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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
25/9/1995 to 22/2/1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to GLPs and OECD Guideline 421.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
There were minor deviations on the study but these did not affect the quality or integrity of the study.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Heptan-2-one
EC Number:
203-767-1
EC Name:
Heptan-2-one
Cas Number:
110-43-0
Molecular formula:
C7H14O
IUPAC Name:
heptan-2-one
Constituent 2
Reference substance name:
606-024-00-3
IUPAC Name:
606-024-00-3
Constituent 3
Reference substance name:
methyl amyl ketone; methyl pentyl ketone; MAK; 1-methylhexanal; butylacetone
IUPAC Name:
methyl amyl ketone; methyl pentyl ketone; MAK; 1-methylhexanal; butylacetone
Details on test material:
Test substance:
-Test substance (as cited in report): Methyl n-Amyl Ketone
-Synonyms: MAK, 2-Heptanone
-Lot number: 4050657
-Physical State and Appearance: Colorless liquid with a pungent/sweet odor
-Supplier: Eastman Chemical Company (Kingsport, TN)
-Purity at study initiation (GC/FID): 99.8%
-Purity at study termination (GC/FID): 99.9%
-Test substance stability: Based on purity analysis, the test substance was considered stable.
-Test substance structure (mass spectrometry): The mass spectrum was consistent with published spectra for this test substance.


Test animals

Species:
rat
Strain:
other: Sprague-Dawley rat (CD®(SD)BR/VAF Plus™)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
-Strain: CD®(SD)BR/VAF Plus™ (Charles River Laboratories, Stone Ridge (Kingston), NY)
-Age (study initiation): 60 days
-Body weight (study start): Males 290 ± 10 g; Females 210 ± 7 g
-Acclimation: 5 days
-Housing: Single housed in stainless-steel wire-mesh cages except during the mating period when the animals were housed one male with one female during non-exposure periods. Housing was washed once a week; cage papers changed daily. On or about Gestation Day 19, solid bottom pans containing bedding material for nesting were placed in the non-exposure cages of the female rats.
-Feed: Purina Rodent Chow #5002, meal (PMI Feed, Inc. Richmond, IN) ad libitum except during inhalation exposure
-Water: Rochester, NY local water system, ad libitum, except during inhalation exposure
-Identification: uniquely-numbered metal ear tags
-Randomization: Animals were chosen using a stratified randomization program based on body weight; body weights did not vary more than 20% from the mean for each sex.

ENVIRONMENTAL CONDITIONS
-Temperature (°C): 21-24
-Humidity (%): 41-63
-Photoperiod: 12 hours light/dark


Study Dates:
-Study Initiation date: September 18, 1995
-Experimental Start date: September 25, 1995
-Experimental Completion date: February 22, 1996

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: filtered compressed air
Details on exposure:
Selection of exposure concentrations:
Exposure concentrations were selected based on data from a 2-week inhalation toxicity study.

Exposure:
Inhalation exposures were conducted in 420 L stainless-steel and glass inhalation chambers. Animals were singly housed during the 6-hour exposure and the animals were rotated on a daily basis to a new cage position within the chamber. The exposure chambers were maintained under negative pressure relative to room air.

Exposure atmosphere generation:
The test atmosphere was generated by metering the test substance into glass distillation columns packed with glass beads, and filtered compressed air was passed through the column to evaporate the test substance. The test substance delivery rate and air flow were adjusted to produce the desired chamber target vapor concentrations. The distillation columns were heated to enhance vaporization. The resultant vapor was directed to a tee upstream of the inhalation chamber where it was mixed with filtered, conditioned outside air to produce airflow of 85-95 Lpm, resulting in 12-14 air changes per hour.

Particle size analysis:
A particle counter (Micro Laser Particle Counter, model µLPC-301, Particle Measuring Systems, Inc., Boulder, CO) was used to measure the number and size of particulates in the chamber. The results indicated that an aerosol of the test substance was not present.

