Pentane-2,4-dione

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Conduction and documentation of study very acceptable.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

1990

Reference 3

Reference Type:

secondary source

Title:

Unnamed

Year:

2001

Materials and methods

Principles of method if other than guideline:

Timed-pregnant Fischer 344 rats were exposed to 2,4-pentanedione vapour by inhalation on gestational days (gd) 6 to 15 at exposure target concentrations of 0, 50, 200 and 400 ppm to evaluate the embryotoxic and fetotoxic (including teratogenic) potential of the test substance administered during organogenesis. The day a copulation plug was found was designated gestational day (gd) 0. Twentyfive plug-positive females were assigned to each experimental group. Clinical observations were recorded daily, and maternal body weights were taken on gd 0, 6, 9, 12, 15 and 18. At scheduled necropsy on gd 21 (CO2 asphyxiation), dams were evaluated for body weight, liver and thymus weights, gravid uterine weight, and status of implantation sites (i.e. resorptions, dead fetuses, live fetuses). Maternal brains were removed, fixed and examined histopathologically. Live fetuses were dissected from the uterus, counted, weighed and sexed and examined for external abnormalities. Approximately one-half of the live fetuses in each litter was examined for visceral abnormalities. These fetuses were then decapitated and their heads fixed in Bouins solution and examined for soft tissue craniofacial malformations. The remaining intact fetuses in each litter were eviscerated, fixed in alcohol, stained with alizarin red S, and examined for skeletal defects and deficits.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

Purity: 99.2%
Lot No.: S-050082

Test animals

Species:

rat

Strain:

Fischer 344

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Harlan Fischer F-344/HarBR
- Age: 81 days
- Housing: single housing in stainless steel wire-mesh cages
- Diet: Certified Rodent Chow, Ralston Purina Co., St. Louis, MO, ad libitum, except during vapour exposures
- Water: ad libitum, except during vapour exposures
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Photoperiod 12 hrs dark / 12 hrs light
- Temperature (°C): 22+/-3
- Humidity (%): 30-70

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Atmospheres were generated by metering liquid 2,4-pentanedione into a heated glass evaporator, maintained at the lowest temperature sufficient to vaporize the liquid (range 44-56°C). The resultant vapour was diluted and carried into the exposure chamber by a countercurrent flow of conditioned air through the evaporator. Different concentrations were produced by varying the rate of metering of 2,4-pentanedione into the evaporator. Each chamber volume was 4320 litres.
- Temperature, humidity, pressure in air chamber: Chamber temperature, relative humidity, and airflow rate were recorded at least 4 times in each 6 hr period.
- Air flow rate: 1000 litres/min
- Air change rate: 14 air changes/hr
- Chamber volume: 4320 L

TEST ATMOSPHERE
- Brief description of analytical method used:
Each chamber atmosphere was analyzed for 2,4-pentanedione concentration every hour during each 6 hr exposure period, and daily nominal concentrations were also calculated for each chamber from a knowledge of amount of 2,4-pentanedione liquid used and the airflow rate. Test substance concentrations were measured using a Perkin-Elmer Model 3920B gas chromatograph equipped with a flame ionization detector. The column was a 10-foot x 1/8 inch stainless steel column packed with 10% SP2100 on 80/100 mesh Supelcoport (Supelco Inc., Bellefonte, Pennsylvania). Calibration of the gas chromatograph was carried out with dynamically generated gas standards of 2,4-pentanedione prepared by syringe injection of the test material into Tedlar gas bags. Gas standards of different concentrations were prepared to encompass the entire range generated in the exposure chambers.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

No decompositional changes or chamber loss of metered 2,4-pentanedione. The mean detected chamber concentrations of 2,4-pentanedione were 52 .7, 202 and 398 ppm for target concentrations of 50, 200 and 400 ppm.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Proof of pregnancy: [vaginal plug] referred to as [day 0] of pregnancy

Duration of treatment / exposure:

Gestational day 6-15

Frequency of treatment:

6 h/day

Duration of test:

Until gestational day 21

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
50, 200, 400 ppm
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
208, 831, 1661 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

25 plug-positive females

Control animals:

yes, sham-exposed

Examinations

Maternal examinations:

Clinical observations were recorded daily, and maternal body weights were taken on gestational day 0, 6, 9, 12, 15 and 18. At scheduled necropsy on gestational day 21 (CO2 asphyxiation), dams were evaluated for body weight, liver and thymus weights. Maternal brains were removed, fixed and examined histopathologically.

Ovaries and uterine content:

Gravid uterine weight and status of implantation sites (i.e. resorptions, dead fetuses, live fetuses).

