC Name:

2-Butanone, 3-methyl

Constituent 4

Reference substance name:

3-methyl-2-butanone

IUPAC Name:

3-methyl-2-butanone

Test material form:

other: liquid

Details on test material:

Colorless to pale yellow liquid
MW = 86.13
Density = 0.08 g/cm3
MP = -92C
BP = 93-95C
Water solubility = 6 g/l

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Sexually mature, virgin female Sprague Dawley [Crl:CD(SD)] rats were used as the test system on this study. This species and strain of animal is recognized as appropriate for developmental toxicity studies. This animal model has been proven to be susceptible to the effects of developmental toxicants. The number of animals selected for this study was based on the United States EPA Health Effects Test guidelines OPPTS 870.3700, Prenatal Developmental Toxicity Study, August 1998 and the OECD Guideline for Testing of Chemicals Guideline 414, Prenatal Developmental Toxicity Study January 2001.

All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and humidity controls were set to maintain environmental conditions of 71°F ± 5°F (22°C ± 3°C) and 50% ± 20%, respectively. Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. Actual mean daily temperature ranged from 71.1°F to 72.1°F (21.7°C to 22.3°C) and mean daily relative humidity ranged from 38.2% to 57.0% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

The test substance or filtered air was administered via a daily 6-hour whole-body exposure during gestation days 0-19. All rats were exposed at approximately the same time each day.

The following table presents the study group assignment:
Group
Number Treatment Target Exposure Level (ppm) Number of Females
1 Filtered Air 0 25
2 MIPK 300 25
3 MIPK 750 25
4 MIPK 1500 25

Exposure levels were based on the results of a previous reproductive screening study in rats at dosage levels of 1.0, 2.5, and 5.0 mg/L (approximate levels of 300, 750, and 1500 ppm, respectively). In this study, concentration-dependent reductions in activity level were noted at 2.5 and 5.0 mg/L and partially closed eyes were noted at 5.0 mg/L. In addition, mean body weights and food consumption were reduced at 2.5 and 5.0 mg/L for the first 2-3 weeks of the pre-mating dosing period and continued to be slightly decreased in the gestational phase at 5.0 mg/L. Developmentally, a decrease in the live litter size and a decrease in postnatal survival from postnatal day (PND) 0-4 were noted at 5.0 mg/L. Therefore, the same exposure levels were chosen for the current study and the dosing regimen was extended (gestation days 0-19) to cover the pre-implantation period (gestation days 0-5) to assess any impact on litter size from exposure during this period.
The selected route of administration for this study was whole-body inhalation exposure because the intended use of the test substance indicated that this would be a likely route of human exposure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyzed exposure concentrations were determined at approximately 30-minute intervals using a gas chromatograph (GC) under the control of the WINH data acquisition software application. Samples were collected from the approximate animal-breathing zone of the inhalation exposure chamber via heated stainless steel sampling lines. Test atmosphere samples were collected automatically using a heated external multi-position valve. For each sample, the chromatograph was displayed and the area under the sample peak was calculated and stored. The concentration in parts per million (ppm) was calculated using a ln-quadratic equation based on the GC calibration curve.

Details on mating procedure:

At the conclusion of the acclimation period, all available females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was placed in a suspended wire-mesh cage with a resident male from the same strain and source for breeding. Resident males were untreated, sexually mature rats utilized exclusively for breeding. These rats were maintained under similar laboratory conditions as the females. A breeding record containing the male and female identification numbers and the dates of cohabitation was maintained. The selected females were approximately 13 weeks old when paired for breeding.
Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day on which evidence of mating was identified was termed gestation day 0 and the animals were separated.
The experimental design consisted of 3 test substance-exposed groups and 1 control group, composed of 25 rats per group. The bred females were assigned to groups using a WTDMS™ computer program which randomized the animals based on stratification of the gestation day 0 body weights in a block design. Animals not assigned to study were transferred to the WIL Research stock colony or euthanized by carbon dioxide inhalation and discarded. Body weight values ranged from 219 g to 286 g on gestation day 0.

