1-butylpyrrolidin-2-one

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-07-19 to 2016-10-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-butylpyrrolidin-2-one

EC Number:

222-437-8

EC Name:

1-butylpyrrolidin-2-one

Cas Number:

3470-98-2

Molecular formula:

C8H15NO

IUPAC Name:

1-butylpyrrolidin-2-one

Test material form:

liquid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test substance was stored at room temperature under a nitrogen blanket, and was considered stable under these conditions.
- Stability under test conditions: Documentation regarding the purity and stability of the test substance is on file with the Sponsor and Charles River.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: No

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:CD(SD)
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 75 days old upon receipt; The selected females were approximately 12 weeks old when paired for breeding.
- Weight at study initiation: Body weight values of the selected females ranged from 230 g to 284 g on Gestation Day 0.
- Fasting period before study: no
- Housing: Upon arrival and until pairing, all rats were individually housed in clean, solid-bottom cages with bedding material (Bed-O'Cobs®; The Andersons, Cob Products Division, Maumee, OH).
- Diet (e.g. ad libitum): ad libitum except during exposure periods (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 Charles River).
- Water (e.g. ad libitum): ad libitum except during exposure periods (Reverse osmosis-purified (on-site) drinking water)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Non-exposure periods: 68°F to 78°F (20°C to 26°C); actual mean daily temperature ranged from 72.3°F to 74.1°F (22.4°C to 23.4°C) during the study. Exposure periods: The mean temperature was between 19°C to 25°C.
- Humidity (%): Non-exposure periods: 30% to 70%; actual mean daily relative humidity ranged from 45.4% to 59.0% during the study. Exposure periods: The mean relative humidity was between 30% to 70%.
- Air changes (per hr): Non-exposure periods: 10. Exposure periods: All chambers were operated under dynamic conditions, at least 12-15 air changes per hour, at a slight negative pressure.
- Photoperiod (hrs dark / hrs light): 12/12

OTHER
- Oxygen content was measured during the method development phase and was 20.9% for Groups 2-4.

IN-LIFE DATES: From: 02 Aug 2016 (animal receipt) To: 02 Sep 2016 (last laparohysterectomy).

Administration / exposure

Route of administration:

inhalation: mixture of vapour and aerosol / mist

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 2.4 - <= 2.7 µm

Geometric standard deviation (GSD):

1.73

Remarks on MMAD:

The target range for MMAD is 1.0 to 3.0 microns and GSD is 1.5 to 3.0. Aerosol particle-size measurements were conducted at least once per week for each test substance chamber. Mean MMAD and GSD were within the target values (table 10). MMAD values were 2.7, 2.4 and 2.4 for the substance-treated groups 2, 3 and 4, respectively. GSD were of 1.81, 1.78 and 1.73 for the substance-treated groups 2, 3 and 4, respectively.

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

- Exposure apparatus: Exposures were conducted using four 1000-L glass and stainless steel whole-body inhalation exposure chambers. One exposure chamber was dedicated for each group for the duration of the study.

- Method of holding animals in test chamber: 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 animal cage batteries were rotated on a daily basis between the 3 battery positions within the chamber to help ensure a similar exposure for all animals within each group over the duration of the exposure period.

- Source and rate of air: Chamber supply air was provided from a HEPA- and charcoal-filtered, temperature- and humidity-controlled source.

- Method of conditioning air: not reported.

- System of generating particulates/aerosols: An individual generation system was used for each test substance exposure chamber. A mixed liquid aerosol/vapor atmosphere of the test substance was generated as follows. During generation trials as part of the range-finding study, it was noted that the test substance was not compatible with a plastic clinical nebulizer, but it was compatible with the glass jar of a Collison nebulizer. The test substance was aerosolized using 1 or more Collison nebulizers. A 6-jet nebulizer was used for Chamber 2 and Chamber 3, and two 6-jet nebulizers were used for Chamber 4. Using a regulator, breathing quality compressed air from the facility in-house air source was delivered to each nebulizer. Test substance was delivered to the nebulizers through the use of an infusion pump.
For Chamber 4, the resulting liquid aerosol/vapor atmosphere from the 6-jet nebulizers were combined at a 4-way ‘cross’-fitting where it was mixed with dry, compressed air. For Chambers 2 and 3, the resulting liquid aerosol/vapor atmosphere from the respective nebulizer was mixed with additional dry, compressed air at a ‘T’-fitting. The amount of compressed air for Chambers 2, 3, and 4 were controlled using a regulator.
In order to produce an exposure atmosphere with an appropriate particle-size, the resulting liquid aerosol/vapor mixture for all test substance chambers was first directed through a glass cyclone in order to reduce the particle-size to under 3 microns. The output from each cyclone was delivered to the chamber inlet, where it was mixed with supply air to achieve the desired target concentration of the test substance.

