Bis(pentane-2,4-dionato)calcium

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Remarks:

The justification for read-across to 2,4-pentanedione is provided in IUCLID section 13

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment. Deficiencies (pointed out by former LR) are evaluted differently now (Details under: Any other information including materials and methods / Conclusions)

Justification for type of information:

Justification for the read-across approach to substance pentane-2,4-dione is provided in IUCLID section 13.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

1990

Reference 2

Reference Type:

other: document of national authority

Title:

Unnamed

Year:

1983

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Food consumption is not reported in Tyl et al 1990.

GLP compliance:

yes

Remarks:

Info in the publication: The study was conducted in full compliance with the Environmental Protection Agency Good Laboratory Practice Standards (EPA, 1983)

Test material

Test material information

Constituent 1

Reference substance name:

Pentane-2,4-dione

EC Number:

204-634-0

EC Name:

Pentane-2,4-dione

Cas Number:

123-54-6

Molecular formula:

C5H8O2

IUPAC Name:

pentane-2,4-dione

Details on test material:

2,4-Pentanedione (CAS No. 123-54-6; 2,4-PO) is a flammable liquid p-diketone of molecular weight 100.13, boiling point 140AoC (760 mm Hg), and vapour pressure 7 torr at 20°C (Hawley, 1977; Sax, 1979). 99.2% purity was used.

Specific details on test material used for the study:

Liquid 2,4-PD of 99.2% purity was used; there was no compositional change
over the study period.

Test animals

Species:

rat

Strain:

other: Fischer 344 inbred albino rats (Harlan Fischer F-344/ HarBR)

Details on test animals or test system and environmental conditions:

Virgin male and female Fischer 344 inbred albino rats (Harlan Fischer F-344/
HarBR) were quarantined for 2 weeks, during which time representative animals
were examined for faecal parasites, bacterial growth in aerobic cultures of lung,
histologic changes in selected visceral tissues and the presence of serum viral
antibody examination for 11 rodent viruses including sialodacryoadenitis. These
quality control data indicated that no rats were positive in these tests and that
the animals were suitable for use. Rats were housed in stainless steel wire-mesh
cages with food (Certified Rodent Chow, Ralston Purina Co., St. Louis, MO)
and water available ad libitum, except during vapour exposures. Animals were
kept on a 12 hr light/dark photo period.

Administration / exposure

Route of administration:

inhalation: vapour

Vehicle:

air

Details on exposure:

Vapour Generation and Test Conditions
Liquid 2,4-PD of 99.2% purity was used; there was no compositional change
over the study period. Atmospheres were generated by metering liquid 2,4-PD
into a heated glass evaporator (Carpenter et aI., 1975), 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-PD into the evaporator. Each
chamber volume was 4320 litres, and the airflow was 1000 litres/min (14 air
changes/hr).

There were three vapour exposure groups (high, intermediate, and low concentration)
and one air-alone control group (0 ppm). Target concentrations were 0, 50, 200 and 400 ppm of 2,4-PD vapour, and
exposures were for 6 hours a day.

Chamber temperature, relative humidity, and airflow rate were recorded at least
4 times in each 6 hr period. Animal cages were rotated daily to compensate for
any variation in chamber exposure conditions. Each chamber atmosphere was
analyzed for 2,4-PD 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-PD liquid used and the airflow rate. 2,4-PD concentrations
were measured using a Perkin-Elmer Model 3920B gas chromatograph
equipped with a fla:me ionization detector. The column was a 1O-foot X
118 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-PD
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.

Chamber temperature and relative humidity for the 2,4-PD and air-control groups are shown in Table 1 (of the publication Tyl et al 1990).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

2,4-PD vapor concentrations for the 2,4-PD and air-control groups are shown in Table 1 (of the publication Tyl et al 1990, see attachment).
All 2,4-PD vapour concentrations measured analytically were within 99.5 to 105.0% of the target
concentrations. Average [Analytical/Nominal] ratios were in the range of 0.93
to 0.96, indicating no decompositional changes, and no significant chamber losses
of metered 2,4-PD.

Details on mating procedure:

Rats were mated one male:one female in stainless steel wire-mesh cages,
and the day a copulation plug was found was designated as gestational day (gd) 0.