Duration of treatment / exposure:

The test substance or filtered air was administered via a daily 6-hour whole-body exposure during gestation days 0-19. All rats were exposed at approximately the same time each day.

Frequency of treatment:

The test substance or filtered air was administered via a daily 6-hour whole-body exposure during gestation days 0-19. All rats were exposed at approximately the same time each day.

Duration of test:

The test substance or filtered air was administered via a daily 6-hour whole-body exposure during gestation days 0-19. All rats were exposed at approximately the same time each day.

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

Treatment from day 0 was used as this study was designed to answer questions regarding information from ACGIH that MiPK exposure results in preimplantation loss. The normal test paradigm of days 6-19 would not account for this observation. While most studies are conducted from days 6-19, the guideline clearly states that the entire period of gestation can be used.

Examinations

Maternal examinations:

All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded daily from gestation days 0 through 20 (prior to exposure during the treatment period). Animals were also observed for signs of toxicity approximately 1 hour following exposure. The absence or presence of findings was recorded for individual animals.
Special attention was given to the state of arousal and response to novel stimuli during exposure (as close as possible to the end of the exposure period each day) by producing a loud-noise stimulus. The noise was produced by allowing an approximately 50 g item to strike the steel side of the exposure chamber at the approximate level of the cage rack. The stimulus item was attached to a length of cotton rope that was held against the steel side of the chamber approximately 45 cm from the item. The stimulus item was raised until the rope was approximately perpendicular to the side of the chamber, and the item was released. Each animal’s response to the stimulus was recorded as: no reaction, slight reaction (ear flick or some evidence that the stimulus was heard), or more energetic response (jump, flinch, and/or vocalization).

Ovaries and uterine content:

Laparohysterectomies and macroscopic examinations were performed blind to treatment group. All females were euthanized on gestation day 20 by carbon dioxide inhalation. The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the postmortem findings were correlated with the antemortem comments, and any abnormalities were recorded. The uterus and ovaries were then exposed and excised. The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.
Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss

Fetal examinations:

Fetal examinations were performed blind to treatment group. Each viable fetus was examined externally, individually sexed, weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region, and tagged for identification. Fetal tags contained the WIL Research study number, the female number, and the fetus number. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Crown-rump measurements and degrees of autolysis were recorded for late resorptions, a gross external examination was performed (if possible), and the tissues were discarded.
Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels. The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development. Heads from approximately one-half of the fetuses in each litter were placed in Bouin’s fixative for subsequent soft-tissue examination by the Wilson sectioning technique. The heads from the remaining one-half of the fetuses were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol.
Following fixation in alcohol, each fetus was macerated in potassium hydroxide and stained with Alizarin Red S and Alcian Blue. Fetuses were then examined for skeletal malformations and developmental variations.
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

Statistics:

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-exposed group to the control group. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Where applicable, the litter was used as the experimental unit.
Mean maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, organ weights (absolute and relative to brain weights), numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett’s test was used to compare the test substance-exposed groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined) and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related lower mean body weight gains and reduced food consumption were noted for the 750 and 1500 ppm groups generally throughout the exposure period (gestation days 0-20). As a result, mean body weights in these groups were up to 11.5% and 12.3% lower than the control group. In addition, mean net body weights, net body weight gains, and gravid uterine weights in the 750 and 1500 ppm groups were lower than the control group. In the 300 ppm group, a test substance-related lower mean body weight gain was noted during gestation days 0-3. Although mean body weight gains in this group were similar to the control group throughout the remainder of the exposure period (gestation days 3-20), the initial lower mean body weight gain was of sufficient magnitude to result in a lower overall mean body weight gain (gestation days 0-20), slightly lower (within 5% of the control group values) mean body weights during gestation days 3-20, and a lower mean net body weight gain. Reduced food consumption was also noted for the 300 ppm group throughout the treatment period. 

