Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

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
Justification for type of information:
CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION

Pre-existing data:
There are no pre-existing data available that would address the pre-natal developmental toxicity study endpoint (GLP or non-GLP). A rat pre-natal developmental toxicity study is not adequate according to the regulation. There are no historical human data that could be used in place of this study.

- (Q)SAR:
There are no accepted QSAR approaches for predicting the outcome of this endpoint. However, there were no Reproductive Toxicity alerts for MiAK.

- In vitro methods
There are no accepted in vitro approaches for predicting the outcome of this endpoint

- Weight of evidence
WoE is not possible as there are not similar substances that could be used for such a comparison.

- Grouping and read-across
The Registrant has not identified any suitable analogues that could be used to provide data for this endpoint. Other similar substances are quite data poor.

- Substance-tailored exposure driven testing
Based on the use patternes of the registered substance in coatings, exposure-based waiving is not possible.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
no data
Specific details on test material used for the study:
MiAK is clear colorless liquid.The purity of the test substance was 99.33%. 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 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 will be conducted using four 1500-L glass and stainless steel whole-body exposure chambers. One chamber will be dedicated for the filtered air group (0 ppm) and three chambers will be dedicated for each of the three test substance-exposed groups.The chambers will be operated under dynamic conditions and at a slight negative pressure. Exposure system air will be supplied from a breathing quality in-house compressed air source,compressed nitrogen source, and/or from a HEPA- and charcoal-filtered supply air source and will provide a minimum of 12 air changes per hour. The airflow rates for each whole-body
chamber may be monitored by measuring the pressure drop between the ports of an orifice plate using a Dwyer Magnehelic® Indicating Transmitter pressure gauge. Each gauge will be calibrated for conversion from pressure to airflow in standard liters per minute.

Average daily temperature and relative humidity of the exposure atmosphere will be 66 ± 5°F (19 ± 3°C) and 50 ± 20%, respectively. Temperature and relative humidity will be monitored with a temperature and humidity transmitter probe for each chamber. Temperature, relative humidity, chamber ventilation rate, and negative pressure within each exposure chamber will be monitored and recorded using the Inhalation Exposure Data Collection System (WINH). The intended schedule for recording these environmental parameters will be approximately every 45 minutes during each exposure. However, recording these parameters
more frequently or recording at additional intervals is acceptable. In the event of a failure of the automated data collection system (e.g., malfunction of a component or of a hardware connection), manual recording will be used. Oxygen content of the exposure atmospheres will be at least 19% and will be documented during method development, and at least once during the exposure phase. The daily exposure duration will be 6 hours. Exposures will be initiated when the generation of test atmospheres is started and will be terminated after 6 hours when the generation is stopped. The test substance chambers will not be opened until at least the T99 period has elapsed. The continued animal exposure during this chamber clearance period is considered to be the reciprocal equivalent of the “under exposure” experienced during the chamber equilibration (concentration build up) period. Therefore, the daily exposure duration will not be adjusted for the calculated T99 for the exposure systems.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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 rabbits will be received by Charles River in 2 cohorts, each cohort will be received from the same facility and be approximately the same age. The animals will arrive 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 / concentrationsopen allclose all
Dose / conc.:
500 ppm
Dose / conc.:
1 250 ppm
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.
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:
clinical signs
body weight and weight gain
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

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.