Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1986-04-26 to 1986-04-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986
Reference Type:
publication
Title:
Unnamed
Year:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
Timed-pregnant Fischer F-344 rats (Harlan Fischer F-344/HarBR) 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 (0, 52.7, 202 and 398 ppm mean analytical concentrations, respectively) to evaluate the embryotoxic and fetotoxic (including tera-togenic) potential of the TS administered during organogenesis. The day a copulation plug was found was designated gestational day (gd) 0. Twenty-five plug-positive fe-males 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 histopa-thologically. Live fetuses were dissected from the uterus, counted, weighed and sexed and examined for external abnormalities. 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

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,4-Pentanedione
- Physical state: liquid
- Stability under test conditions: stable

Test animals

Species:
rat
Strain:
Fischer 344
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Indianapolis, IN, USA
- Age at study initiation: 81 days (males) and 88 days (females)
- Weight at study initiation: 154 to 168 g
- Fasting period before study: not reported
- Housing: three (during breeding) single (plug positive females)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: two weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): approx. 14
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:piston pump to meter liquid 2,4-Pentanedione
- Method of holding animals in test chamber: cage chambers
- Source and rate of air: not reported
- Temperature, humidity in air chamber: 74 to 75 °F, 43 to 46 %
- Air flow rate: 1000 l/min
- Air change rate: approx. 14



TEST ATMOSPHERE
- Brief description of analytical method used: Gas Chromatograph



Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gas Chromatograph
Details on mating procedure:
- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation:
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- Verification of same strain and source of both sexes: [yes / no (explain)]
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy
- Any other deviations from standard protocol:
Duration of treatment / exposure:
gestational days (GD) 6-15
Frequency of treatment:
6 h/day consecutive days
Duration of test:
13 days (treatment), animals were sacrificed on GD 21
Doses / concentrationsopen allclose all
Dose / conc.:
50 ppm (nominal)
Remarks:
210 mg/m3
Dose / conc.:
200 ppm (nominal)
Remarks:
840 mg/m3
Dose / conc.:
400 ppm (nominal)
Remarks:
1680 mg/m3
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: pretest on toxicity
- Rationale for animal assignment (if not random):random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: every day
- Cage side observations checked in table were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 6, 9, 12, 15, 18, 21


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: liver, thymus, gravid uterus weight
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [ half per litter ]
- Skeletal examinations: Yes: [ half per litter ]
- Head examinations: Yes: [ half per litter ]
Statistics:
Qunatitative continuous variables were intercompared by use of Levene's test, analysis of variances and t-tests-. When Levene's test indicated homogeneous variances, and ANOVA was significant, the pooled t-test was used for pairwise comparisons. When Levene's test indicated inhomogeneous variances all goups were compared by an ANOVA for uneual variances followed where necessary by a separate variance t-test for pairwise comparisonss.
Nonparametric data obtained following laparohysterectomy were statistically treated using the Kruskal-Wallis test followed by the Mann-Whitney U-testwhen appropriate, Incidence data were compared using Fisher's exact test. For all statistical tests the fiducial limit of 0.05 (two-tailed) was used as the criterion for significance.
Indices:
no data
Historical control data:
no data

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significant decrease in body weight gain in the HD group. During exposure period (GD 6-15) the body weight gain in the HD group was less than 50% compared to the control, i.e. 9.9 +/-7% in the HD group vs. 20.4 +/-5.6% in the control group. At sacrifice on GD 21 there was no effect efident on maternal body weight, i.e. retardation was compensated during the the last 6 days of exposure.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Absolute and relative liver weight was elevated at 200 but not at 400 ppm
Neuropathological findings:
no effects observed
Description (incidence and severity):
Histologic examination of the maternal brains indicated no effecs of exposure on any of the sections examined, including the regions of the basal ganlia and deep cerebellar nuclei.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
severely reduced body weight gain

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEC
Effect level:
200 ppm
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEC
Effect level:
400 ppm
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:
reduced fetal weights; reduced fetal ossification in the 200 and 400 ppm group

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
LOAEC
Effect level:
400 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduced fetal weights; and as a variation: reduced fetal ossification
Dose descriptor:
LOEC
Effect level:
200 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
changes in litter size and weights
Key result
Dose descriptor:
NOAEC
Effect level:
400 ppm
Based on:
test mat.
Basis for effect level:
other: embryotoxicity

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
200 ppm
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

Applicant's summary and conclusion

Conclusions:
Based on a significantly reduced body weight gain in the 400 ppm exposure group the NOAEC/LOAEC derived for maternal toxicity is 200 and 400 ppm, respectively. The NOEC/LOEC for developmental toxicity is 50 and 200 ppm, respectively, which is based on slightly but significantly reduced fetal weights in male fetuses at 200 ppm (<5%) and in male and female fetuses at 400 ppm and a consistent pattern of reduced fetal ossification at 400 ppm. Delayed ossification is considered as a result of the compensation of the dalay in maternal body weight gain after cessation of exposure. The NOAEC for embryotoxicity and teratogenicity is 400 ppm (highest dose tested).
Executive summary:

Groups of 25 timed-pregnant Fischer F-344 rats (Harlan Fischer F-344/HarBR) 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 (0, 52.7, 202 and 398 ppm mean analytical concentrations, respectively) 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. Twenty-five 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. 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.

There was no maternal mortality in this study. Significantly reduced body weight gain at 400 ppm; liver weight significantly increased at 200 ppm but no further significant effects were determined. Maternal toxicity was indicated by reduced body weights on 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. For fetal toxicity 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 were observed. 17 out of 79 observed skeletal variation 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 were observed among the groups in the incidence of external, visceral or skeletal malformations; no further treatment related effects. 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 2,4-pentanedione vapour by inhalation to timed-pregnant Fischer F-344 rats during organogenesis at 0, 50, 200 and 400 ppm resulted in maternal toxicity at 400 ppm. Fetotoxicity was observed at 200 and 400 ppm in terms of reduced fetal weights per litter (approximately 3 and 10 %, respectively) and at 400 ppm in terms of a consistent pattern of reduced fetal ossification. There was no evidence of embryotoxicity or teratogenicity at any exposure concentrations employed, including those which produced maternal toxicity. Based on a significantly reduced body weight gain in the 400 ppm exposure group the NOAEC/LOAEC derived for maternal toxicity is 200 and 400 ppm, respectively. The NOEC/LOEC for developmental toxicity is 50 and 200 ppm, respectively, which is based on reduced fetal weights  in male fetuses at 200 ppm (<5%) and in male and female fetuses at 400 ppm and a consistent pattern of reduced fetal ossification at 400 ppm. The NOAEC for embryotoxicity and teratogenicity is 400 ppm (highest dose tested).