- Temperature, humidity, pressure in air chamber: Chamber ventilation rate and negative pressure within the chambers were continually monitored and recorded approximately every 45 minutes during the 6-hour exposure periods. Temperature and relative humidity within each exposure chamber were determined at least 3 times during each period. The mean temperature and mean relative humidity were to be between 19°C to 25°C and 30% to 70%, respectively. Oxygen content was measured during the method development phase and was 20.9% for Groups 2-4.

- Air flow rate: The airflow rates for each whole-body chamber were monitored by measuring the pressure drop between the ports of an orifice plate using a Dwyer Magnehelic® Indicating Transmitter pressure gauge. Each gauge was calibrated for conversion from pressure to airflow in standard liters per minute through the use of a Fox Gas Mass Flowmeter Transmitter (model no. FT2, Fox Thermal Instruments; Marina, CA). Sample flow through the sample train was controlled using a needle valve connected to the facility vacuum source. Prior to each sample collection, the sample flow rate was measured using a Mini-Buck Calibrator (model no. M-5, A.P Buck Inc.; Orlando, FL). An approximate sample flow rate was 2.0 L/min for 8, 2.5, and 1.5 minutes for Chambers 2, 3, and 4, respectively. Chamber ventilation rate (airflow) and negative pressure within each exposure chamber were continually monitored and recorded at approximately 45-minute intervals through the use of the Inhalation Toxicology Data Acquisition System (WINH) and a personal computer.

- Air change rate: All chambers were operated under dynamic conditions, at least 12-15 air changes per hour, at a slight negative pressure.

- Method of particle size determination: Aerosol particle-size measurements were conducted at least once per week for each test substance chamber. Aerosol particle-size measurements were conducted using a 7-stage stainless-steel cascade
impactor (model no. 02-100-2L, IN-TOX Products; Moriarty, NM). Pre-weighed, 22-mm stainless-steel collection substrates were used for stage 1 to 7. A pre-weighed, 25-mm glass fiber filter (Type A/E, PALL Corporation) was used as the final collection substrate. Aerosol particle-size measurements were conducted at least once per week for each test substance chamber. Samples of the mixed liquid aerosol/vapor atmosphere of test substance were collected at an approximate sample flow rate of 2.0 LPM for 8, 2.5, and 1.5 minutes for Chambers 2, 3, and 4, respectively. Sample flow rates were measured using a Mini-Buck Calibrator (model no. M-5). Following sample collection, the aerosol retained on the filters was determined by re-weighing the filters and the particle-size was calculated based on the impactor stage cut-offs. The particle-size was expressed as the mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD).

- Treatment of exhaust air: All test substance atmosphere chamber exhaust passed through an activated-carbon drum prior to passing through the facility exhaust system, consisting of redundant exhaust blowers preceded by activated-charcoal and HEPA-filter units.

TEST ATMOSPHERE
- Brief description of analytical method used: Following each sample collection, aerosol content was measured using standard gravimetric methods and the vapor concentration was determined using analytical techniques (liquid-phase injection gas chromatograph (GC) with flame ionization detection). The filter was re-weighed and the aerosol concentration (mg/L) was calculated by dividing the gravimetrically determined mass of test substance aerosol by the sample volume.
- Samples taken from breathing zone: yes (the description please see below in "Details on analytical verification of doses or concentrations".

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 used during each generation period and the total volume of air that passed through the chamber during exposure. Test substance usage was determined by weighing pre-filled syringes prior to and at the termination of each generation. Total air volume was calculated by multiplying the daily mean ventilation rate by the duration of generation.

Samples of the exposure atmospheres were collected using a sampling train consisting of a pre-weighed, glass fiber filter in-line with a 30-mL midget impinger at approximately 60-minute intervals. The filter was held in a filter holder and placed in the animal-breathing zone of the chamber. The impinger was filled with approximately 10 mL of isopropanol as the trapping liquid. Prior to each sample collection, the sample flow rate was measured using a calibrator.
Aerosol concentrations
Following each sample collection, aerosol content was measured using standard gravimetric methods and the vapor concentration was determined using analytical techniques. The filter was re-weighed and the aerosol concentration (mg/L) was calculated by dividing the gravimetrically determined mass of test substance aerosol by the sample volume.
Vapor Concentrations
Analyzed concentrations of the test substance vapor in the exposure atmospheres were determined using a liquid-phase injection gas chromatograph (GC) with flame ionization detection.
Following sample collection, a portion of the impinger liquid was transferred to a 2-mL vial and analyzed using a GC. Liquid sample injection onto the chromatography column occurred via manual injection (approximately 2 μL) into a split/splitless injector, the chromatograph was displayed, and the area under the sample peak was calculated and recorded. Test substance concentration within the impinger liquid, in μg/mL, was calculated using the ln-quadratic equation based on the GC calibration curve. The total amount of vaporized test substance recovered was determined by multiplying the impinger liquid concentration by the impinger solvent volume (10 mL). The vapor concentration was determined by dividing the amount of test substance recovered by the sample volume.
Total Test Substance Concentrations
Total test substance (combined aerosol and vapor) was calculated by addition of the gravimetrically and analytically determined concentrations.

