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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted in a GLP facility to OECD guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
MPK
Batch/Lot no. TD14042663
Exp. date: 11-Dec-2016
CAS no. 107-87-9
WIL ID no. 14034C
Clear, colorless liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Sexually mature, virgin female Sprague Dawley [Crl:CD(SD)] rats were used as the test system on this study. Crl:CD(SD) rats (125 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC, on 06-Aug-2015. The animals were approximately 80 days old upon receipt. Each female was examined by a qualified biologist on the day of receipt. The day following receipt, all animals were weighed and clinical observations were recorded. The animals were housed for a minimum of 12 days for acclimation purposes. During the acclimation period, the rats were observed twice daily for mortality and changes in general appearance and behavior.

Upon arrival, all rats were housed 2-3 per cage in clean, solid-bottom cages with bedding material (Bed-O'Cobs®; The Andersons, Cob Products Division, Maumee, OH). The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the females were individually housed in clean, solid-bottom cages with bedding material.
Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research.
Municipal water supplying the facility was sampled for contaminants according to WIL Research SOPs. Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the acclimation period and during the study, except during the exposure periods when water and food were withheld.

All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and relative 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 70.5°F to 82.3°F (21.4°C to 27.9°C) and mean daily relative humidity ranged from 43.3% to 64.8% 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:
Animal exposures were conducted using four 1000-L glass and stainless steel whole-body inhalation exposure chambers. One exposure system was dedicated for each group for the duration of the study. A HEPA filter and an activated charcoal bed were used to pre-treat room air prior to delivery to the chambers. All test substance atmosphere chamber exhaust passed through the facility exhaust system consisting of redundant exhaust blowers preceded by activated-charcoal and HEPA-filter units. The exposure period was 6 hours per day from gestation days 6 through 19. Animals were housed in a normal animal colony room during non-exposure hours. Food and water were withheld during each exposure period. For each day’s exposure, the animals were transferred to exposure caging in the colony room, transported to the exposure room, exposed for the requisite duration, and returned to their home cages in the animal colony room. The cage batteries were rotated on a daily basis between the 3 battery positions within the chambers to help ensure a similar exposure for all animals within each group over the duration of the exposure period.
The exposure chamber mean temperature and relative humidity were to be between 19°C to 25°C and 30% to 70%, respectively. All chambers were operated with at least 12 to 15 air changes per hour (200 to 250 standard liters per minute) and at a slight negative pressure. Oxygen content was 20.9% for Chambers 1 and 2, 20.6% for Chamber 3, and 20.5% for Chamber 4.
The control exposure system (Group 1, 0 ppm) was operated as follows. Humidified supply air was delivered to the exposure system from the WIL Research Inhalation Department supply air source. To maintain the relative oxygen content in the exposure system at approximately the same level as within the test substance exposure systems, compressed nitrogen was delivered to the inlet where it mixed with supply air. The rate of nitrogen was monitored using a regulator and controlled using a rotameter-type flowmeter.
MPK vapors were generated using J-type glass-bead column vaporization systems. The column (1-inch ID x 22-inch height) for each test substance exposure chamber was filled with various sized glass beads and heated to approximately 110ºC. An FMI Lab Pump was used to deliver the liquid test substance from a reservoir to the top of the bead column. Nitrogen was metered to the bottom of the bead column using a regulator and a rotameter-type flowmeter. Vaporization occurred as the test substance flowed over the surface of the heated beads while the nitrogen flowed up through the column. The concentrated MPK vapors were directed to each glass exposure chamber inlet where the concentration was reduced by mixing with humidified supply air prior to entering each exposure chamber.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Nominal exposure concentrations were calculated for each test substance exposure chamber from the total amount of test substance consumed during the exposure (as weighed prior to and at the termination of the generation) and the total volume of air passed through the chamber during exposure. Total air volume was calculated by multiplying the daily mean ventilation rate by the duration of generation. This value included the ventilation flow through the chamber and the nitrogen through the generation system. Analyzed exposure concentrations were determined at approximately 45-minute intervals using a gas chromatograph (GC). Samples were collected from the approximate animal-breathing zone of the exposure chamber via 1/4-inch polyethylene tubing. Under the control of the WINH system, sampling and analyses were performed as follows. An external multi-position valve permitted sequential sampling from the exposure room and each exposure chamber. Gas sampling injection onto the chromatography column occurred via an internal gas-sampling valve with a sample loop, the chromatograph was displayed and the area under the sample peak was calculated and stored. The system then acquired the stored peak area data and used a ln-quadratic equation based on the GC calibration curve to calculate the measured concentration in ppm.
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 solid-bottom cage with bedding material 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-treated 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 colony or euthanized by carbon dioxide inhalation and
discarded. Body weight values ranged from 217 g to 287 g on gestation day 0.
Duration of treatment / exposure:
MPK or humidified, filtered air (control group) was administered via whole-body
inhalation exposures for 6 hours per day from gestation days 6 through 19.
Frequency of treatment:
MPK or humidified, filtered air (control group) was administered via whole-body
inhalation exposures for 6 hours per day from gestation days 6 through 19.
Duration of test:
MPK or humidified, filtered air (control group) was administered via whole-body
inhalation exposures for 6 hours per day from gestation days 6 through 19.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1500 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
750 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
250 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
Methyl n-propyl ketone (MPK) was administered via whole-body inhalation exposure to
3 groups (Groups 2-4) of 25 bred female Crl:CD(SD) rats for 6 hours per day from
gestation days 6 through 19. Target exposure concentrations were 250, 750, and
1500 ppm for Groups 2, 3, and 4, respectively. A concurrent control group (Group 1)
composed of 25 bred females was exposed to humidified, filtered air on a comparable
regimen. The females were approximately 14 weeks of age at the initiation of exposure.
All animals were observed twice daily for mortality and moribundity. Clinical
observations, arousal response observations, body weights, and food consumption were
recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was
performed on each female and select organs were 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.

