5-methylhexan-2-one

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

5-methyl-2-hexanone

IUPAC Name:

5-methyl-2-hexanone

Constituent 2

Reference substance name:

5-methylhexan-2-one

EC Number:

203-737-8

EC Name:

5-methylhexan-2-one

Cas Number:

110-12-3

Molecular formula:

C7H14O

IUPAC Name:

5-methylhexan-2-one

Constituent 3

Reference substance name:

MIAK; Isoamyl methyl ketone

IUPAC Name:

MIAK; Isoamyl methyl ketone

Details on test material:

no data

Specific details on test material used for the study:

MiAK is clear colorless liquid. The test substance was stored at room temperature (18 °C to 24 °C), protected from light, and was considered stable under these conditions.

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Time-mated female New Zealand White [Hra:(NZW)SPF] rabbits were used as the test system on this study. This species and strain of animal is recognized as appropriate for developmental toxicity studies. The number of animals selected for this study (24 females/group) was based on the US EPA Health Effects Test Guidelines OPPTS 870.3700, Prenatal Development Toxicity Study, Aug 1998 and the OECD Guidelines for the Testing of Chemicals: Guideline 414, Prenatal Developmental Toxicity Study, Jan 2001, which recommends evaluation of approximately 20 females with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or test substance-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 20 females/group at termination. Time-mated female New Zealand White rabbits were received in 2 shipments; each shipment was received from the same facility and was approximately the same age. One hundred four time-mated female New Zealand White rabbits in total (52/shipment) were received in good health from Covance Research Products, Inc., Denver, PA, on 21 Apr 2017 and 19 May 2017. The time-mated rabbits were received on Gestation Day 1, 2, or 3. The animals were approximately 7 months old upon receipt. Each animal was uniquely identified using a microchip (BMDS system) which was implanted subcutaneously in the dorsoscapular region. The rabbits were observed twice daily for mortality and general changes in appearance and behavior.

All rabbits were housed during the study in an environmentally controlled room. The room temperature and relative humidity controls were set to maintain environmental conditions of 61 °F to 71 °F (16 °C to 22 °C) and 30 % to 70 %, 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 65.4 °F to 68.8 °F (18.6 °C to 20.4 °C) and mean daily relative humidity ranged from 45.3 % to 67.7 % during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

Exposures were conducted using four 1500-L glass and stainless steel whole-body inhalation exposure chambers. One exposure chamber was dedicated to the filtered air control group and one exposure chamber was dedicated to each of the test substance-exposed groups for the duration of the study.
Air supplied to the whole-body chambers was provided from an in-house nitrogen source and a HEPA- and charcoal-filtered, temperature and humidity-controlled supply air source. All chamber exhaust passed through the facility exhaust system, which consists of redundant exhaust blowers preceded by activated-charcoal and HEPA-filtration units.
Chamber temperature and relative humidity were monitored using a humidity and temperature transmitter probe. Chamber airflow rates were monitored using a sharp edge orifice meter and Dwyer Magnehelic® Indicating Transmitter pressure gauge. Each gauge was calibrated for conversion from pressure to airflow in standard liters per minute (SLPM) through the use of a Fox Gas Mass Flowmeter Transmitter. Chamber temperature, relative humidity, ventilation rate, and negative pressure within each chamber was continually monitored and recorded at approximately 45-minute intervals.
Oxygen content of the exposure atmosphere was measured during the method development phase and during the study phase on 03 May 2017 (Gestation Day 13, 14, or 15) for Phase I and 14 Jun 2017 (Gestation Day 27, 28, or 29) for Phase II, and was at least 20.2 % and 20.5 % for all chambers for Phase I and II, respectively.
Test substance chambers were monitored for aerosol formation during the method development phase. No aerosol was detected.
All animals were housed in a normal animal colony room during non-exposure periods. Prior to each exposure, the animals were transferred to exposure caging and transported to the exposure room. Animals were then exposed for the requisite duration and then returned to their home cages. Animals were housed individually in standard exposure batteries of appropriate size for the whole-body exposure chamber in use during exposure periods. Food and water were withheld during the animal exposure periods. The cage batteries were rotated on a daily basis between the battery positions of 1–3 within the chambers to help ensure a similar exposure for all animals within each group over the duration of the exposure period.

