Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
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
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Twelve male and 12 female (nulliparous and non-pregnant) Sprague-Dawley rats [Crl:CD(SD)IGS BR] obtained from Charles River Laboratories, Stone Ridge (Kingston), NY were randomly assigned to each exposure group. The male and female rats were 63 or 66 days of age and weighed 324 ± 9 or 226 ± 9 grams (mean± SD), respectively, at the start of the study. Rats were chosen for these study because they are a common representative species for inhalation and developmental toxicity studies. They have a high fecundity and this strain is routinely used in rodent reproductive and developmental toxicity studies in our laboratory. Also, the rat is one of the two primary rodent species recommended for use in developmental toxicity
studies in the OECD Test Guideline

Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited vivarium in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). During nonexposureperiods, rats were singly housed in stainless-steel, wire-mesh cages in a room separate from the exposure room. No other study was housed in the same room as this study. Exposure cages were washed daily. Housing cages and racks were washed once a week. Absorbent paper, used to collect excreta, was changed daily.

The study room was maintained at 21.0- 24.0°C and 43.5- 68.5% relative humidity. A photoperiod of 12 hours light from 6 a.m. to 6 p.m. was maintained. Animals were isolated upon arrival and allowed to acclimate for a period of at least five days prior to assignment to this study. Animals were judged to be healthy prior to testing. Certified Rodent Diet [Purina Rodent Chow #5002, meal (P~ Feed, Inc. Richmond, IN] was available ad libitum except during exposure. Feed containers were cleaned and refilled at least once a week. No known contaminants which would interfere with the
outcome of this study were present in the feed. Water was available ad libitum, except during exposure, through an automatic watering
system. The source of the water was the local public water system. There have been no contaminants identified in periodic water analyses that would be expected to interfere with the conduct of the study. Upon arrival, all rats were identified by uniquely-numbered metal ear tags. Ear tags
which were lost during the study were replaced. During randomization, study-specific animal numbers were assigned to each animal. Cage cards, color-coded for each group, contained the study-specific animal number and the ear tag number.
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
Exposure concentrations were selected based on test results of a four-day probe study during which 3 male and 3 female rats per group were exposed to 5.0, 2.5, 1.0, or 0.0 mg/L of the test substance 6 hours/day for four days. All exposure conditions were as· described below. Significant toxicity was observed (minimal to minor reductions inactivity during exposure for the 5.0 mg/L group, reduced fecal volumes on Day 2 for
the 5.0 mg/L female rats, lower mean feed consumption between Days 0 and 1 for the 2.5 and 5.0 mg/L female rats) suggesting that these exposure concentrations could be tolerated during the main study without confounding interpretation of the study results, particularly with respect to pregnant animals. Therefore, exposure concentrations of 5.0, 2.5, 1.0, or 0.0 mg/L were selected for the reproduction/developmental toxicity screening
test.

The inhalation exposures were conducted in 590 L stainless-steel and glass inhalation chambers at target vapor concentrations of 5.0, 2.5, 1.0, and 0.0 mg/L. Animals were singly housed during the 6-hour exposures. The exposure chambers were maintained under negative pressure relative to room air. The air flow, temperature, and humidity were recorded approximately every 30 minutes. Chamber vapor concentrations were recorded at least once each hour. The test atmosphere was generated by metering the test substance into glass distillation columns packed with glass beads. Filtered, compressed air was passed through the glass bead-packed columns to evaporate the test substance. The test substance delivery rate and air flow rate were adjusted to produce the desired chamber target vapor concentration. The resultant vapor was directed via glass tubing to a tee just upstream of the inhalation chamber where it was mixed with filtered, conditioned outside air to produce a total airflow of 116 to 212 Lpm (12 to 22 air changes per hour). A Micro Laser Particle Counter (model JlLPC-301, Particle Measuring Systems, Inc., Boulder, CO) was used to measure the number and size of particulates in the chamber. The results indicated that an aerosol of the test substance was not present.