Fetal examinations:

Live fetuses were dissected from the uterus, counted, weighed and sexed and examined for external abnormalities. Approximately one-half of the live fetuses in each litter was examined for visceral abnormalities. These fetuses were then decapitated and their heads fixed in Bouins solution and examined for soft tissue craniofacial malformations. The remaining intact fetuses in each litter were eviscerated, fixed in alcohol, stained with alizarin red S, and examined for skeletal defects and deficits.

Statistics:

The unit for comparison was the pregnant female or the litter. Results of quantitative analytical variables (eg. maternal body weights, organ weights, foetal body weights) were intercompared for the 2,4-pentanedione-exposed groups and air-alone group by the use of the Levene test for equal variances, analysis of variance (ANOVA), and t-tests with Bonferroni probabilities. When Levene's test indicated homogeneous variances and the ANOVA was significant, the pooled t-test was used. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by the separate variance t-test. Students t-test was employed for analysis of incidence data, with the number of litters (with one or more affected foetuses) being the unit of comparison. Nonparametric data obtained following laparohysterectomy were treated statistically using the Kruskal-Wallis test, followed by the Mann-Whitney U test when appropriate. For all statistical tests, a fiducial limit of 0.05 (two-tailed) was used as the criterion for significance.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
There was no maternal mortality in this study. Maternal toxicity was indicated by reduced body weights on gestational day (gd) 9, 12, 15, and 18 but not on gd 21, and reduced weight gain for the intervals gd 6-9, 6-12, 6-15 (exposure period) and gd 6-18, but not for the post-exposure period (gd 15-21). There were no treatment-related effects on maternal liver, thymus or gravid uterine weight, or on body weight (absolute or corrected for gravid uterus) at sacrifice; histologic examination of the maternal brains showed no pathological effects related to treatment. There were also no effects of treatment on the number of ovarian corpora lutea, of total, non-viable or viable implantations per litter, or on pre- or post-implantation loss or on sex ratio. There were no maternal deaths, early deliveries or abortions. Pregnancy rate was high and equivalent across all treatment groups. One dam each at 0, 50 and 200 ppm carried a totally resorbed litter on gd 21. Two dams at 400 ppm had totally resorbed litters on gd 21. Clinical observations were made daily throughout the study. Most of the observations were limited to the eyes, nose and blood at the vaginal orifice and only in a few dams per group. In addition, urogenital area wetness was present in a few dams only at 0, 50, 200 ppm (not at 400 ppm). At sacrifice on gd 21, there was no effect of exposure on maternal body weight, maternal body weight corrected for gravid uterine weight or on absolute or relative (to corrected body weight) thymus weight. Absolute and relative liver weight was elevated at 200 but not at 400 ppm. Administration of test substance vapour by inhalation to timed pregnant Fischer F-344 rats during organogenesis at 0, 50, 200 and 400 ppm resulted in matemal toxicity at 400 ppm.
Based on a significantly reduced body weight gain in the 400 ppm exposure group the NOAEL/LOAEL derived for maternal toxicity is 200 and 400 ppm, respectively.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

831 mg/m³ air

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

LOAEL

Effect level:

1 661 mg/m³ air

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Significant reduction in female body weight per litter at 400 ppm (all fetuses, males and females approximately 10 %) and at 200 ppm (all fetuses, and males but not females approximately 3%). One visceral variation (partial fetal atelectasis) significantly increased at 400 ppm. 17 out of 79 observed skeletal variations exhibited significant changes in incidence and indicated a consistent pattern of reduced ossification in the 400 ppm group (for example poorly or unossified phalanges, unossified cervical or poorly ossified thoracic centrum). No differences among the groups in the incidence of external, visceral or skeletal malformations. No further treatment related effects. The NOAEL/LOAEL for developmental toxicity is 50 and 200 ppm, respectively, which is based on reduced fetal weights in male fetuses at 200 ppm and in male and female fetuses at 400 ppm and a consistent pattern of reduced fetal ossification at 400 ppm.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

1 661 mg/m³ air

Based on:

test mat.

Basis for effect level:

other: teratogenicity

2

Dose descriptor:

NOAEL

Effect level:

208 mg/m³ air

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

3

Dose descriptor:

LOAEL

Effect level:

831 mg/m³ air

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 1: Body Weight of Foetuses Removed on Gestational Day 21 from 2,4-pentanedione - Vapour-exposed and Air-alone Control Fischer 344 Rats.