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 passed through the camber during exposure. Test substance usage was determined by weighing the test substance container prior to and after the termination of generation. Total volume of air (including nitrogen from the vapor generation) was calculated by multiplying the daily mean ventilation rate by the duration of generation.

Analyzed exposure concentrations were determined at approximately 45 minute intervals using a gas chromatograph (GC). Samples were collected from the approximate animal-breathing zone of the exposure chamber via 1/8-inch heated stainless steel tubing. Tubing was heated to approximately 60 °C using heat tapes, J-type thermocouples, and temperature controllers. Under the control of the WINH system, sampling and analyses was performed as follows. The program controls an external multi-position valve that permits sequential sampling from the exposure room and each exposure chamber. The multi-position valve was heated to approximately 65 °C using a disc heater and was controlled using a temperature controller and J-type thermocouple. Gas sampling injection onto the chromatography column occurs via an internal gas-sampling valve with a sample loop, the chromatograph is displayed and the area under the sample peak was calculated and stored.The WINH system then acquires the stored peak area data and uses an ln-quadratic equation based on the GC calibration curve to calculate the measured concentration in ppm.

Details on mating procedure:

Time-mated female New Zealand White rabbits were received in 2 shipments; each shipment was received from the same facility and was approximately the same age. One hundred four time-mated female New Zealand White rabbits in total (52/shipment) were received in good health from Covance Research Products, Inc., Denver, PA, on 21 Apr 2017 and 19 May 2017. The time-mated rabbits were received on Gestation Day 1, 2, or 3.

Duration of treatment / exposure:

gestation day 7 through 28

Frequency of treatment:

Daily

Duration of test:

6 hours per day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 ppm

Doses / concentrations 2

Dose / conc.:

1 250 ppm

Doses / concentrations 3

Dose / conc.:

2 500 ppm

No. of animals per sex per dose:

24 per group

Control animals:

yes

Details on study design:

This study was conducted in 2 phases (Phase I and Phase II) due to the limitations in the total number of animals that can be placed in the stainless steel whole-body exposure chambers at one time. For each phase, methyl isoamyl ketone (MiAK) was administered via whole-body inhalation exposure for 6 hours per day to 3 groups of 12 time-mated female New Zealand White [Hra:(NZW)SPF] rabbits from Gestation Days 7–28. Target exposure concentrations were 500, 1250, and 2500 ppm. Overall mean analyzed exposure concentrations were 508, 1257, and 2493 ppm for Phase I and 499, 1262, and 2497 ppm for Phase II for the 500, 1250, and 2500 ppm groups, respectively. Each phase also contained a concurrent control group, composed of 12 time-mated females, that was exposed to humidified, filtered air on a comparable regimen. The females for both phases were approximately 7 months 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 29, a laparohysterectomy was performed on each female. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

Examinations

Maternal examinations:

All rabbits were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded daily from the day of receipt through Gestation Day 29 (prior to exposure during the treatment period). Animals were also observed for signs of toxicity at approximately the midpoint of exposure, the end of the exposure period, and 1–2 hours following exposure.

Ovaries and uterine content:

Gravid uterine weight was collected and net body weight (the Gestation Day 29 body weight exclusive of the weight of the uterus and contents) and net body weight change (the Gestation Day 0–29 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

Fetal examinations:

External, visceral, and skeletal findings were recorded .Viable fetus with Crown-rump measurements, degrees of autolysis and gross examinations, if possible, were recorded for late resorptions.

Statistics:

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1 % and 5 %, comparing each test substance-exposed group to the control group. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from
statistical analyses. Due to the use of sig. figures & 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 & net), body weight changes (absolute & net), food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, & viable fetuses, & fetal body weights (separately by sex & combined) were subjected to a parametric one-way ANOVA to determine intergroup differences. Mean litter proportions (% per litter) of prenatal data (viable & nonviable fetuses, early & late resorptions, total resorptions, pre- & postimplantation loss, & fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, & 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 ANOVA or the nonparametric ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's test or Dunn’s test was used to compare the test substance-exposed groups to the control group, respectively.