Once each week (Days 1, 8, 15, 22, 29, 36, 43, and 49), samples of chamber test atmosphere were collected into Tedlar bags. These samples were analyzed using a GC/FID. Daily MIRAN Vapor Concentration Determination Chamber vapor concentrations were monitored with a multipositional air sampling and analysis system. The system consisted of a single :MIRAN IA infrared gas analyzer (Wilks Foxboro Analytical, South Norwalk, CT) and a computer-operated four-port sampling valve (Valco Instruments, Houston, TX).Chamber vapor samples were continuously collected from each chamber through TEFLON tubing (0.48 mrn i.d.). The valve position was periodically changed to sample from each chamber at least once each hour. The voltage output of the MIRAN and chamber concentrations were printed in real-time and captured on electronic media. Voltage data were converted to concentration by linear interpolation between the calibration data points immediately on each side of the sampled data. A test to determine variations in concentration at different positions within the exposure chambers was conducted prior to study initiation. The air from the breathing zones of cage positions 1, 3, 7, 9, 14, 19, and 21 was sampled and compared to the concentration at a fixed reference position (cage 15). Based on deviations from the reference position of less than 10%, the chamber atmosphere was considered to be homogeneous.

Total chamber air flow was a combination of filtered, compressed air, which was used to vaporize the test substance and to carry the vapor from the generation system to the inhalation chamber, and additional filtered, compressed air dilution air. The air flow rate was continuously monitored using calibrated flowmeters (Gilmont Instruments, Barrington, IL). The oxygen content of the chamber exposure atmosphere was measured during exposure from the reference position using a MAS Mini OX Monitor (MAS Instrument Division, Pittsburgh, PA). The oxygen content of the chamber exposure atmosphere was~ 20%. Chamber temperature and humidity were measured using wet/dry bulb hygrometers and were recorded approximately every 30 minutes during exposure.
Details on mating procedure:
Male and female rats were mated 1:1 within the same dose group for 1 to 14 days. The study and ear tag numbers of the mated pairs were recorded. Copulation was verified through identification of sperm in vaginal smears or by appearance of a cqpulation plug. The morning of the day copulation was verified was considered Day 0 of gestation. Following copulation, the male and female rats were separated and housed individually until study termination. For female rats that showed no evidence of mating, the expected delivery date was calculated from the last day of possible copulation with a male.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Once each week (Days 1, 8, 15, 22, 29, 36, 43, and 49), samples of chamber test atmosphere were collected into Tedlar bags. These samples were analyzed using a GC/FID. Chamber vapor concentrations were monitored with a multipositional air sampling and analysis system. The system consisted of a single :MIRAN IA infrared gas analyzer (Wilks Foxboro Analytical, South Norwalk, CT) and a computer-operated four-port sampling valve (Valco Instruments, Houston, TX).
Chamber vapor samples were continuously collected from each chamber through TEFLON tubing (0.48 mrn i.d.). The valve position was periodically changed to sample from each chamber at least once each hour. The voltage output of the MIRAN and chamber concentrations were printed in real-time and captured on electronic media. Voltage data were converted to concentration by linear interpolation between the calibration data points immediately on each side of the sampled data.
Duration of treatment / exposure:
35-48 days for females (depending on the time for mating) and 51 days for males.
Frequency of treatment:
Daily
Details on study schedule:
The study consisted of four phases: pre-mating (14 days); mating (1 to 14 days); pregnancy (21 to 22 days); and early lactation (4 days). The female rats were exposed, 6 hours/day, through Day 19 of gestation (approximately 35-48 consecutive exposures). Females that delivered a litter, and their offspring, were euthanatized on Days 4 postpartum. Females showing no evidence of copulation were euthanatized 23 days after the last day of the mating period. Male rats were exposed, 6 hours/day, throughout the entire study (until all dams had littered or had been euthanatized due to non-delivery of a litter) for a total of 51 consecutive exposures. All male rats were euthanatized the following day. Mating male and female rats were mated 1: 1 within the same dose group for 1 to 14 days. The study and ear tag numbers of the mated pairs were recorded. Copulation was verified
through identification of sperm in vaginal smears or by appearance of a cqpulation plug. The morning of the day copulation was verified was considered Day 0 of gestation. Following copulation, the male and female rats were separated and housed individually until study termination. For female rats that showed no evidence of mating, the expected delivery date was calculated from the last day of possible copulation with a male.
Remarks:
Doses / Concentrations:
5 mg/l
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
2.5 mg/l
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0 mg/l
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
1 mg/l
Basis:
analytical conc.
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
The study consisted of four phases: pre-mating (14 days); mating (1 to 14 days); pregnancy (21 to 22 days); and early lactation (4 days). The female rats were exposed, 6 hours/day, through Day 19 of gestation (approximately 35-48 consecutive exposures). Females that delivered a litter, and their offspring, were euthanatized on Days 4 postpartum. Females showing no evidence of copulation were euthanatized 23 days after
the last day of the mating period. Male rats were exposed, 6 hours/day, throughout the entire study (until all dams had littered or had been euthanatized due to non-delivery of a litter) for a total of 51 consecutive exposures. All male rats were euthanatized the following day.
Positive control:
None
Parental animals: Observations and examinations:
Male and female rats were mated 1:1 within the same dose group for 1 to 14 days. The study and ear tag numbers of the mated pairs were recorded. Copulation was verified through identification of sperm in vaginal smears or by appearance of a cqpulation plug. The morning of the day copulation was verified was considered Day 0 of gestation. Following copulation, the male and female rats were separated and housed individually
until study termination. For female rats that showed no evidence of mating, the expected delivery date was calculated from the last day of possible copulation with a male.
Sperm parameters (parental animals):
On the day of necropsy, motility was determined for the right epididymis. The epididymis was placed in a petri dish containing 1% bovine serum albumin in phosphate buffered saline pre-warmed to ~38°C. The epididymis was pierced three times with a scalpel blade followed by a period of at least 3 minutes to allow the sperin to swim out. A sample of the mixture in the petri dish was then loaded into the pre-warmed stage of the Hamilton Thorne IVOS automated sperm analyzer. Five fields were automatically selected by the analyzer and each motion image was recorded and stored on the optical disk. The images were subsequently analyzed and the percent motility was determined for each animal.