Chamber vapor homogeneity:
The test substance concentration in the air from 13 different cage positions was compared to the concentration at a fixed reference position within the inhalation chamber. Based on the data, the test atmosphere was considered homogeneous.

Air flow measurement:
Total chamber air flow, a combination of compressed air and dilution air, was continuously monitored using a flowmeter (Omega Air Velocity Transducer (FMA-602-V-S) and Ratemeter (DPF66-RS232)).

Oxygen level:
During pre-study tests, the oxygen content of the chamber exposure atmosphere was measured from a reference position using a Model K Oxygen Indicator (Johnson-Williams Products, Bacharach Instrument Co., Mountain View, CA), and the oxygen content was ≥ 20%.

Chamber temperature and humidity:
Chamber temperature and humidity were measured using wet/dry bulb hygrometers and were recorded approximately every 30 minutes.
Details on mating procedure:
Male and female rats were paired 1:1 within the same dose group for 1-14 days. Copulation was verified through identification of sperm in vaginal smears or by appearance of a copulatory plug. The morning of the day copulation was verified was considered Gestation Day 0. Following copulation, animals were separated and housed individually until study termination.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Purity, structure, and stability determination:
-Purity was measured using GC/FID at study initiation (99.8%) and study termination (99.9%).
-Based on the purity determinations, the test substance was considered stable over the test period.
-Structure of the test substance was determined by mass spectrometry and the results were consistent with MAK.

Vapor concentration determination:
Chamber vapor concentrations were monitored with a single MIRAN IA infrared gas analyzer (Wilks Foxboro Analytical, South Norwalk, CT) and a computer-operated four-port sampling valve. Chamber vapor samples were analyzed from each chamber at least once an hour.

Nominal concentration determination:
Nominal concentration was calculated by dividing the amount of test substance consumed by the total chamber air flow.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
7 days/week
Details on study schedule:
The study consisted of four phases: pre-mating (14 days), mating (1-14 days), gestation (21-24 days), and early lactation (4 days). The females were exposed 6 hours/day through Gestation Day 19 (34-47 consecutive exposures). Females that delivered a litter and their offspring were euthanized on Lactation Day 4, 5 or 6. Females that showed no evidence of copulation were euthanized 23 days after the last day of the mating period. Males were exposed 6 hours/day throughout the entire study (until all dams had littered or were euthanized due to non-delivery) for a total of 50 consecutive daily exposures. All males were euthanized the following day.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
80 ppm
Basis:
other: target
Remarks:
Doses / Concentrations:
400 ppm
Basis:
other: target
Remarks:
Doses / Concentrations:
1000 ppm
Basis:
other: target
No. of animals per sex per dose:
12 animals/sex/group
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
Clinical observations:
Before and after each exposure, each rat was removed from its cage and examined. Clinical observations included, but were not limited to, inspection of the hair, skin, eyes, mucous membranes, motor activity, feces, urine, respiratory system, circulatory system, autonomic nervous system, central nervous system, and behavior patterns. During inhalation exposure, rats visible through the chamber windows were observed for clinical signs. Tapping sounds were made on the outside of the chamber to assess the animals’ activity levels. Beginning on Day 20 of gestation (during the non-exposure period), hands-on clinical examinations were conducted daily on all females.

Body weight:
Body weights for male rats were measured prior to exposure on Days 0, 7, and weekly thereafter. For female rats, body weights were measured prior to exposure on Days 0, 7, 13 and 21 (premating), Gestation Day 0, 7, 14, and 20, and Lactation Days 0 and 4. Terminal body weights for males and females were measured after exsanguination, but prior to necropsy.

Feed consumption:
Feed consumption was measured for males between Days 0-7, 7-13, 27-35, 35-42, and 42-49. Male rats were fasted overnight prior to necropsy. Feed consumption was measured for females between Days 0-7, 7-13 (premating), Gestation Days 0-7, 7-14, and 14-20, and Lactation Days 0-4.
Oestrous cyclicity (parental animals):
Not determined in the study.
Sperm parameters (parental animals):
Not determined in the study.
Litter observations:
Clinical Observations:
All pups were observed for clinical abnormalities.