Historical control data:

yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Maternal toxicity was apparent in the 2500 ppm group as evidenced by adverse clinical observations (general irritation-related findings of partial closure of the eyes and/or clear material around the mouth and/or nose) noted at the midpoint of exposure, end of the daily exposure, and/or postexposure observations.

Dermal irritation (if dermal study):

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A test substance-related lower mean body weight gain was noted in at 2500 ppm group during entire exposure period (Gestation Days 7–29).

Description (incidence and severity):

Mean food consumption in the 2500 ppm group during Gestation Days 7–10, 10–13, and 13–20, and when the entire exposure period (Gestation Days 7–29) wre lower in 2500 ppm group when compared to the control.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption, evaluated as g/animal/day and g/kg/day, was significantly (p < 0.01) lower in the 2500 ppm group during Gestation Days 7–10, 10–13, and 13–20, and when the entire exposure period (Gestation Days 7–29) was evaluated when compared to the control group, which corresponded to the lower mean body weight gains or body weight losses. The differences were considered adverse.

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

no effects observed

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

Gross pathological findings:

no effects observed

Description (incidence and severity):

At the scheduled necropsy on Gestation Day 29, no test substance-related internal findings were noted at exposure levels of 500, 1250, and 2500 ppm.

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

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Intrauterine growth and survival were unaffected by test substance exposure at exposure levels of 500, 1250, and 2500 ppm. Parameters evaluated included postimplantation loss, number and percentage of viable fetuses, mean fetal body weights and fetal sex ratios. Mean number 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

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

Data tables are provided in the "attached background materials" section, as fidelity reasons prevent the copying of .PDF tables to IUCLID.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 250 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

The heart and great vessel associated visceral malformations retroesophageal aortic arch, Tetralogy of Fallot, interventricular septal defect, and bulbous aorta in the 2500 ppm group were considered test substance-related and adverse.

Other effects:

no effects observed

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEC

Effect level:

1 250 ppm

Based on:

test mat.

Basis for effect level:

visceral malformations

Fetal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

visceral/soft tissue: cardiovascular

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

2 500 ppm

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Any other information on results incl. tables

 

Overall Mean Nominal Exposure Concentration Phase I

Exposure Chamber:	2	3	4
Target Concentration (ppm):	500	1250	2500
Nominal Concentration (ppm)	544	1351	2716
Standard Deviation:	34.4	54.6	142.2
N:	24	24	24

Overall Mean Nominal Exposure Concentration Phase II

Exposure Chamber:	2	3	4
Target Concentration (ppm):	500	1250	2500
Nominal Concentration (ppm)	480	1211	2764
Standard Deviation:	8.5	71.4	64.8
N:	24	24	24

Overall Mean Exposure Concentration Phase I

Exposure Chamber:	1	2	3	4
Target Concentration (ppm):	0	500	1250	2500
Mean Concentration (ppm)	0	508	1257	2493
Standard Deviation:	0.0	20.5	33.0	64.4
N:	24	24	24	24

Overall Mean Exposure Concentration Phase II

Exposure Chamber:	1	2	3	4
Target Concentration (ppm):	0	500	1250	2500
Nominal Concentration (ppm)	0	499	1262	2497
Standard Deviation:	0.0	7.1	26.1	29.2
N:	24	24	24	24

 

Discussion

The objectives of the study were to determine the potential of the test substance, methyl isoamyl 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 level (NOAEL) for maternal and developmental toxicity.

Maternal toxicity was apparent in the 2500 ppm group as evidenced by adverse clinical observations (general irritation-related findings of partial closure of the eyes and/or clear material around the mouth and/or nose) noted at the midpoint of exposure, end of the daily exposure, and/or postexposure observations. In addition, a test substance-related lower mean body weight gain was noted in this group when the entire exposure period (Gestation Days 7–29) was evaluated, which generally correlated with an approximate 30 % decrease in food consumption and increased incidences of decreased defecation noted at the daily examinations during the same period.

Developmental toxicity, in the presence of maternal toxicity, was apparent in the 2500 ppm group, as evidenced by higher mean litter proportions of heart and great vessel visceral malformations, including retroesophageal aortic arch, Tetralogy of Fallot, and an interventricular septal defect. The mean litter proportions of these malformations also exceeded the maximum mean values of the Charles River Ashland developmental historical control data.