Testicular homogenization-resistant spermatid head counts were performed on the frozen left testes. After the tissue was thawed, the tunic was removed from the testis, the tissue was weighed, homogenized, and then vortexed. A 100 ul sample was transferred to a violet reaction vial containing a dye which uniquely stains the head of the sperm. A sample of stained sperm was placed into a 20 um deep glass slide which was loaded into
the Hamilton Thome IVOS automated sperm analyzer. Twenty fields were automatically selected by the analyzer for each animal and total sperm counts were determined. Head counts were reported on an absolute and relative (to testes weight) basis. Epididymal homogenization-resistant spermatozoan counts were performed on the frozen left epididymides. The epididymal tissue was processed in a similar manner as with the testicular tissue, except that the caudal left epididymis was used. Head counts were reported on an absolute and relative (to epididymal weight) basis.
Postmortem examinations (parental animals):
Adult male rats were fasted overnight, anesthetized with carbon dioxide and exsanguinated by severing the posterior vena cava on Day 51. Adult female rats that showed no evidence of delivery of a litter were euthanatized on Day 23 or 24 of the gestation phase. All other adult female rats were anesthetized with carbon dioxide and exsanguinated by severing the posterior vena cava on Day 4 post-partum. Following exsanguination, the adult animals were weighed and subjected to a necropsy and gross examination. Special attention was paid to the organs of the reproductive system. The uteri from all adult female rats were examined, and implantation sites were counted. The following tissues were collected from adult animals and fixed in 10% buffered formalin: ovaries, vagina, uterus, fallopian tubes, and male accessory sex glands, and gross lesions. The right testis and right epididymis (after motility analysis) were fixed in Bouin's fixative; after approximately 24 hours, these tissues were rinsed twice with 50% ethyl alcohol then stored in 70% ethyl alcohol. The left testis and left epididymis were placed into individual containers, frozen, with dry ice, and stored at -70°C.
Statistics:
Mean values were calculated for time-weighted average atmospheric concentration, chamber temperature, chamber relative humidity, chamber airflow, chamber nominal concentration, adult body weight and body weight change, male pup and female pup body weight and body weight change, feed consumption, organ weights, organ-to-body weight ratios, gestation period, litter size, and pup percent survival. Homogeneity of adult body weight, feed consumption, and organ weight data were evaluated using Bartlett's test (p < 0.01). Adult body weight, feed consumption, and organ weight data were evaluated using a one-way analysis of variance (ANOVA) (p < 0.05) and Duncan's multiple range test (p < 0.05). Adult body weight change, male pup and female pup body weight and body weight change, gestation period, litter size, and pup percent survival were evaluated using Bartlett's test (p < 0.01), one-way analysis of variance
(ANOVA) (p < 0.05), and Dunnett's t-test (p < 0.05) to indicate statistical significance (MINITAB Statistical Software, State College, PA). When the variances of the means were not considered equal by the Bartlett's test (p <0.01), the data were evaluated using a Kruskal-Wallis H-test (p < 0.05) followed by Mann-Whitney U-test (p < 0.05) (MINITAB Statistical Software, State College, PA). The reproductive performance of the dams was evaluated in a contingency table, using a Chi-square test (p <0.05).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
minimal reductions in activity level at 5 mg/l only. Reduced activity is defmed as less movement, decreased alertness, and slower response to tapping on the chamber wall compared with activity levels exhibited by control animals.
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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
During exposure, the adult male and female rats exposed to 5.0 mg/L had minimal reductions in activity level. Reduced activity is defmed as less movement, decreased alertness, and slower response to tapping on the chamber wall compared with activity levels exhibited by control animals. All other adult animals appeared normal during exposure. No other test substance-related clinical abnormalities were observed. Incidental lesions consisted of alopecia, red discoloration of urine, superficial skin wounds, eschar formation, malocclusion, ocular or nasal porphyrin discharge, red discoloration of hair, and sialorrhea. One 2.5 mg/L male rat (#526) had severe hematuria, detected using a Multistix (a clinical dipstick designed for evaluation of urine samples), from Day 10 through study termination. At necropsy, the kidneys from Rat #526 appeared larger than normal. Microscopic evaluation of this animal's kidneys revealed hydronephrosis~ prominent granular deposits within the renal pelvis, as well as a focus of transitional epithelial mineralization.