Details on mating procedure:

- Impregnation procedure: cohoused
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.
- If cohoused:
- M/F ratio per cage: 1
- Length of cohabitation: not reported.
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

6 hours

Frequency of treatment:

once daily

Duration of test:

during Gestation Days 6–19.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.3 mg/L air (nominal)

Remarks:

Mean analyzed exposure concentrations was 0.29 mg/L and corresponded to mean estimated inhaled (delivered) dosage level 76.2 mg/kg/day.

Doses / concentrations 2

Dose / conc.:

0.6 mg/L air (nominal)

Remarks:

Mean analyzed exposure concentrations was 0.58 mg/L and corresponded to mean estimated inhaled (delivered) dosage level 152.6 mg/kg/day.

Doses / concentrations 3

Dose / conc.:

1.2 mg/L air (nominal)

Remarks:

The highest exposure concentration of 1.2 mg/L was the maximum achievable concentration that corresponded to a respirable particle size (< 3 microns, as required by the OPPTS 870.3465 and OECD 413 guidelines). The mean analyzed exposure concentration was 1.2 mg/L and corresponded to mean estimated inhaled (delivered) dosage levels of 315.8 mg/kg/day.

No. of animals per sex per dose:

24

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: 2-week range-finding study.
In that study, the test substance was administered via whole-body inhalation exposure, 6 hours per day, for 14 consecutive days to 3 groups of 5 female Crl:CD(SD) rats. As part of the generation trials for this study, attempts were made to produce a maximum achievable concentration that corresponded to a respirable particle size (< 3 microns, as required by the OPPTS 870.3465 and OECD 413 guidelines). It was estimated that the highest exposure concentration that met this criteria would be 1.0 mg/L. Therefore, target exposure concentrations were 0.25, 0.5, and 1.0 mg/L for Groups 2, 3, and 4, respectively. Mean exposure concentrations (total test substance; aerosol plus vapor) over the duration of the exposure period were 0.29, 0.64, and 1.23 mg/L for the same respective groups. Minor adjustments to generation settings (nebulizer pressure and/or dilution airflow) allowed the actual achieved concentration to be approximately 1.2 mg/L and consistently maintain a particle size under 3 microns (achieved 2.6 microns).
All animals survived to the scheduled necropsy. There were no test substance-related effects on body weights, food consumption, macroscopic pathology, or lung weights in any of the test substance-exposed groups. Clinical observations noted in the 1.0 mg/L group included partial closure of the right and left eyes, impaired equilibrium, and increased respiration rate. Test substance-related clinical observations of rales were noted in the 0.5 and 1.0 mg/L groups. Observations of rales were noted beginning as early as Study Day 3 in the 1.0 mg/L group and continued through the remainder of the study period. An animal in the 0.5 mg/L group was observed with rales beginning on Study Day 4.
Based on the lack of dose-limiting toxicity observed at up to 1.2 mg/L, exposure concentrations of 0.3, 0.6, and 1.2 mg/L were chosen for this study. The selected route of administration for this study was inhalation exposure because this is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.
- Rationale for animal assignment (if not random): 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 Charles River rat colony.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual clinical observations were recorded daily during Gestation Days 0–20 (prior to exposure during the treatment period). Animals were also observed for signs of toxicity at approximately the midpoint of each 6-hour exposure and approximately 1 hour following exposure. The absence or presence of findings was recorded for all animals.

BODY WEIGHT: Yes
- Time schedule for examinations: 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.
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.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes. Individual food consumption was recorded on Gestation Days 0 and 6–20 (daily).
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined:
The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the postmortem findings were correlated with the antemortem observations, and any abnormalities were recorded. Wet lung weights were recorded for all females at the scheduled necropsy, and the lungs were subsequently placed in 10% neutral-buffered formalin for possible future histopathological examination. Other maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings; 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:

The ovaries and uterine content was examined after termination: Yes
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.
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

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.

- External examinations: Yes: all per litter. 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.

- Soft tissue examinations: Yes: all per litter. 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.

- Skeletal examinations: Yes: all per litter. Following fixation in alcohol, each fetus was stained with Alizarin Red S7 and Alcian Blue8. Fetuses were then examined for skeletal malformations and developmental variations.