 

CONCLUSIONS

Based on the adverse clinical findings of eye, nasal, and oral irritation, and adverse effects on maternal food consumption with correspondingly lower mean body weight gain during Gestation Days 7 through 20 in the 2500 ppm group, the no-observed-adverse-effect level (NOAEL) for maternal toxicity was 1250 ppm when methyl isoamyl ketone was administered by whole-body inhalation exposure to time-mated New Zealand White rabbits. Based on heart and great vessel visceral malformations at the 2500 ppm exposure level, 1250 ppm was considered to be the NOAEL for embryo/fetal developmental toxicity.

Applicant's summary and conclusion

Conclusions:

Based on the adverse clinical findings of eye, nasal, and oral irritation, and adverse effects on maternal food consumption with correspondingly lower mean body weight gain during Gestation Days 7 through 20 in the 2500 ppm group, the no-observed-adverse-effect level (NOAEL) for maternal toxicity was 1250 ppm when methyl isoamyl ketone was administered by whole-body inhalation exposure to time-mated New Zealand White rabbits. Based on heart and great vessel visceral malformations at the 2500 ppm exposure level, 1250 ppm was considered to be the NOAEL for embryo/fetal developmental toxicity.

Executive summary:

Methyl isoamyl ketone (MiAK) was administered via whole-body inhalation exposure for 6 hours per day to 3 groups of 12 time-mated female New Zealand White [Hra:(NZW) SPF] rabbits from Gestation Days 7–28. Target exposure concentrations were 500, 1250, and 2500 ppm. Overall mean analyzed exposure concentrations were 508, 1257, and 2493 ppm for Phase I and 499, 1262, and 2497 ppm for Phase II for the 500, 1250, and 2500 ppm groups, respectively. All females survived to the scheduled necropsy. Test substance-related increased incidences of decreased defecation were noted for 12 of 24 females in the 2500 ppm group at the daily examinations. At the midpoint of exposure, end of the daily exposure, and/or postexposure observations, test substance-related, adverse general irritation-related findings of partial closure of the eyes and/or clear material around the mouth and/or nose were noted for females in the 2500 ppm group. Clear material around the nose was also noted sporadically for 7 of 24 females in the 1250 ppm group at the midpoint of exposure; however, this finding was transient and not observed at the postdosing observation, and therefore was not considered adverse. No other test substance-related clinical observations were noted at any exposure level. Test substance-related lower mean body weight gains or body weight losses, with correspondingly reduced mean food consumption, were noted in the 2500 ppm group during Gestation Days 7–10, 10–13, and 13–20, resulting in a lower mean body weight gain when the entire exposure period (Gestation Days 7–29) was evaluated compared to the control group. These decreases in body weight gain generally correlated with an approximate 30 % decrease in food consumption during this time period and were considered adverse. Mean gravid uterine weights, mean net body weights and net body weight changes in the 500, 1250, and 2500 ppm groups were unaffected by test substance exposure. No test substance-related maternal macroscopic findings were noted at the scheduled necropsy at any exposure level. Intrauterine growth and survival were unaffected by maternal test substance exposure. Heart and great vessel visceral malformations were noted in 3 fetuses from different litters within the 2500 ppm group, including retroesophageal aortic arch, Tetralogy of Fallot, an interventricular septal defect, and bulbous aorta, were considered test substance-related and adverse. There were no fetal skeletal malformations noted in the 2500 ppm group and fetal morphology was unaffected by test substance exposure in the 500 and 1250 ppm groups. Based on the adverse clinical findings of eye, nasal, and oral irritation, and adverse effects on maternal food consumption with correspondingly lower mean body weight gain during Gestation Days 7 through 20 in the 2500 ppm group, the no-observed-adverse-effect level (NOAEL) for maternal toxicity was 1250 ppm when methyl isoamyl ketone was administered by whole-body inhalation exposure to time-mated New Zealand White rabbits. Based on heart and great vessel visceral malformations at the 2500 ppm exposure level, 1250 ppm was the NOAEL for embryo/fetal developmental toxicity.