Acetone

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study similar to guideline study, detailed study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Acetone

EC Number:

200-662-2

EC Name:

Acetone

Cas Number:

67-64-1

Molecular formula:

C3H6O

IUPAC Name:

propan-2-one

Details on test material:

- Analytical purity: 100 %

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Raleigh, NC, USA
- Age at study initiation: 13 w
- Housing: 5-6 rats/cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 30 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 75 +- 3 °F
- Humidity (%): 55 +- 15 %
- Air changes (per hr): average air flow 12-18 CFM
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

whole body

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 2.3 cubicm Batelle-designed inhalation exposure chambers (Harford System; Lab Products Onc., Aberdeen, MD)
- Method of holding animals in test chamber: caging
- Source and rate of air: HEPA- and charcoal-filtered air
- Temperature, humidity, pressure in air chamber:
- Air flow rate: 15 cubic ft/min
- Air change rate: 15 air changes per hour

TEST ATMOSPHERE
- Brief description of analytical method used: continuous monitoring with an HP5840 GC system
- Samples taken from breathing zone: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Continuous monitoring by gas chromatography (mean concentrations +- SD): 0.08 +- 0.05, 439 +- 27, 2200 +- 25, 11000 +- 137 ppm

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/3 or 1/2
- Length of cohabitation: up to 5 nights
- Proof of pregnancy: sperm-positive vaginal lavage smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

- Groups A and B - mated dams: from gestation day 6-19, 6 h/d, 7 d/w
- Group C - virgin rats: 14 days, 6 h/d, 7 d/w

Frequency of treatment:

daily

Duration of test:

- Groups A and B - mated dams: sacrifice on gestation day 20
- Group C - virgin rats: sacrifce after 15 days

No. of animals per sex per dose:

- Group A: 30-31 mated female rats for investigation of standard teratogenicity study protocol and urine parameters
- Group B: additional 7 mated female rats for monitoring of ketone levels in plasma. These dams and their fetuses were subjected to the investigations of the standard teratogenicity study protocol except for teratological evaluation. Only, a gross examination of external effects was performed.
- Group C: 10 virgin rats for observation of toxicity and body weight development independent of gestation

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: chosen with the goal of observing mild maternal toxicity at hightest dose level and a NOEL at the lowest dose level

Examinations

Maternal examinations:

Types of examinations performed in Groups A, B, C:

CAGE SIDE OBSERVATIONS (A, B, C): Yes
- Time schedule: daily

BODY WEIGHT (A, B, C): Yes
- Time schedule: gestation day 0, 6, 9, 12, 15, 18

GRAVID UTERINE WEIGHT (A, B): Yes
- At sacrifice

EXTRA-GESTATIONAL WEIGHT GAIN (A, B): Yes
- Maternal body weight gain at sacrifice: (Maternal body weight at sacrifice) - (gravid uterine weight) - (maternal body weight on gestation day 0)

POST-MORTEM EXAMINATION (A, B, C): Yes
- Sacrifice on gestation day 18 (A, B) or 1 day post exposure (C)
- Organs examined: maternal liver and kidney weight, uterine contents

OTHER:
- Examination of urine sample on the morning of sacrifice (A, B, C): pH, protein, glucose, ketones, bilirubin, blood and urobilinogen via urine dip-stick
- Determination of ketone bodies in plasma (B): blood was collected from the additional dams (N=7/group) 30 minutes post exposure on gestation days 7 (2nd day of exposure), 14 and 19 and again one hour prior to start of exposure on the following day. Plasma was analyzed for the levels of acetone, acetoacetic acid, and ß-hydroxybutyrate by headspace-gas chromatography after conversion of acetoacetic acid and ß-hydroxybutyrate to acetone.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included (A, B):
- Gravid uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Live / dead fetuses: yes
- Fetal weight: Yes
- Fetal sex: Yes

Fetal examinations:

- External examinations (A, B): Yes: all per litter
- Soft tissue examinations (A): Yes: half per litter
- Skeletal examinations (A): Yes: all per litter
- Head examinations (A): Yes: half per litter

Statistics:

ANOVA: body weight, incidence data; Tukey's test: for comparison of control and exposed groups; orthogonal trend test (Winer, 1971): dose-response relationships

Historical control data:

Contemporary control data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Group A - 11000 ppm: significantly reduced weight gain on gestation days 14, 17 and 20, p<0.05; significant reduction of body weight, gravid uterine weight, and extra-gestational weight gain (see Table 1)

Effect levels (maternal animals)

1

Dose descriptor:

NOAEC

Effect level:

2 200 ppm

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

LOAEC

Effect level:

11 000 ppm (analytical)

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Group A - 11000 ppm:
- Fetotoxicity:
percentage of litters with resorptions was increased in comparison to controls (77 vs. 50 %), no significant effect;
fetal weights significantly reduced (see Table 2)
- Fetal malformations:
no significant increase of incidence of malformations

Effect levels (fetuses)

1

Dose descriptor:

NOAEC

Effect level:

2 200 ppm

Basis for effect level:

other: fetotoxicity

2

Dose descriptor:

LOAEC

Effect level:

11 000 ppm

Basis for effect level:

other: fetotoxicity

3

Dose descriptor:

NOAEC

Effect level:

11 000 ppm

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Group A:

Table 1: Maternal toxicity data

Concentration (ppm)	0	440	2200	11000
N	26	27	29	26
Body weight on GD 20 (g) ^b	401.2 +- 29.5	398.5 +- 27.9	390.8 +- 28.1	371.3 +- 29.1 ^a
Uterine weight (g)  ^b	83.2 +- 19.0	79.7 +- 14.5	74.5 +- 16.3	67.1 +- 13.9  ^a
Extra-gestational weight gain  ^b	45.3 +- 16.7	45.1 +- 13.2	43.2 +- 15.2	29.7 +- 14.4  ^a

 ^a significantly different from 0-ppm group, p<0.05

 ^b significantly correlated with exposure concentration, p<0.05

Table 2: Fetal toxicity data

Concentration (ppm)	0	440	2200	11000
N (Litter examined)	26	27	28	26
Fetal weight  (g) : mean of both sexes	3.6 +- 0.4	3.7 +- 0.2	3.5 +- 0.3	3.1 +- 0.3 ^a
male	3.7 +- 0.4	3.8 +- 0.2	3.6 +- 0.3	3.1 +- 0.3 ^a
female	3.5 +- 0.3	3.6 +- 0.2	3.4 +- 0.3	3.0 +- 0.3 ^a

^a significantly different from 0-ppm group, p0.05

 ^b significantly correlated with exposure concentration, p0.05

Fetal malformations:

The percent of litters with at least one malformed pup was significantly increased at 11000 ppm: 11.5 % vs. 3.8 % in concurrent control group, 6.3 % in contemporary control group.

Mean percent of live fetuses per litter with at least one malformation: increased at 11000 ppm: 2.8 % vs. 0.9 % in concurrent control group, no significant effect

Types of malformations: none of the major malformations observed in the highest exposure group had been observed in control litters: e.g. cleft sternum, ectopic heart, major vessel malformations, edema, unilateral arhinia, microstomia, vertebral agenesis, missing tail. Several fetuses with multiple malformations.

Group B:

Maternal weights and reproductive data were consistent with data of Group A except for extra-gestational weight gain which was lower than in Group A.

Plasma levels of ketone bodies:

- Acetone (see Table 3): 30 min post exposure: increase of plasma level correlating with exposure concentration; at 440 and 6600 ppm return to control level within 17 h post-exposure; at 11000 ppm still slightly elevated at 17 h post exposure. However, there was no indication of accumulation as levels on gestation day 19 were not significantly greater than on gestation day 7 (2nd day of exposure).