- Head examinations: Yes: all per litter. 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.

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

Indices:

Gestation Day 20 Laparohysterectomy
Intrauterine data were summarized using 2 methods of calculation. An example of each method of calculation follows:
1. Group Mean Litter Basis:
Postimplantation Loss/Litter = (No. Dead Fetuses, Resorptions (Early/Late)/Group)/ No. Gravid Females/Group

2. Proportional Litter Basis:
Summation Per Group (%) = Sum of Postimplantation Loss/Litter (%)/ No. Litters/Group;

Where:
Postimplantation Loss/Litter (%) = (No. Dead Fetuses, Resorptions (Early/Late)/Litter)/ No. Implantation Sites/Litter x 100

Fetal Morphological Examination
The fetal developmental findings were summarized by:
1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and
2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as follows:
Summation per Group (%) = (Sum of Viable Fetuses Affected/Litter (%))/ No. Litters/Group

Where:
Viable Fetuses Affected/Litter (%) = (No. Viable Fetuses Affected/Litter)/ No. Viable Fetuses/Litter x 100

Historical control data:

Charles River has historical data on the background incidence of fetal malformations and developmental variations in the Crl:CD(SD) rat (Number of Datasets in Historical Control: 79-80). This animal model has been proven to be susceptible to the effects of developmental toxicants.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related, increased incidences of red material around the nose, mouth, and forelimbs was noted in the 0.6 and 1.2 mg/L groups generally throughout the treatment period; these observations were noted primarily at approximately 1 hour following exposure at 0.6 mg/L and at the daily examinations and approximately 1 hour following exposure at 1.2 m/L. The aforementioned observations were considered adverse at 1.2 mg/L; because these findings were generally resolved by the following daily examination in the 0.6 mg/L group, they were considered nonadverse at this exposure level. In the 1.2 mg/L group, an increased incidence of clear material on the ventral neck was noted primarily during Gestation Days 16–20 at the daily examinations and/or approximately 1 hour following exposure and was considered test substance-related and adverse. No other test substance-related clinical observations were noted at the daily examinations, midpoint of exposure, or approximately 1 hour following exposure at any exposure level. Observations noted in the exposed groups, including hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not exposure-related.

Mortality:

no mortality observed

Description (incidence):

All females in the control, 0.3, 0.6, and 1.2 mg/L groups survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 1.2 mg/L group, test substance-related mean body weight losses or lower mean body weight gains were noted throughout the exposure period (Gestation Days 6–9, 9–12, 12–15, and 15–20) and resulted in a lower mean body weight gain when the entire exposure period (Gestation Days 6–20) was evaluated compared to the control group; differences were generally significant (p < 0.05 or p < 0.01). In addition, mean body weights in this group were lower (5.4% to 11.5%; significant at p < 0.01) than the control group during Gestation Days 8–20. Lower mean net body weight and net body weight gain were noted in the 1.2 mg/L group compared to the control group; differences were significant (p < 0.01). In addition, a significantly (p < 0.01) lower mean gravid uterine weight was noted in the 1.2 mg/L group compared to the control group. The aforementioned effects noted in the 1.2 mg/L group were considered adverse.
In the 0.3 and 0.6 mg/L groups, significantly (p < 0.01) lower mean body weight gains were noted during Gestation Days 6–9 compared to the control group. Mean body weight gains in these groups were similar to the control group for the remainder of the exposure period (Gestation Days 9–12, 12–15, and 15–20). Due the decrements in mean body weight gain noted at the beginning of the exposure period, a significantly (p < 0.01) lower mean body weight gain was noted in the 0.6 mg/L group compared to the control group when the entire exposure period (Gestation Days 6–20) was evaluated; mean body weight gain in the 0.3 mg/L group was similar to the control group for this interval. The lower mean body weight gains noted in the 0.3 and 0.6 mg/L groups were not of sufficient magnitude to affect mean body weights at these exposure levels, and therefore were considered nonadverse. Mean maternal net body weights, net body weight gains, and gravid uterine weights in the 0.3 and 0.6 mg/L groups were unaffected by test substance exposure.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test substance-related, lower mean maternal food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 1.2 mg/L group throughout the exposure period (Gestation Days 6–9, 9–12, 12–15, and 15–20) and resulted in lower mean food consumption in this group compared to the control group when the entire exposure period (Gestation Days 6–20) was evaluated; differences were significant (p < 0.01). The lower mean food consumption in the 1.2 mg/L group correlated with the mean body weight losses and lower mean body weight gains and was considered adverse.
Test substance-related, lower mean food consumption was noted during Gestation Days 6–9 in the 0.3 mg/L group and during Gestation Days 6–9, 9–12, and 12–15 in the 0.6 mg/L group compared to the control group; differences were generally significant (p < 0.05 or p < 0.01). Mean food consumption was similar to the control group during Gestation Days 9–12, 12–15, and 15–20 at 0.3 mg/L and during Gestation Days 15–20 at 0.6 mg/L. As a result of the initial decrements in mean food consumption, significantly (p < 0.05 or p < 0.01) lower mean food consumption was noted in these groups compared to the control group when the entire exposure period (Gestation Days 6–20) was evaluated. However, the differences noted in the 0.3 and 0.6 mg/L groups were not of sufficient magnitude to affect mean body weights in these groups, and therefore were considered nonadverse.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test substance-related effects on mean lung weights were noted in the 0.3, 0.6, and 1.2 mg/L groups. Differences from the control group were slight and not statistically significant.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At the scheduled necropsy on Gestation Day 20, no test substance-related internal findings were observed at exposure levels of 0.3, 0.6, and 1.2 mg/L. Macroscopic findings observed in the test substance-exposed groups occurred infrequently and/or in a manner that was not exposure-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance exposure at exposure levels of 0.3, 0.6, and 1.2 mg/L.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance exposure at exposure levels of 0.3, 0.6, and 1.2 mg/L. Parameters evaluated included postimplantation loss, live litter size, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