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):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
In this study, reduced reactivity to noise stimulus, lower mean body weights and body weight gains, and corresponding reduced mean food consumption were noted for females in the 300, 750, and 1500 ppm groups.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

LOAEC

Effect level:

< 300 ppm (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

not determinable

Remarks:

no NOAEC identified

2

Dose descriptor:

NOAEC

Effect level:

> 1 500 ppm (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEC

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

skeletal malformations

visceral malformations

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

1 500 ppm

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

In this study, reduced reactivity to noise stimulus, lower mean body weights and body weight gains, and corresponding reduced mean food consumption were noted for females in the 300, 750, and 1500 ppm groups. Based on these results, an exposure level of less than 300 ppm was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Based on the absence of developmental toxicity at any exposure level tested, 1500 ppm, the highest exposure level tested, was considered to be the NOAEL for embryo/fetal development when MIPK was administered by whole-body inhalation to bred Crl:CD(SD) rats.

Executive summary:

Objective

The objective of the study was to determine the potential of the test substance to induce developmental toxicity after maternal exposure from conception to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity. 

Design

Methyl isopropyl ketone (hereafter referred to as MIPK) was administered via whole body inhalation exposure to 3 groups of 25 bred female Crl:CD(SD) rats for 6 hours daily from gestation days 0 through 19. Target exposure levels were 300, 750, and 1500 ppm, corresponding to mean measured exposure concentrations of 304, 757, and 1529 ppm, respectively. A concurrent control group composed of 25 bred females was exposed to filtered air on a comparable regimen. The females were approximately 13 weeks of age at the initiation of exposure. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each female and selected organs were collected and weighed. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

Results 

All females survived to the scheduled euthanasia. Test substance-related reduced reactivity to a noise stimulus near the end of exposure was noted for females in all test substance‑exposed groups when compared to the control group. No test substance‑related clinical findings were noted for females in the 300, 750, or 1500 ppm groups at the daily examinations or approximately 1 hour following exposure. 

Test substance-related lower mean body weight gains and reduced food consumption were noted for the 750 and 1500 ppm groups generally throughout the exposure period (gestation days 0-20). As a result, mean body weights in these groups were up to 11.5% and 12.3% lower than the control group. In addition, mean net body weights, net body weight gains, and gravid uterine weights in the 750 and 1500 ppm groups were lower than the control group. In the 300 ppm group, a test substance-related lower mean body weight gain was noted during gestation days 0-3. Although mean body weight gains in this group were similar to the control group throughout the remainder of the exposure period (gestation days 3-20), the initial lower mean body weight gain was of sufficient magnitude to result in a lower overall mean body weight gain (gestation days 0-20), slightly lower (within 5% of the control group values) mean body weights during gestation days 3-20, and a lower mean net body weight gain. Reduced food consumption was also noted for the 300 ppm group throughout the treatment period. 

No test substance-related macroscopic findings were noted at any exposure level. 

Test substance-related higher mean adrenal gland weights (absolute and relative to brain weight) were noted at 1500 ppm when compared to the control group. Mean adrenal weights at 300 and 750 ppm and mean liver and kidney weights at 300, 750, and 1500 ppm were unaffected by test substance exposure. 

Intrauterine growth and survival in the 300, 750, and 1500 ppm groups were unaffected by test substance exposure. There were no test substance-related effects on fetal morphology noted at any exposure level. 

Conclusion

In this study, reduced reactivity to noise stimulus, lower mean body weights and body weight gains, and corresponding reduced mean food consumption were noted for females in the 300, 750, and 1500 ppm groups. Based on these results, an exposure level of less than 300 ppm was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Based on the absence of developmental toxicity at any exposure level tested, 1500 ppm, the highest exposure level tested, was considered to be the NOAEL for embryo/fetal development when MIPK was administered by whole-body inhalation to bred Crl:CD(SD) rats.