Body weight:
Body weights were measured as a group, by sex, on Postnatal Days 0 and 4.
Postmortem examinations (parental animals):
Euthanasia:
Male rats were fasted overnight beginning after the last inhalation exposure, anesthetized with carbon dioxide, and euthanized by exsanguination. The male rats were necropsied in a random order based on a computer-generated list. The adult females were anesthetized with carbon dioxide and euthanized by exsanguination. Females that showed no evidence of copulation were euthanized on Day 23 of the gestation phase. All other females were euthanized on Day 4, 5, or 6 post-partum.

Necropsy:
All adult animals were weighed and subjected to a necropsy and gross examination. Special attention was paid to the organs of the reproductive system. The uteri from all adult females were examined, and implantation sites were counted.

The following tissues were collected from adult animals and fixed in 10% buffered formalin: ovaries, vagina, uterus, Fallopian tubes, and male accessory sex glands. The right testis and epididymis were fixed in Bouin’s fixative. The left testis and epididymis were placed in individual containers, frozen with dry ice, and stored at -70 °C for possible evaluation of spermatid (sperm) head counts.

Organ weights:
The testes and epididymides were weighed; paired organs were weighed together.

Histopathology:
All tissues were embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin (H&E) stains. Sections of the right testes, right epididymis, and ovaries from the control and the 1000 ppm exposure group were examined for histopathology.
Postmortem examinations (offspring):
Euthanasia:
Litters were evaluated for viability and pups were examined for the presence of external abnormalities.
Statistics:
Mean values were calculated for the study exposure parameters including time-weighted average atmospheric concentration, chamber temperature, chamber relative humidity, nominal concentrations, adult body weight, body weight change, male and female pup body weight, pup body weight change, feed consumption, organ weights, organ-to-body weight ratio, gestation period, litter size, and pup percent survival data. Body weight, body weight change and organ-to-body weight ratios were evaluated using Bartlett’s test (p ≤ 0.01), one-way analysis of variance (ANOVA) (p ≤ 0.05) and Duncan' multiple range test (p ≤ 0.05), or Dunnett’s t-test (p ≤ 0.05) (body weight change only) to indicate statistical significance. When the variances of the means were not considered equal by the Bartlett’s test (p ≤ 0.01), the data were evaluated using a Kruskal-Wallis H-test followed by the Mann-Whitney U-test.

The reproductive performance of the dams and the fertility and fecundity indices were evaluated in contingency tables using a Chi-square test (p ≤ 0.05).
Reproductive indices:
No data provided in study report.
Offspring viability indices:
No data provided in study report.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

Mortality:
No mortality occurred during the study.

Clinical observations:
Adult animals exposed to 1000 ppm appeared to have reduced activity levels during exposure, as observed through the inhalation chamber windows. The animals appeared to acclimate to the test substance, with the severity decreasing from moderate during the first two exposures, to predominantly minor for the remainder of the first two weeks, to predominantly minimal during the remainder of the study. Reduced activity levels (minimal to minor) were seen at 400 ppm only during the first exposure and on two incidences thereafter. Animals in the 80 ppm and control groups appeared normal during exposure. In these observations, reduced activity was defined as less movement, decreased alertness and slower response to tapping on the chamber wall compared with activity levels exhibited by control animals. All other sporadic signs were not considered treatment-related.

Body weight:
Mean body weights and weight change for all adult male exposure groups and mean body weights for all adult female exposure groups were comparable to control throughout the study. The mean body weight change for the 400 ppm adult female rats between Gestation Day 0 and 7 was significantly lower than the control. All other body weight change determinations for adult female rats were comparable to the control group.