see above

Early or late resorptions:

no effects observed

Description (incidence and severity):

see above

Dead fetuses:

no effects observed

Description (incidence and severity):

see above

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

With the exception of 2 and 1 females in the control and 0.6 mg/L groups, respectively, all females were determined to be gravid.

Other effects:

not specified

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

0.6 mg/L air (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean male, female, and combined fetal weights in the 1.2 mg/L group (3.5 g, 3.3 g, and 3.4 g, respectively) were significantly (p < 0.01) lower (approximately 13%) than the control group values (4.0 g, 3.8 g, and 3.9 g, respectively) and were also below the minimum mean values in the Charles River Ashland historical control data (version 2016.03; 3.682 g, 3.458 g, and 3.573 g, respectively). The lower mean fetal weights at 1.2 mg/L correlated with decrements in maternal mean body weight gain and food consumption noted in this group during the fetal period (Gestation Days 15–20), which is the time of greatest fetal growth, and were considered adverse.
Lower (approximately 5%) mean male, female, and combined fetal weights were noted in the 0.3 and 0.6 mg/L groups; differences were generally significant (p < 0.05 or p < 0.01). However, the values at 0.3 and 0.6 mg/L (3.8 g for males, 3.6 g for females, and 3.7 g for combined) were greater than the minimum mean values and only 2% to 3% lower than the mean (3.898 g for males, 3.697 g for females, and 3.801 g for combined) and median (3.883 g for males, 3.696 g for females, and 3.792 g for combined) values within the Charles River Ashland historical control data. The concurrent control group values for fetal body weight (4.0 g for males, 3.8 g for females, and 3.9 g for combined) were 2% to 3% heavier (approximately equal to the 75th quartile; 3.971 g for males, 3.761 g for females, and 3.861 g for combined) than the mean values in the Charles River Ashland historical control data. Therefore, the lower mean fetal body weights in the 0.3 and 0.6 mg/L groups were due to a combination of slightly heavier fetuses in the concurrent control group and slightly lighter fetuses in the test substance-treated groups. The absence of a dose-response relationship between the 0.3 and 0.6 mg/L group fetuses also suggests a lack of effect at these exposure concentrations. In addition, the magnitude of change in these groups compared to the control group was not as severe as the difference in the 1.2 mg/L group. Therefore, the lower mean fetal weights at 0.3 and 0.6 mg/L were not considered test substance-related.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Intrauterine survival was unaffected by test substance exposure at exposure levels of 0.3, 0.6, and 1.2 mg/L. Parameters evaluated included postimplantation loss, live litter size, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