 

Foetal weight per litter (g) (Means± SD)	2,4-pentanedione analytical concentration (ppm)
	0	53	202	398
Number of litters	22	20	23	20
All foetuses	4.57 ± 0.20	4.55 ± 0.23	4.42 ± 0.14 e	4 .10 ± 0.14 f
Male fetuses	4.71 ± 0.18 b	4.68 ± 0.24 c	4.57 ± 0.19 e	4.24 ± 0.14 d,f
Female fetuses	4.40 ± 0.19 b	19b 4.41 ± 0.21	4.28 ± 0.17	3 .95 ± 0.14 d,f

 

b: N = 21 because one litter contained only female foetuses and one litter contained only male foetuses at 0

ppm

c: N = 19 because one litter contained only female foetuses at 53 ppm

d: N = 19 because one litter contained only female foetuses and one litter contained only male foetuses at 398 ppm

e: p < 0.05 (compared to controls)

f: p < 0.001 (compared to controls)

Table 2: Significant Variations Observed in Foetuses and Litters: Dams Exposed to 2,4-Pentanedione Vapour on Gestational Days 6 through 15^a.

	Foetuses				Litters
Group (ppm)	0	53	202	398	0	53	202	398
Number examined externally, b
No significant findings	183	162	195	167	22	20	23	20
Number examined Viscerally, c	96	87	104	90	22	20	23	20
Partial Foetal Atelectasis	8	15	21	14	5	10	9	12, f
	8.3	17.2	20.2	15.6	22.7	50.0	39.1	60.0
Number examined Skeletallv, d	87	75	91	77	22	20	23	19, e
Cervical centrum #6 unossified	2	2	2	10	2	1	2	9, f
	2.3	2.7	2.2	13.0	9.1	5.0	8.7	47.4
Cervical centrum #6 bilobed	18	13	7	1	14	9	6, f	1, g
	20.7	17.3	17.7	1.3	63.6	45.0	26.1	5.3
Cervical centrum #5 unossified	6	5	10	34	6	4	10	16, g
	6.9	6.7	11.0	44.2	27.3	20.0	43.5	84.2
Cervical centrum #5 bilobed	9	6	4	1	8	6	4	1, f
	10.3	8.0	4.4	1.3	36.4	30.0	17.4	26.3
Thoracic centrum # 1 poorly ossified	0	0	4	7	0	0	4	5, f
	0.0	0.0	4.4	9.1	0.0	0.0	17.4	5.3
Premaxillary poorly ossified	2	0	2	10	2	0	2	8, f
	2.3	0.0	2.2	13.0	9.1	0.0	8.7	42.1
Some proximal Phalages (forelimb) poorly ossified	41	46	67	72	16	15	21	19, f
	47.1	61.3	73.6	93.5	72.7	75.0	91.3	100.0
Majoriry proximal phalages (hindlimb) poorly ossified	46	41	52	13	19	17	19	8, g
	52.9	54.7	57.1	16.9	88.4	85.0	82.6	42.1
Some proximal phalanges (hindlimb unossified)	58	55	68	16	20	18	21	10, f
	66.7	73.3	74.7	20.8	90.9	90.0	91.3	52.6
Majority proximal phalanges (hindlimb) unossified	8	11	9	19	5	8	8	11, f
	9.2	14.7	9.9	24.7	22.7	40.0	34.8	42.9
All proximal phalanges (hindlimb) Unossified	9	4	11	42	8	4	9	16, g
	10.3	5.3	12.1	54.5	36.4	20.0	39.1	84.2
Some metatarsals (hindlimb) poorly ossified	6	1	7	33	5	1	7	15, g
	6.9	1.3	7.7	42.9	22.7	5.0	30.4	78.9
Yiphoid process (sternebra #6) poorly ossified	7	3	7	30	5	3	7	15,g
	8.0	4.0	7.7	39.0	22.7	15.0	30.4	78.9
Number with external variations	14	16	20	13	10	10	11	9
Percent with external variations	7.7	9.9	10.3	7.8	45.5	50.0	47.8	45.0
Number with soft tissue variations	64	70	94	75	20	20	23	19
Percent with soft tissue variations	66.7	80.5	90.4	83.3	90.9	100.0	100.0	95.0
Number with skeletal variation s	87	75	91	77	22	20	23	19
Percent with skeletal variations	100.0	100.0	100.0	100.0	100.0	100.0	100.0	100.0
Total number with variations	152	146	186	153	22	20	23	19
Total percent with variations	83.1	90.1	95.4	91.6	100.0	100.0	100.0	95.0

^aFor all fïndings, the number (of foetuses affected or litters with one or more affected foetuses) is presented on top and the percentage of the total (foetuses of litters) examined is presented beneath. A single foetus may be represented more than once in listing individual defects. Only live foetuses were examined.

^bAll Foetuses were examined externally.

^cApproximately 50% of each litter were examined viscerally and for soft tissue craniofacial defects.

^dApproximately 50% of each litter were examined for skeletal defects after staining with alizarin red S.

^eOne litter consisted of only one live foetus. By convention in this laboratory, this foetus was examined for visceral and craniofacial alterations.

^fp < 0.05

^gp < 0.01