Duration of treatment / exposure:

Timed-pregnant Fischer 344 rats were exposed on gestational days (gd) 6 to 15.
Sacrifice on gd 21 by CO2 asphyxiation.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

53 ppm (analytical)

Remarks:

50 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

202 ppm (analytical)

Remarks:

200 ppm (nominal)

Doses / concentrations 4

Dose / conc.:

398 ppm (analytical)

Remarks:

400 ppm (nominal)

No. of animals per sex per dose:

Twenty-five plug-positive females were assigned by stratified randomization by body
weight to each experimental group on gd 0, and housed individually throughout the study.

Control animals:

yes, concurrent vehicle

Details on study design:

The choice of concentrations was based on a range-finding study in timed-pregnant Fischer 344 exposed to 100, 200,400 and
700 ppm on gestational days (gd) 6 through 15 for 6 hr/day. All dams exposed to 700 ppm died prior to gd 21. Maternal body weight gain was decreased at
400 and 700 ppm (gd 6-9) and clinical signs of toxicity were observed at all exposure concentrations. Foetal body weight was reduced at 400 ppm, but the
reduction was not statistically significant.

Examinations

Maternal examinations:

Maternal body weights were measured on gd, 0, 6 (pre-exposure period), 9, 12, 15 (post-exposure period); and 18 and 21 (post-exposure period).

Maternal brains and thymuses were removed and fixed in buffered neutral 10% formalin. Maternal body cavities
were opened by midline thoracolaparotomy. Maternal thymus, liver and gravid uterine weights were determined.

Brain Histology
Maternal brains, removed at sacrifice, were fixed in neutral 10% formalin and processed for paraffin embedding; 5 urn sections were stained with haematoxylin and eosin. Each brain was divided into 6 to 9 coronal sections which included the basal ganglia and deep cerebellar nuclei.

All in-life necropsy and postnecropsy evaluations were performed without knowledge of the exposure concentrations of the dams or foetuses.

Ovaries and uterine content:

The gravid uterus, ovaries, cervix, vagina, and abdominal and thoracic cavities were examined by gross inspection.
The uteri were immediately ligated at their cervical end to prevent expulsion of
conceptuses by myometrial peristalsis. Ovarian corpora lutea were counted. The uteri
were examined externally and dissected longitudinally to expose their contents.

Fetal examinations:

The numbers of live and dead fetuses and early and late resorption sites were
recorded, and uteri from females that appeared nongravid were placed in 10%
ammonium sulphide solution for detection of early resorptions.

All foetuses were weighed, sexed, and examined for external malformations
including cleft palate, and variations.

One half of the foetuses in each litter were examined for thoracic and abdominal visceral abnormalities using a modification
of the method of Staples (1974). These foetuses were decapitated and their heads
were fixed in Bouin's solution for examination of craniofacial structures by sectioning
methods modified from Wilson (1965, 1973) and van Julsingha and Bennett
(1977).

The remaining intact foetuses in each litter were eviscerated,
processed for skeletal staining with alizarin red S (Dawson, 1926; Peltzer and
Schardein, 1966), and then examined for skeletal malformations and variations.

All in-life necropsy and postnecropsy evaluations were performed without knowledge
of the exposure concentrations of the dams or foetuses.

Statistics:

The unit for comparison was the pregnant female or the litter (Weil, 1970).
Results of quantitative analytical variables (eg. maternal body weights, organ
weights, foetal body weights) were intercompared for the 2,4-PD-exposed groups
and air-alone group by the use of the Levene (1960) 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 (Brown and Forsythe, 1974).
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 (Sokal and Rholf, 1969).
For all statistical tests, a fiducial limit of 0.05 (two-tailed) was used as the criterion for significance.

Historical control data:

See also under Any other information on materials and methods incl. tables.

No historical control data in Tyl et al 1990 presented.