see above

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

see above

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

The numbers of fetuses (litters) available for morphological evaluation were 323(22), 358(24), 339(23), and 350(24) in the control, 0.3, 0.6, and 1.2 mg/L groups, respectively. Malformations were observed in 2(2), 2(2), 1(1), and 4(3) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin.
No test substance-related external malformations were observed for fetuses at any exposure level. Fetal anasarca was noted for 1 fetus each in the control (No. 6130-16) and 0.3 (No. 6082-07) mg/L groups. In the 0.3 mg/L group, Fetus No. 6094-16 was noted with anophthalmia (bilateral; confirmed skeletally as orbits smaller than normal). The aforementioned malformations were noted in single fetuses, similarly in the control group, and were not observed in an exposure-related manner; therefore, they were not considered test substance-related.
No external developmental variations were observed in fetuses in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related skeletal malformations were noted for fetuses at any exposure level. In the 1.2 mg/L group, 2 fetuses (Nos. 6025-10 and 6113-02) were noted with costal cartilage anomaly (bifurcated or fused costal cartilage) and 1 fetus (No. 6113-07) was noted with 8 cervical vertebrae (with normal presacral vertebrae count). Fetus No. 6065-03 in the 1.2 mg/L group was noted with severely malaligned sternebrae; this finding was also noted for 1 fetus (No. 6089-14) in the control group. Sternoschisis (no. 4 or no. 6 sternal bands not joined) was noted for 1 fetus each in the control (No. 6130-16) and 0.6 (No. 6076-04) mg/L groups. The aforementioned skeletal malformations were observed in single fetuses, similarly in the control group, were not observed in an exposure-related manner, and/or the differences in the mean litter proportions were not statistically significantly different from the concurrent control group and the values were within the ranges of the Charles River Ashland historical control data; therefore, they were not considered test substance-related.
Test substance-related skeletal developmental variations were noted in the 1.2 mg/L group. In the 1.2 mg/L group, a significantly (p < 0.01) lower mean litter proportion of cervical centrum no. 1 ossified was noted compared to the control group (3.4% versus 27.3% per litter). In addition, a higher (not statistically significant) mean litter proportion of sternebra(e) nos. 5 and/or 6 unossified was noted in the 1.2 mg/L group (23.1% per litter) compared to the control group (9.4% per litter); the value at 1.2 mg/L exceeded the maximum mean value in the Charles River Ashland historical control data (19.86% per litter). The aforementioned findings noted at 1.2 mg/L were considered indicators of developmental delay and correlated with the reduced fetal weights and decrements in maternal food consumption and body weight gain noted at this exposure level.
No other test substance-related skeletal developmental variations were noted at any exposure level. Other findings observed in the test substance-exposed groups were noted infrequently, similarly in the control group, were not observed in an exposure-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.

Visceral malformations:

no effects observed

Description (incidence and severity):

No test substance-related visceral malformations were observed for fetuses at any exposure level. In the 0.3 mg/L group, Fetus No. 6094-16 was noted with an interventricular septal defect (a <1 mm in diameter opening in the anterior portion of the septum); this fetus was also noted with an external malformation. Because this finding was noted in a single fetus and did not occur in an exposure-related manner, this finding was not considered test substance-related. In the control group, Fetus No. 6130-16 was noted with a bulbous aorta (ascending and aortic arches).
No test substance-related visceral developmental variations were noted. Findings observed in the test substance-exposed groups were noted infrequently, similarly in the control group, were not observed in an exposure-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.
Renal papilla(e) not fully developed (Woo and Hoar Grade 1) was noted for 2 (Nos. 6020-12 and 6071-13), 1 (No. 6095-14), 1 (No. 6047-06), and 1 (No. 6110-13) fetuses in the control, 0.3, 0.6, and 1.2 mg/L groups, respectively. This finding was not classified as either a malformation or developmental variation, was not included on the summary tables, and was not considered to be test substance-related because it occurred infrequently, at similar frequencies in the control group, and in a manner that was not exposure-related.

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

The numbers of fetuses (litters) available for morphological evaluation were 323(22), 358(24), 339(23), and 350(24) in the control, 0.3, 0.6, and 1.2 mg/L groups, respectively. Malformations were observed in 2(2), 2(2), 1(1), and 4(3) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Fetal malformations, when observed in the test substance-exposed groups, occurred infrequently or at a frequency similar to that in the control group, did not occur in an exposure-related manner, and/or were within the Charles River Ashland historical control data ranges. Based on these data, no fetal malformations were attributed to the test substance.
Test substance-related fetal skeletal developmental variations were noted in the 1.2 mg/L group. A higher mean litter proportion of sternebra(e) nos. 5 and/or 6 unossified and a lower mean litter proportion of cervical centrum no. 1 ossified were noted in the 1.2 mg/L group. These findings were considered secondary to the reduced fetal weights and decrements in maternal food consumption and body weight gain noted at this exposure level. No other test substance-related developmental variations were noted at any exposure level.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

0.6 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

other: Test substance-related fetal skeletal developmental variations

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Localisation:

skeletal: sternum

Description (incidence and severity):

Variations: In the 1.2 mg/L group, a test substance-related higher mean litter proportion of sternebra(e) nos. 5 and/or 6 unossified and lower mean litter proportion of cervical centrum no. 1 ossified were noted compared to the control group; these findings were considered indicators of developmental delay and correlated with the reduced fetal weights and decrements in maternal food consumption and body weight gain noted at this exposure level.