Mean feed consumption values for all test substance-exposed male and female groups were comparable to those of the control groups throughout the study.

Mean body weights and body weight changes for all adult male test substance-exposed groups and mean body weights for all adult female exposed groups were comparable to those of the control groups throughout the study. The mean body weight change, between Days 0 and 14 of the pre-mating phase for the 1.0 mg/1 adult female group, was higher (p < 0.05) when compared with the control group. All other mean body weight changes for adult female exposed groups were comparable to those of the control group. The mean absolute, but not relative, epididymides weight for the 5.0 mg/l adult male group was higher (p < 0.05) when compared with the control group. Mean terminal body weights for adult male and female test substance-exposed groups and the mean absolute and relative testes weights for adult male exposed groups were comparable to those of the control group. No test substance-related lesions were observed at necropsy or on microscopic examination of collected tissues. See the pathologist's report beginning on page 60 for details of the gross and histopathology examinations.
Dose descriptor:
NOEC
Effect level:
2.5 mg/L air
Sex:
male/female
Basis for effect level:
other: NOEC was based upon reduced activity at 5 mg/l
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
For all examined parameters (rc~productive performance; gestation length; pup survival; prenatal loss; numbers of implants, live and dead pups, and male and female pups; and .pup body weight and pup body weight change), 1he test substance-exposed groups were comparable to the control
group. Abnormalities were observed in pups from all groups. These abnormalities included bruises under the skin, superficial skin wounds, bleeding from the toes, dried blood on the skin of the toes, eschar formation, purple tinge to the skin tone, and little or no milk in the stomach.
Additionally, pups were occasionally missing (presumably cannibalized) or found dead. These observations were distributed across all groups, and were not considered to be treatment-related.
Dose descriptor:
NOEC
Generation:
F1
Effect level:
5 mg/L air
Sex:
male/female
Basis for effect level:
other: The reproductive NOEC was based on no findings at the highest dose concentration in the P of F1 animals.
Reproductive effects observed:
not specified
Conclusions:
Exposure to the test substance resulted in reduced activity during exposure, for animals exposed to 5.0 mg/L. However, no adverse effects on reproduction or development were observed. Therefore, the no-observed-effect level (NOEL) for toxicity to adult animals was 2.5 mg/L and the NOEL for reproductive and developmental toxicity was determined to be 5.0 mg/L.
Executive summary:

The potential toxicity of the test substance was evaluated using a reproduction/developmental toxicity screening test. The test consisted of four phases: pre-mating (14 days), mating (1 to 14 days), gestation (21 to 22 days), and early lactation (4 days). Male and female Sprague-Dawley rats were exposed to concentrations of 5.0, 2.5, 1.0, or 0.0 mg/L of the test substance for six hours

per day, 7 days per week for a total of 35 to 48 exposures for female rats (through Day 19 of gestation) and 51 exposures for male rats. Exposure concentrations were selected based upon the results of a 4 day probe study that demonstrated minimal to minor reductions in activity during exposure for the 5.0 mg/L group, reduced fecal volume on Day 2 for the 5.0 mg/L female group,

and lower mean feed consumption between Days 0 and 1 for the 2.5 and 5.0 mg/L female rats. Both the mean analytical (GC/FID) and mean daily time-weighted average (MIRAN) concentrations were within 1.0 % of the target concentrations. The temperature and relative humidity inside the chambers during exposure were 21.2- 24.0°C and 52- 83%, respectively. All adult animals survived to study termination. Adult animals were observed for signs of toxicity prior to exposure, once per hour during exposure, and 30 minutes to one hour after exposure. Beginning on Day 20 of gestation (during the non-exposure period), each female rat was