Food consumption:
Mean feed consumption for the 1000 ppm adult male and female rats was significantly lower than the controls on Day 7 of the pre-mating phase. All other feed consumption measurements throughout the study were comparable to those of control.

Organ weights:
No test substance-related changes were observed.

Gross pathology and Histology:
No test substance-related changes were observed.

Effect levels (P0)

Dose descriptor:
NOEC
Effect level:
1 000 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: Exposure to MAK resulted in acute signs of reduced activity during exposure at 400 and 1000 ppm and a transient reduction in feed consumption at 1000 ppm, but no adverse effects on reproduction or development were observed.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

Litter data:
The mean gestation length was statistically longer for the 80 ppm group (22.3 ± 0.5 days) when compared to the control group (21.7 ± 0.5 days).

Observations:
Pup abnormalities were distributed across all groups and were not considered to be treatment-related.

Body weight:
The mean male and female pup weight change and the mean percent male pup weight change for the 80 ppm group between Postnatal Days 0 and 1 were significantly higher when compared to control.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Exposure conditions:

-The overall time-weighted average analytical concentrations of 79, 406, and 1023 ppm were within 10% of the target analytical concentrations of 80, 400, and 1000 ppm, respectively.

-Daily mean temperatures and relative humidity inside the chambers during inhalation exposure were 21.1-24.0 °C and 41.6-66.8%, respectively. 

Applicant's summary and conclusion

Conclusions:
There were no test material-related effects on reproductive organs, mating performance or fertility in a reproductive and developmental toxicity screening test in which adult male and female rats were exposed via inhalation to target concentrations of 0, 80, 400 or 1000 ppm methyl n-amyl ketone for 6 hours/day, 7 days/week during premating, mating, gestation and early lactation for a total of 50 exposure days for males and 34 to 47 exposure days for females. There were no dose-related statistically significant differences from control in reproductive success, mean gestation day, mean number of live pups, mean number of implants, mean percent prenatal loss, mean percent survival of delivered pups, mean number of male and female pups, and mean male and mean female pup weight. There were also no adverse effects on mean number of pups dying between Days 0-4, mean percent pup survival between Days 0-4, and mean male and mean female pup body weight changes over Days 0-4. In addition, no pup abnormalities were reported. The NOEC for reproductive and neonatal toxicity was considered to be 1000 ppm, the highest exposure level tested.


In a well-conducted reproductive and developmental toxicity screening study in rats, there was no clear evidence of test substance-related effects on reproduction of F0 males and females or on early survival and development of F1 pups. Based on this information, methyl n-amyl ketone is not a reproductive toxicant and it is not selectively toxic to the developing fetus. Methyl n-amyl ketone is not classified for “Developmental or Reproductive Toxicity” according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) 1272/2008 or the UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS). The test material 2-heptanone (Methyl n-amyl ketone) is not classified for reproductive or developmental effects in Annex I of Directive 76/548/EEC This substance is also not classified for effects on or via lactation.
Executive summary:

In a reproductive and developmental toxicity screening study, methyl n-amyl ketone (MAK) was administered to groups of 12 Sprague-Dawley (CD®(SD)BR/VAF Plus™) rats/sex via inhalation at concentrations of 0, 80, 400 or 1000 ppm for 6 hr/day, 7 days/wk. Females were exposed to the test substance until Gestation Day 19 (34-47 total exposures) and males were exposed for 50 days. Reduced activity only during inhalation exposure, statistically significant changes to body weights, and food consumption were observed in animals exposed to 400 and 1000 ppm MAK. However, changes in the severity of activity decreased with continued inhalation exposure; body weight was reduced in the 1000 ppm groups only at one observation point, and no dose-related changes in feed consumption were observed in either sex. There were no effects on reproductive organs or reproductive or developmental endpoints in the study. Based on these findings it can be concluded that under the conditions of the present study the NOEC for developmental and reproductive inhalation toxicity for methyl n-amyl ketone was 1000 ppm, the highest exposure concentration tested.