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

1.2 mg/L air (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Table 4. Overall Nominal Exposure Concentrations

Exposure Chamber:	2	3	4
Target Concentration (mg/L):	0.30	0.60	1.2
Nominal Concentration (mg/L):	1.0	1.8	4.9
Standard Deviation:	0.11	0.12	1.13
N:	17	17	17

The overall mean nominal concentrations for each exposure group are presented below. Due to the use of glass cyclones, a large amount of liquid test substance was removed from the generation atmosphere prior to entering the exposure chamber. This resulted in a greater calculated nominal concentration.

The overall mean analyzed exposure concentrations for each group are presented below:

Table 5. Overall Mean Exposure Concentrations

Exposure Chamber:	1	2	3	4
Target Concentration (mg/L):	0	0.3	0.6	1.2
Mean Concentration (mg/L):	0	0.29	0.58	1.2
Standard Deviation:	0.0	0.031	0.035	0.05
N:	5	17	17	17

The overall mean aerosol particle size for each test substance-treated group is presented below:

Table 6. Mean Aerosol Particle-Size

Exposure Chamber:	2	3	4
Mean MMAD (microns):	2.7	2.4	2.4
Mean GSD:	1.81	1.78	1.73
N:	3	3	3

The mean estimated inhaled (delivered) dose and the mean deposited dose for each interval and the overall exposure period are presented below. The deposited dose will be estimated at 10% of the estimated inhaled dose.

Table 7. Mean Estimated Delivered Dosage

Exposure Chamber:	1	2	3	4
Target Concentration (mg/L):	0	0.3	0.6	1.2
Mean Concentration (mg/L):	0	0.29	0.58	1.2
Females (mg/kg/day):	0.0	76.2	152.6	315.8
Deposited Dose (mg/kg/day):	0.0	7.62	15.26	31.58

Applicant's summary and conclusion

Conclusions:

Target exposure concentrations for the current study were 0.3, 0.6, and 1.2 mg/L; the highest exposure concentration of 1.2 mg/L was the maximum achievable concentration that corresponded to a respirable particle size (< 3 microns, as required by the OPPTS 870.3465 and OECD 413 guidelines).
Maternal toxicity was evidenced by adverse clinical observations and mean body weight losses and lower mean body weight gains with corresponding lower mean food consumption in the 1.2 mg/L group throughout the gestation treatment period. Developmental effects were noted at 1.2 mg/L as evidenced by lower mean fetal weights, which correlated with a lower mean gravid uterine weight and decrements in maternal food consumption and body weight gain during the fetal period (Gestation Days 15-20). Lower mean fetal weights were also noted in the 0.3 and 0.6 mg/L groups; however, mean fetal weight values in these groups were greater than the minimum mean values and only slightly lower than the mean and median values in the Charles River Ashland historical control data. In addition, the concurrent control group values for fetal body weight were 2% to 3% heavier (approximately equal to the 75th quartile) than the mean values in the Charles River Ashland historical control data. Therefore, the lower mean fetal body weights at 0.3 and 0.6 mg/L were due to a combination of slightly heavier fetuses in the concurrent control group and slightly lighter fetuses in the test substance-treated groups, and were not considered test substance-related. Test substance-related fetal skeletal developmental variations (higher mean litter proportion of sternebra(e) nos. 5 and/or 6 unossified and lower mean litter proportion of cervical centrum no. 1 ossified) were noted in the 1.2 mg/L group, which corresponded with the lower mean fetal weights noted in this group. No adverse maternal or developmental effects were noted at 0.3 and 0.6 mg/L. Based on these results, an exposure level of 0.6 mg/L was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal development when the test substance was administered via whole-body inhalation exposure to bred Crl:CD(SD) rats. Test substance exposure concentrations of 0.3, 0.6, and 1.2 mg/L corresponded to mean estimated inhaled (delivered) dosage levels of 76.2, 152.6, and 315.8 mg/kg/day, respectively.

Executive summary:

The objectives of the study were to determine the potential of the test substance 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 level (NOAEL) for maternal toxicity and developmental toxicity. The test substance was administered via whole-body inhalation exposure to 3 groups of 24 bred female Crl:CD(SD) rats for 6 hours per day from Gestation Days 6–19. Based on a range-finding study in nonpregnant female rats, target exposure concentrations for the current study were 0.3, 0.6, and 1.2 mg/L; the highest exposure concentration of 1.2 mg/L was the maximum achievable concentration that corresponded to a respirable particle size (< 3 microns, as required by the OPPTS 870.3465 and OECD 413 guidelines). Mean analyzed exposure concentrations were 0.29, 0.58, and 1.2 mg/L for the same respective groups. Test substance exposure concentrations of 0.3, 0.6, and 1.2 mg/L corresponded to mean estimated inhaled (delivered) dosage levels of 76.2, 152.6, and 315.8 mg/kg/day, respectively. A concurrent control group composed of 24 bred females were exposed to humidified, filtered air on a comparable regimen. The females were approximately 13 weeks of age at the initiation of exposure. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On Gestation Day 20, a laparohysterectomy was performed on each female and the lungs 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, 0.3, 0.6, and 1.2 mg/L groups survived to the scheduled necropsy. Test substance-related, increased incidences of red material around the nose, mouth, and forelimbs were noted in the 0.6 and 1.2 mg/L groups generally throughout the treatment period at the daily examinations and approximately 1 hour following exposure and was considered adverse at 1.2 mg/L; these observations were generally resolved by the following daily examination in the 0.6 mg/L group, and were therefore considered nonadverse at this exposure level. In the 1.2 mg/L group, a test substance-related increased incidence of clear material on the ventral neck was noted during the latter half of gestation at the daily examinations and/or approximately 1 hour following exposure and was considered adverse. No other test substance-related clinical observations were noted at the daily examinations, midpoint of exposure, or approximately 1 hour following exposure at any exposure level. In the 1.2 mg/L group, test substance-related mean body weight losses or lower mean body weight gains with corresponding reductions in mean food consumption were noted throughout the exposure period and resulted in a lower mean body weight gain and food consumption when the entire exposure period (Gestation Days 6–20) was evaluated compared to the control group. In addition, mean body weights in this group were 5.4% to 11.5% lower than the control group during Gestation Days 8-20. Test substance-related lower mean net body weight, net body weight gain, and gravid uterine weight were also noted in the 1.2 mg/L group. The aforementioned body weight effects noted in the 1.2 mg/L group were considered adverse. In the 0.3 and 0.6 mg/L groups, lower mean body weight gains were noted during Gestation Days 6–9 compared to the control group; mean body weight gains in these groups were similar to the control group for the remainder of the exposure period. Lower mean food consumption was noted during Gestation Days 6–9 in the 0.3 mg/L group and during Gestation Days 6–15 in the 0.6 mg/L group compared to the control group and resulted in lower mean food consumption in these groups when the entire treatment period (Gestation Days 6–20) was evaluated. However, the aforementioned differences in body weight gain and food consumption noted at 0.3 and 0.6 mg/L were not of sufficient magnitude to affect mean body weights at these exposure levels, and therefore were considered test substance-related but nonadverse. Mean maternal body weights, net body weights, net body weight gains, and gravid uterine weights in the 0.3 and 0.6 mg/L groups were unaffected by test substance exposure. No test substance-related macroscopic findings or changes in mean lung weights were noted for females at any exposure level at the scheduled necropsy on Gestation Day 20.

Mean fetal weights (male, female, and combined) in the 0.3, 0.6, and 1.2 mg/L groups were up to 5.3%, 5.3%, and 13.2% lower, respectively, than the control group. The differences in mean fetal weights noted in the 1.2 mg/L group correlated with decrements in maternal mean body weight gain and food consumption noted in this group during the fetal period (Gestation Days 15–20), which is the time of greatest fetal growth, and were considered adverse. When compared to the Charles River Ashland historical control data, the mean fetal weight values in the 0.3 and 0.6 mg/L groups were greater than the minimum mean values and only slightly lower than the mean and median values. In addition, the concurrent control group values for fetal body weight were 2% to 3% heavier (approximately equal to the 75th quartile) than the mean values in the Charles River Ashland historical control data. Therefore, the lower mean fetal body weights at 0.3 and 0.6 mg/L were due to a combination of slightly heavier fetuses in the concurrent control group and slightly lighter fetuses in the test substance-treated groups. Additionally, the magnitude of change in these groups compared to the control group was not as severe as the difference in the 1.2 mg/L group and there was no dose-response relationship between the 0.3 and 0.6 mg/L groups. Therefore, the lower mean fetal weights at 0.3 and 0.6 mg/L were considered unrelated to treatment. Intrauterine survival was unaffected by test substance exposure at exposure levels of 0.3, 0.6, and 1.2 mg/L. A test substance-related, higher mean litter proportion of sternebra(e) nos. 5 and/or 6 unossified and a lower mean litter proportion of cervical centrum no. 1 ossified was noted in the 1.2 mg/L group compared to the control group. The aforementioned findings noted at 1.2 mg/L were considered indicators of developmental delay and secondary to the reduced fetal weights and decrements in maternal food consumption and body weight gain noted at this exposure level. There were no test substance-related malformations noted at 0.3, 0.6, and 1.2 mg/L or developmental variations noted at 0.3 and 0.6 mg/L.