Historical control data may add value to the presented information in some cases. However, the experimental design of a study with pre-determined group sizes is in general set-up conclusively in itself and before the start of the treatments. If one accepts historical control data one also accepts the extension of the control group while not extending the treatment groups to the same extent or with the analogous volume of data. Furthermore, the historical control data is added in retrospect (unevenly only for the control group) with data generated at least at partially different conditions and although the actual study on a particular substance has already been concluded.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Details on maternal toxic effects:
There were no maternal deaths, early deliveries, or abortions. Pregnancy rate
was high and equivalent across the groups, and the number of totally resorbed
litters seen on gd 21 was low (Table 2, Tyl et al 1990).
There were no concentration-related clinical signs of toxicity in the dams during
exposure or in the post-exposure period. Maternal toxicity was indicated by
reduced body weights at 398 ppm on gd 9, 12, 15 and 18, but not on gd 21.
Also, maternal weight gain was reduced at 398 ppm for the intervals gd 6-9, 6-
12,6-15, and 6-18, but no difference was present for the postexposure gd inten:al,
gd 15-21. There was a very slight, nonstatistically significant, reduction
in body weights at 202 ppm for gd 6·9, 6-12, and 6-15 (Table 3, Tyl et al 1990). At sacrifice on
gd 21 there were no effects of 2,4-PD vapour concentration on maternal body
weight (total or corrected for gravid uterine weight), or on absolute or relative
(to corrected body weight) thymus weight. Absolute and relative liver
weights were increased at 202 but not 398 ppm (Table 4, Tyl et al 1990). Light microscopical
examination of maternal brains revealed no evidence of gliosis, and no treatment-related
differences in the low incidence of other neuropathological findings (Table 5, Tyl et al 1990).

Detailed information is provided in tabular form in Tyl et al 1990, see attachment.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEC

Effect level:

202 ppm (analytical)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Conversion of NOAEC [ppm] into NOAEC [mg/m3] was calculated (see below).

2

Key result

Dose descriptor:

NOAEC

Effect level:

827 mg/m³ air (analytical)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Conversion NOAEC: NOAEC [mg/m3] = NOAEC [ppm] x Molecular mass [100.12 g/mol] / Molar volume [24.45 L]

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
There was no effect of 2,4-PD vapour exposure on the number of corpora lutea,
on the number of total, nonviable or viable implantations per litter, on the
percent pre- or post-implantation loss, on the percent live foetuses per litter, or
on the foetal sex ratio (Table 6, Tyl et al 1990). On gd 21, foetal body weight per litter was
significantly reduced at 398 ppm for males, females, and all foetuses and at 202
ppm for males and all foetuses. Female foetal weight was also reduced at 202
ppm, but the difference was not statistically significant relative to that in the airalone
control group (Table 7, Tyl et al 1990).

The incidence of external variations was unaffected by treatment. The incidence
of one visceral variation (out of 46 observed), partial foetal atelectasis, was
increased at 398 ppm relative to that of the controls (Table 8, Tyl et al 1990). A total of 17
skeletal variations (out of 79 observed) exhibited statistically significant changes
in incidence in the 398 ppm group, suggesting a consistent pattern of fetotoxicity
at the high concentrations. These skeletal variations are shown in Table 8 (Tyl et al 1990). There
appeared to be a slightly increased number of foetuses (but not litters) exhibiting
visceral and total variations at aIl2,4-PD vapor concentrations, but the incidences
were not dose related and were not statistically significantly different from those
of the control. There were no differences among the groups in the incidence of
individual malformations, malformations by category (external, visceral and skeletal),
or total malformations (Table 9, Tyl et al 1990).

Detailed information is provided in tabular form in Tyl et al 1990, see attachment.

Effect levels (fetuses)

1

Key result

Dose descriptor:

NOAEC

Effect level:

53 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: fetal weight

Remarks on result:

other: Conversion of NOAEC [ppm] into NOAEC [mg/m3] was calculated (see below).

2

Key result

Dose descriptor:

NOAEC

Effect level:

217 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: fetal weight

Remarks on result:

other: Conversion NOAEC: NOAEC [mg/m3] = NOAEC [ppm] x Molecular mass [100.12 g/mol] / Molar volume [24.45 L] = 217.0 mg/m3

Remarks:

// molar volume at 1 atm and 25°C is 24.45 L

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

827 mg/m³ air (analytical)

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

yes

Any other information on results incl. tables

Please find details on results in the attached pdf file (= Tyl et al 1990).