removed from its cage and examined at least once daily. Pups were observed once daily. During exposure, reduced activity was observed for the adult 5.0 mg/L group. Adult animals from the 2.5, 1.0, and 0.0 mg/L groups appeared normal during exposure. No test substance-related clinical signs were observed when parental animals were examined outside of the chambers. No test substance-related changes in mean feed consumption, body weights, or body weight change were observed. At study termination, animals were anesthetized with carbon dioxide and exsanguinated. No test substance-related changes in mean terminal body weight or in reproductive organ weights were observed. Mean sperm motility and mean epididymal spermatozoan and testicular spermatid counts were comparable among the groups. No test substance-related gross pathology was observed for adult animals from any group. No exposure related changes were observed during histological examination of the reproductive organs of any of the test substance-exposed animals. No reproductive or developmental toxicity was observed and no test substance-related clinical

abnormalities were observed for the pups. In conclusion, exposure to the test substance resulted in reduced activity during exposure for animals exposed to 5.0 mg/L. However, no adverse effects on reproduction or development were observed. Therefore, the no-observed-effect level (NOEL) for toxicity to adult animals was 2.5 mg/L and the NOEL for reproductive and developmental toxicity was determined to be 5.0 mg/L.

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
5 000 mg/m³
Study duration:
subacute
Species:
rat
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 421 Reproductive Developmental Screen::

The potential toxicity of the test substance was evaluated using a reproduction/developmental toxicity screening test. The test consisted of four phases: pre-mating (14 days), mating (1 to 14 days), gestation (21 to 22 days), and early lactation (4 days). Male and female Sprague-Dawley rats were exposed to concentrations of 5.0, 2.5, 1.0, or 0.0 mg/L ofthe test substance for six hours

per day, 7 days per week for a total of 35 to 48 exposures for female rats (through Day 19 of gestation) and 51 exposures for male rats. Exposure concentrations were selected based upon the results of a 4 day probe study that demonstrated minimal to minor reductions in activity during exposure for the 5.0 mg/L group, reduced fecal volume on Day 2 for the 5.0 mg/L female group,

and lower mean feed consumption between Days 0 and 1 for the 2.5 and 5.0 mg/L female rats. Both the mean analytical (GC/FID) and mean daily time-weighted average (MIRAN) concentrations were within 1.0 % of the target concentrations. The temperature and relative humidity inside the chambers during exposure were 21.2- 24.0°C and 52- 83%, respectively. All adult animals survived to study termination. Adult animals were observed for signs of toxicity prior to exposure, once per hour during exposure, and 30 minutes to one hour after exposure. Beginning on Day 20 of gestation (during the non-exposure period), each female rat was

removed from its cage and examined at least once daily. Pups were observed once daily. During exposure, reduced activity was observed for the adult 5.0 mg/L group. Adult animals from the 2.5, 1.0, and 0.0 mg/L groups appeared normal during exposure. No test substance-related clinical signs were observed when parental animals were examined outside of the chambers. No test substance-related changes in mean feed consumption, body weights, or body weight change were observed. At study termination, animals were anesthetized with carbon dioxide and exsanguinated. No test substance-related changes in mean terminal body weight or in reproductive organ weights were observed. Mean sperm motility and mean epididymal spermatozoan and testicular spermatid counts were comparable among the groups. No test substance-related gross pathology was observed for adult animals from any group. No exposure related changes were observed during histological examination of the reproductive organs of any of

the test substance-exposed animals. No reproductive or developmental toxicity was observed and no test substance-related clinical

abnormalities were observed for the pups. In conclusion, exposure to the test substance resulted in reduced activity during exposure for animals exposed to 5.0 mg/L. However, no adverse effects on reproduction or development were observed. Therefore, the no-observed-effect level (NOEL) for toxicity to adult animals was 2.5 mg/L and the NOEL for reproductive and developmental toxicity was determined to be 5.0 mg/L.

2 -Generation Reproductive study:

In accordance with Annex IX of REACH, a 2 -generation study is not required at this tonnage unless existing testing indicates a need, and that need has not been demonstrated.

Effects on developmental toxicity

Link to relevant study records
Reference
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
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
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.
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.
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.
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.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No maternally toxic effects
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
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
5 284 mg/m³
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

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.


Justification for selection of Effect on developmental toxicity: via inhalation route:
The study was done in a GLP facility to OECD guidelines

Justification for classification or non-classification

Available test data does not support classification