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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)

Data source

Reference
Reference Type:
publication
Title:
2,4-Pentanedione: 9-Day and 14-Week Vapor Inhalation Studies in Fischer-344 Rats
Author:
Dodd et al.
Year:
1986
Bibliographic source:
Fundam. Appl. Toxicol. 1986, 7, 329-339.

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
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

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female Fischer 344 rats, 24 to 38 days of age, were obtained from Charles River Breeding Laboratories, Inc., Kingston, New York.
They were confirmed to be free from intestinal parasites, and on the basis of histologic examination of tissues from selected animals, did not have any common rodent diseases. They were clinically normal upon receipt and during a 2-week acclimatization period prior to the start of exposures.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
not specified
Details on inhalation exposure:
Chambers with a volume of 4.3 m3 operated at an airflow rate of 1000 liters/min were used. The chambers were constructed of stainless steel and had glass observation ports. Temperature and relative humidity were measured at least four times during each exposure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations of 2,4-PD vapor were analyzed approximately once every 33 min during the 6-hr exposure periods using a Perkin-Elmer Model 3920B gas chromatograph equipped with a flame ionization detector.

Analytically determined concentrations of 2,4-PD vapor in the exposure chambers were close to target values:

Target concentration (ppm)
Exposure week 100 300 650

1 103 ± 10* 282 ± 23 605 ± 39
2 101 ± 2 308 ± 4 651 ± 5
3 103 ± 1 316 ± 2 663 ± 7
4 102 ± 2 310 ± 2 654 ± 4
5 103 ± 1 313 ± 6 661± 2
6 104 ± 2 311 ± 8 663 ± 9
7 99 ± 2 313 ± 4 653 ± 24
8 99 ± 1 312 ± 3 661 ± 4
9 99 ± 1 306 ± 3 650 ± 4
10 98 ± 0 307 ± 3 644 ± 5
11 99 ± 1 307 ± 2 660 ± 15
12 99 ± 2 305 ± 3 645 ± 4
13 99 ± 2 302 ± 1 641 ± 5
14 99 ± 1 302 ± 2 640 ± 6
Mean ± SD of
weekly means 101 ± 2 307 ± 8 650 ± 15

*Weekly mean ± SD concentration in ppm.

The analytical/nominal concentration ratios for the three test atmospheres ranged from 0.89 to 0.95, indicating no decompositional changes or significant chamber losses of metered 2,4-PD.
Duration of treatment / exposure:
Exposures were for 6 hr/day, 5 days a week for 14 weeks, with some animals being sacrificed during the 14th week.
Frequency of treatment:
The number of 6-hr exposures was 67 for male rats and 68 for females rats.
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
air control group
Dose / conc.:
100 ppm
Dose / conc.:
300 ppm
Dose / conc.:
650 ppm
No. of animals per sex per dose:
20 males and 20 females for each exposure group, and an additional 10 males were added to the high dose and control groups for glutaraldehyde perfusion and subsequent transmission electron microscopic examination of sciatic and tibial nerves.
Control animals:
yes, sham-exposed
Details on study design:
Dose selection rationale:
- Based on results obtained in previous 9-day study.

