Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The 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
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Heptan-2-one
EC Number:
203-767-1
EC Name:
Heptan-2-one
Cas Number:
110-43-0
Molecular formula:
C7H14O
IUPAC Name:
heptan-2-one
Constituent 2
Reference substance name:
MAK
IUPAC Name:
MAK
Constituent 3
Reference substance name:
methyl n-amyl ketone
IUPAC Name:
methyl n-amyl ketone
Details on test material:
Batch/lot no. TD15017200
Clear, colorless translucent liquid

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. 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. Each animal was uniquely identified using a programmable microchip (BMDS system) which was implanted subcutaneously in the dorsoscapular region during the acclimation period. The animals were housed for a minimum of 14 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). Enrichment devices were provided to all animals as appropriate throughout the study (except during the exposure periods) for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.

The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent
LabDiet® 5002, was a certified. Feed lots used during the study were documented in the study records. The feeders were changed and sanitized once per week. 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 70.7°F to 71.5°F (21.5°C to 21.9°C) and mean daily relative humidity ranged from 39.7% to 49.1% 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 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 245 g to 320 g on gestation day 0.
The test substance or humidified, filtered air were administered via a daily 6-hour
whole-body exposure during gestation days 6-19.

The following table presents the study group assignment:

Group Treatment Target Exposure Number of Females
Concentration (ppm)

1 Filtered Air 0 25
2 MAK 300 25
3 MAK 600 25
4 MAK 1200 25


Exposure levels were selected based on data from similar materials (methyl iso-amyl ketone and methyl propyl ketone). In a previous prenatal developmental toxicity study, rats were exposed to methyl isoamyl ketone (MiAK) 6 hours per day at exposure levels of 0, 250, 750, and 1500 ppm from gestation days 6-19, inclusively. All rats survived to the scheduled euthanasia. Lower mean body weight, body weight gain, and corresponding lower food consumption were noted in the 1500 ppm group. In another previous prenatal developmental toxicity study, rats were exposed to methyl n-propyl ketone (MPK) for 6 hours per day at exposure levels of 0, 250, 750, and
1500 ppm from gestation days 6-19, inclusively. All rats survived to the scheduled
euthanasia. Mean body weight and food consumption were generally similar across
groups.
Based on these results, exposure levels of 0, 300, 600, and 1200 ppm were selected for
the current study. The selected route of administration for this study was inhalation exposure as this is the potential route of exposure for humans
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 1/8-inch 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 WINH 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 245 g to 320 g on gestation day 0.
Duration of treatment / exposure:
14 days
Frequency of treatment:
daily
Duration of test:
20 days
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle

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 0-1 hour following exposure. The absence or
presence of findings was recorded for all animals. A separate computer protocol was
used to record any observations noted outside of the above-specified intervals.

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 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).

Laparohysterectomies and macroscopic examinations were performed blind to treatment group. All rats 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 subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss.
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.
Ovaries and uterine content:
Laparohysterectomies and macroscopic examinations were performed blind to treatment group. All rats 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 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 Harrison’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 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-treated 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 from those listed in the report data tables. 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 to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-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 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. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.

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:
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):
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
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:no effects

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEC
Effect level:
1 200 ppm
Based on:
test mat.
Basis for effect level:
other: all measurements
Key result
Dose descriptor:
NOAEC
Effect level:
1 200 ppm
Based on:
test mat.
Basis for effect level:
other: All measurements

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:
1 200 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: all measurements

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Executive summary:

The objectives of the study were to determine the potential of the test substance, methyl

n-amyl 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.

 

The test substance, methyl n-amyl ketone (MAK), 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

300, 600, and 1200 ppm for Groups 2, 3, and 4, respectively. Mean analyzed exposure

concentrations were 303, 613, and 1251 ppm for the same respective groups. 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 selected 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, 300, 600, and 1200 ppm groups survived to the scheduled

necropsy. Increased incidences of red material around the nose and/or mouth were noted in the 1200 ppm group compared to the vehicle control group at 0-1 hour following exposure during gestation days 7-19; however, these findings generally did not persist to the daily examinations, and therefore were considered test substance-related but nonadverse. No other test substance-related clinical observations were noted at the daily examinations or 0-1 hour following exposure at any exposure level.

In the 1200 ppm group, an increased number of females with no reaction to the arousal

response stimulus was noted at the end of the exposure period and corresponded to a

slight decrease in the incidence and number of females with a slight reaction or more

energetic response to the stimulus compared to the control group; these observations were considered test substance-related but nonadverse due to the lack of any other adverse toxicological effects on the females in this group. Arousal response was unaffected by test substance exposure at 300 and 600 ppm.

 

A lower mean body weight gain was noted in the 1200 ppm group during gestation

days 6-9 and resulted in a slightly lower mean body weight gain in this group compared

to the control group when the entire treatment period (gestation days 6-20) was evaluated. A lower mean net body weight gain was also noted in the 1200 ppm group compared to the control group. In addition, mean food consumption in the 1200 ppm group was lower than the control group throughout the treatment period. However, the aforementioned differences at 1200 ppm were not of sufficient magnitude to affect mean body weights or net body weight in this group, and therefore were considered test substance-related but nonadverse. Mean gravid uterine weight in the 1200 ppm group was similar to the control group. Mean maternal body weights, body weight gains, net body weights, net body weight gains, gravid uterine weights, and food consumption in the 300 and 600 ppm groups were unaffected by test substance exposure.

No test substance-related macroscopic findings were noted at the scheduled necropsy on gestation day 20. There were no test substance-related effects on brain or liver weights noted at 300, 600, and 1200 ppm.

 

Intrauterine growth and survival were unaffected by test substance exposure at all

exposure concentration levels. No test substance-related external, visceral, or skeletal

malformations or developmental variations were noted in the 300, 600, and 1200 ppm

groups.

 

There were no adverse effects of the test substance on maternal animals at 300, 600, or 1200 ppm. In addition, there were no adverse test substance-related effects on

intrauterine growth and survival and fetal morphology at any exposure level. Based on

these results, an exposure level of 1200 ppm, the highest exposure level tested, was

considered to be the no-observed-adverse-effect concentration (NOAEC) for maternal

toxicity and embryo/fetal development when methyl n-amyl ketone was administered via

whole-body inhalation for 6 hours daily to bred Crl:CD(SD) rats.