- Acetoacetate and ß-hydroxybutyrate: no alterations of plasma levels

Table 3: Plasma concentrations of acetone in mg/L (in mM) in pregnant rats

Concentration (ppm)		0	440	2200	11000
30 min post-exposure	GD 7	< 0.6  (<0.01)	38.3 (0.66)	296.2 (5.1)	2149.0 (37)
	GD 14	<0.6 (<0.01)	43.0 (0.74)	273.0 (4.7)	2090.9 (36)
	GD 19	1.7 (0.03)	52.9 (0.91)	296.2 (5.1)	1974.7  (34)
17 h post-exposure	GD 7	<0.6 (<0.01)	<4.1 (<0.07)	<0.6 (<0.01)	174.2 (3)
	GD 14	<0.6 (<0.01)	<0.6 (<0.01)	0.6 (0.01)	174.2 (3)
	GD 19	<0.6 (<0.01)	<1.7 (<0.03)	23.2 (0.40)	348.5 (6)

Of the 7 female rats in the 440-ppm group only 3 were pregnant. There did not appear to exist a significant difference between pregnant and non-pregnant females with respect to plasma levels of any of the three ketone bodies.

Group C: reduction of body weight (- 6 %) as in group A, no statistical significance due to higher standard deviation

Applicant's summary and conclusion

Conclusions:

Exposure of pregnant rats to 0, 440, 2200 and 11000 ppmacetone did not result in selective developmental toxicity. Maternal toxicity was evident at the high exposure level of 11000 ppm as decreases in body and uterine weight. At this dose level a significant decrease of fetal weight indicated fetal toxicity. There was no indication of a teratogenic potential (NOAEL 11000 ppm).

Executive summary:

The potential for acetone to cause developmental toxicity was assessed in pregnant Sprague-Dawley rats exposed to 0, 440, 2200 and 11000 ppm acetone vapours (0, 1,060, 5,300, 26,500 mg/m³) in exposure chambers (whole-body exposure) from gestation days 6 to 19 (6 h/d, 7 d/w). The test protocol for Group A (N=31/dose level) was comparable to OECD Guideline 414. In Group B plasma levels of the ketone bodies acetone, acetoacetate and ß-hydroxybutyrate were analyzed in 7 additional dams/dose level on gestation days 7, 14 and 19 both 30 min and 17 h post exposure. These dams and there fetuses were subjected to the same examinations as Group A except for an evaluation of malformations (only gross examination for external effects). For comparison, toxicity and body weight development was observed in a third group, Group C, with 10 virgin females per dose level. Urine samples from all groups were examined on the morning of sacrifice for pH, protein, glucose, ketones, bilirubin, blood and urobilinogen via urine dip-sticks.

Pregnant rats (Groups A and B) did not exhibit overt signs of maternal toxicity other than statistically significant reductions for the 11000 -ppm group in body weight, cumulative weight gain from gestation day 14 onwards, uterine weight and in extragestational weight gain. Mean body weights of treated virgins (Group C) were also reduced, but not significantly. No significant effect was observed in the mean absolute or relative liver or kidney weights, the number of implantations, the mean percent of live pups/litter, the mean percent of resorptions/litter, or the fetal sex ratio. However, fetal weights were significantly reduced for the 11000 -ppm group (A und B). The incidence ot fetal malformations was not significantly increased, although the percent of litters with at least one pup exhibiting malformations was greater for the 11000 -ppm group than for the control group (11.5 % vs. 3.8%). The diversity of malformations was greater than that found in the lower dose groups or the controls. There was no increase in the incidence of fetal variations, reduced ossification sites, or in the mean incidence of fetal variations per litter. These changes are not interpreted as an indication of a substance-specific teratogenic potential of acetone. Analysis of plasma samples (Group B) showed an increase in plasma acetone levels which correlated with increasing exposure concentration at 30 min post exposure. Acetone levels dropped to control levels within 17 h post exposure except for a slight increase in the 11000 -ppm group. However, no accumulation of acetone was found over the entire exposure period and there was no increase of the other ketone bodies.