Detailed results are provided in Tyl et al 1990 allocated to the treatment/control groups (0 ppm, 53 ppm, 202 ppm, 398 ppm) in tabular form for the following examined parameters:

Maternal body weights and body weight gain of pregnant Fischer 344 rats exposed to 2,4-pentanedione vapour on gestational days 6 through 15

Pregnancy and litter data for vapour-exposed groups and air-alone control group

Maternal body and organ weights at sacrifice on gestational day 21 for Fischer 344 rats exposed to 2,4-pentanedione vapour on gestational days 6 through 15

Histological findings in brains removed from 2,4 -pentanedione vapour-exposed and air-alone control Fischer 344 rats after sacrifice on gestational day 21

Gestational findings in 2,4-pentanedione vapour-exposed and air-alone control Fischer 344 rats

Body weight of fetuses removed on gestational day 21 from 2,4-pentanedione vapour-exposed and air-alone control Fischer 344 rats

Significant variations observed in foetuses and litters; dams exposed to 2,4-pentanedione vapour on gestational days 6 through 15

Malformations observed in foetuses and litters; dams exposed to 2,4-pentanedione vapour on gestational days 6 through 15

Applicant's summary and conclusion

Conclusions:

The present study has shown that exposure of pregnant Fischer 344 rats to 398 ppm 2,4-PD vapour for 6 hours a day during the period of organogenesis resulted
in maternal toxicity in the form of reduced body weight and body weight gain. The depression of body weight was observed during the exposure period, but
there was full recovery by sacrifice on gd 21. No such significant maternal toxicity occurred at 202 ppm. These findings accord with those in a 9-day repeated
exposure study with 2,4-PD vapour, in which decreases in body weight and body weight gain occurred in both male and non-pregnant female Fischer 344 rats
during exposure to 418 ppm but not at 197 ppm 2,4-PD (Dodd et al., 1986).

Foetotoxicity, considered as nonspecific, was seen at 398 ppm 2,4-PD vapour as reduced foetal body weight and a consistent pattern of reduced skeletal ossification, and at 202 ppm (= 827.16 mg/m3) as reduced foetal body weight.
The reduction in skeletal ossification variations were located in those skeletal districts which ossify last, and are considered to be a sensitive indication of foetotoxicity (Aliverti et al., 1979).
Neither embryotoxicity nor teratogenicity that could be considered specific to 2,4-PD was seen at any exposure concentration employed in this study.

In the current developmental toxicity study, no clinical or morphological evidence for neurotoxicity was found in pregnant Fischer 344 rats exposed to
398 ppm 2,4-PD vapour for 6 hours a day over 10 consecutive days.

---------

Statement of former lead registrant:
In conclusive. Insufficient data. Reduced body weights and effects on ossificaton wrere noted, in the high dose group in the presence reduced maternal body weights as well as reduced fetal weights in the mid-dose group. The effects on ossification not necessarily considered adverse.

Evaluation and statement of the present lead registrant:
The data on pentane-2,4-dione gives valuable information on the developmental toxicity of this substance and is regared as meaningful and suitable to fulfil the REACH information requirements for the substance Bis(pentan-2,4-dionato)calcium (CAS No 19372-44-2). The read-across justification is provided in IUCLID section 13. The results are regarded as conclusive but not sufficient for classification.

Executive summary:

Timed-pregnant Fischer 344 rats were exposed on gestational days (gd) 6 to 15 inclusive to analytically measured concentrations (as mean ± SD) of 53 ± 1.6, 202 ± 4.7 and 398 ± 5.7 ppm 2,4-PD vapour. At sacrifice (gd 21) foetuses were examined for external, visceral and skeletal variations and malformations. There was no maternal mortality, and body weight was reduced only at 398 ppm. Histological examination of maternal brains from the 398 ppm group showed no abnormalities. No treatment-related effects were seen on number of corpora lutea; total, nonviable or viable implants per Litter; pre-or post-implantation losses; or foetal sex ratio. Reduced foetal body weight per litter was seen at 398 ppm (males and females and all foetuses) and 202 ppm (males and all foetuses). There was no concentration-related, or statistically significant, increase in the incidence of individual malformations, malformations by category (external, visceral or skeletal), or total malformations. Partial foetal atelectasis was increased at 398 ppm, and the increased incidence of 17 skeletal variants (out of 79 observed) indicated a consistent pattern of foetotoxicity at 398 ppm. In summary, at 398 ppm there was maternal toxicity (reduced body weight) and foetotoxicity (reduced body weight and ossification) and at 202 ppm there was foetotoxicity (reduced body weight). Embryotoxicity or teratogenicity were not seen at any concentration. The no-observable-effects concentration was 53 ppm for developmental toxicity and 202 ppm for maternal toxicity.