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 dose administration during the treatment period). Animals were also observed for signs of toxicity 0-1 hour following exposure. The absence or presence of findings was recorded for all animals.

Weekly during the exposure period, 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 on a weekly basis) by producing a loud-noise stimulus. The noise was produced by allowing an approximately 50-g PVC or stainless steel pipe 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. The response to the stimulus was recorded for animals visible in the chamber as: not observed; no reaction; slight reaction (ear flick or some evidence that the stimulus was heard); or more energetic response (jump, flinch and/or vocalization). Individual maternal body weights were recorded on gestation days 0 and 6-20 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 6-9, 9-12, 12-15, 15-20, and 6-20 (entire exposure period).

Gravid uterine weight was collected and net body weight (the gestation day 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation day 0-20 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

Individual food consumption was recorded on gestation days 0 and 6-20 (daily). Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. When food consumption could not be determined for an animal during a given interval (due to weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable for a given animal were designated as “NA” on the individual report tables.
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 cranial, 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 observations, 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 subsequentlyplaced in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964).
The brain, liver, and maternal tissues with gross lesions were preserved in 10% neutral-buffered formalin for possible future histopathologic examination. For gross lesions, representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded. Brain and liver were weighed from all animals at the scheduled necropsy: Absolute organ weights and organ to brain weight ratios were calculated
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.
Nonviable fetuses (if the degree of autolysis was minimal or absent) were examined, the crown-rump length measured, weighed, sexed, and tagged individually. 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 (Stuckhardt and Poppe, 1984). The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development (Woo and Hoar, 1972). Heads from approximately one-half of the fetuses in each litter were placed in Harrison’s fixative for subsequent soft-tissue examination by the Wilson sectioning technique (Wilson, 1965). 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 stained with Alizarin Red S (Dawson, 1926) and Alcian Blue (Inouye, 1976). 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. Data obtained from nongravid animals were excluded from statistical analyses. Where applicable, the litter was used as the experimental unit. Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, organ weights (absolute and organ-to-brain weight ratios), 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 (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test article-treated 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 post-implantation 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 (Kruskal and Wallis, 1952) to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test article-treated groups to the control group.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No maternally toxic effects

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEC
Effect level:
1 500 ppm
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
1 500 ppm
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No developmental toxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
There were no adverse effects on maternal animals or effects on intrauterine growth,
survival, and fetal morphology at any exposure level. Therefore, an exposure level of
1500 ppm (the highest exposure level evaluated) was considered to be the
no-observed-adverse-effect concentration (NOAEC) for maternal toxicity and
embryo/fetal development when methyl n-propyl ketone was administered via
whole-body inhalation exposure for 6 hours per day from gestation days 6 through 19 to
bred Crl:CD(SD) rats.
Executive summary:

The objectives of this study were to determine the potential of the test substance, methyl n-propyl ketone, to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect concentration (NOAEC) for maternal and developmental toxicity.

Methyl n-propyl ketone (MPK) was administered via whole-body inhalation exposure to 3 groups (Groups 2-4) of 25 bred female Crl:CD(SD) rats for 6 hours per day from gestation days 6 through 19. Target exposure concentrations were 250, 750, and 1500 ppm for Groups 2, 3, and 4, respectively. A concurrent control group (Group 1) composed of 25 bred females was exposed to humidified, filtered air on a comparable regimen. The females were approximately 14 weeks of age at the initiation of exposure. All animals were observed twice daily for mortality and moribundity. Clinical observations, arousal response observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each female and select organs were 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.

All females in the control and test substance-exposed groups survived to the scheduled necropsy on gestation day 20. A test substance-related increase in the incidence of red material around the nose and/or mouth was noted in all test substance-treated groups when compared to the control group at the 0-1 hour post-exposure observation throughout the exposure period. These findings did not persist until the pre-exposure observations on the following day and were not considered adverse.

There was no discernible change or shift in the absolute numbers or pattern of arousal response at any exposure level.

There were no test substance-related effects on mean body weights, body weight gains, net body weights, net body weight gains, gravid uterine weights, or food consumption in any test substance-exposed group.

There were no remarkable macroscopic findings or test substance-related effects on brain or liver weights in any exposure group. Intrauterine growth and survival were unaffected by test substance exposure at all exposure levels. Fetal morphology (external, visceral, and skeletal) was unaffected by test substance exposure at all exposure levels.

There were no adverse effects on maternal animals or effects on intrauterine growth, survival, and fetal morphology at any exposure level. Therefore, an exposure level of 1500 ppm (the highest exposure level evaluated) was considered to be the no-observed-adverse-effect concentration (NOAEC) for maternal toxicity and embryo/fetal development when methyl n-propyl ketone was administered via whole-body inhalation exposure for 6 hours per day from gestation days 6 through 19 to bred Crl:CD(SD) rats.