Examinations

Observations and examinations performed and frequency:
Animals were observed daily for signs of toxicity. Body weights were measured the morning before the first exposure, weekly during the exposure period, and immediately before sacrifice.
Individual animal food and water consumption was measured for approximately 15 h in metabolism cages with 10 males and 10 females from each exposure concentration; measurements were made during exposure week 14.
Serum chemistry and hematologic evaluations were performed on blood samples collected from survivors at the end of the 14-week exposure or 4-week recovery periods.
Animals that were moribund or found dead and survivors sacrificed at the end of the exposure regimen and recovery periods were subjected to necropsy.
Sacrifice and pathology:
Animals that were moribund or found dead and survivors sacrificed at the end of the exposure regimen and recovery periods were subjected to necropsy. Organ weights were measured for liver, lungs, brain, heart, thymus, kidneys, and testes in survivors at scheduled sacrifices. Selected tissues for histologic evaluation were as follows: nasal turbinates, larynx, trachea, lungs, epididymides, testes, spleen, thymus, urinary bladder, adrenal glands, brain (5 sections), thyroid, parathyroids, heart, kidney, pituitary, skeletal muscle (gastrocnemius), sternal bone, spinal cord (lumbosacral region), and liver.
Other examinations:
For ultra-structural evaluation of the sciatic and tibial nerves, five high concentration and five control male rats were sacrificed at the end of the 14-week exposure and perfused through the left ventricle with saline followed by a buffered mixture of 4% formaldehyde and 1% glutaraldehyde.
Statistics:
Results of quantitative continuous variables were intercompared among the three concentration groups and the one control group by use of analysis of variance, Bartlett's homogeneity of variance, and Duncan's multiple range tests. The latter was used to delineate which exposure groups differed from the control, when F from the analysis of variance was significant. If Bartlett's test indicated heterogenous variances, all groups were compared by an analysis of variance for unequal variances followed, if necessary, by t tests. Corrected Bonferroni probabilities were used for t test comparisons. A fiducial limit of 0.05 (two-tailed) was used as the critical level of significance for all comparisons.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
General signs of toxicity and sensory irritancy, seen at 650 ppm, were complete or partial eyelid closure, periocular, perinasal, and perioral encrustation, wetness around the urogenital area, hypoactivity, lack of coordination, paresis, ataxia, irregular gait, hypothermia, and emaciation.
At 650 ppm, all 20 females and 10 of 30 males died between the second and sixth weeks of exposure. These animals had acute degenerative changes in the deep cerebellar nuclei, vestibular nuclei and corpora striata, and acute lymphoid degeneration in the thymus.
Mortality:
mortality observed, treatment-related
Description (incidence):
At 650 ppm, all 20 females and 10 of 30 males died between the second and sixth week of exposure. The earliest death was after 9 days of exposure.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of the 650-ppm males were reduced throughout the 14-week exposure period, but there was considerable weight increase during the 4-week recovery period. However, final weights were still statistically significantly below those for the control males. Before death, body weights of the female rats of the 650-ppm group were considerably reduced. Also, females of the 307-ppm group had slight, but statistically significant, decreased body weight gains for study Days 45 through 121. However, at the conclusion of the 4-week recovery period, the difference between the absolute body weight means for the control and 307-ppm female rats was not statistically significant. Body weights of the 101-ppm-exposed rats were similar to the controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Even though test substance was administered via inhalation, no treatment-related alterations in food and water intake were observed at the conclusion of the 14 week exposure regimen.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Even though test substance was administered via inhalation, no treatment-related alterations in food and water intake were observed at the conclusion of the 14 week exposure regimen.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
For the 650-ppm males and the 307-ppm females, the respective mean erythrocyte counts were 11 and 4% below control values. The decrease in red blood cell count was accompanied with slight increases in MCV and MCH and a mild decrease in hematocrit. The white blood cell count was increased in the 650-ppm male rats, due to an increase in lymphocytes. There was recovery from these effects by the end of the 4-week postexposure period.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Clinical chemistry determinations which deviated from controls were an increase in urea nitrogen and alkaline phosphatase activity, and a decrease in creatinine, calcium, and aspartate aminotransferase (AST) activity in the 650-ppm males. Serum calcium was also decreased in males and females of the 307-ppm group. Although a mild increase in alkaline phosphatase activity persisted in the 650-ppm recovery male rats, the remaining serum chemistry values were similar to control values, indicating reversibility of effects.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urinalysis showed minimal alterations in males, which included low pH (6.0 vs 7.0 in controls) at 650 ppm, and slightly increased bilirubin and urobilinogen at 650 and 307 ppm. These alterations in urine parameters were not found at the end of the postexposure observation period.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Neurobehavioural effects were observed (see neuropathological findings).
Immunological findings:
effects observed, treatment-related
Description (incidence and severity):
Thymic lesions, consisting of acute lymphoid degeneration and atrophy, were observed in 7 of 10 male and 13 of 20 female rats that died during the 14-week exposure regimen. They were not present in survivors. Dermatitis, dermal necrosis, and in a few cases, cellulitis were seen in the skin and subcutis of the heads of 5 male and 8 female rats that died, but not in survivors. The inflammatory infiltrate was predominately of polymorphonuclear leukocytes and mast cells.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
After 14-weeks of 2,4-PD vapor exposure a statistically significant decrease in most absolute organ weights was observed for the 650-ppm males, but organ weights relative to body weight had increased. Organ weights of the 101- and 307-ppm groups were unaltered, except for absolute lung weights of the females which were slightly increased at 101 and slightly decreased at 307 ppm.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Thymic lesions, consisting of acute lymphoid degeneration and atrophy, were observed in 7 of 10 male and 13 of 20 female rats that died during the 14-week exposure regimen. They were not present in survivors. Dermatitis, dermal necrosis, and in a few cases, cellulitis were seen in the skin and subcutis of the heads of 5 male and 8 female rats that died, but not in survivors.
Neuropathological findings:
effects observed, treatment-related
Description (incidence and severity):
The brain lesions observed in 7 of 10 male and 18 of 20 female rats that died during the exposure regimen took the form of acute degenerative changes in the vestibular nuclei, deep cerebellar nuclei, and corpora striata. The lesions consisted of peracute to acute areas of pallor and vacuolation in the neuropil. Within these foci were nuclear pyknosis and karyorrhexis, and the presence of macrophages with vacuolated cytoplasm. The degree of vacuolation and macrophage infiltration was greatest in the deep cerebellar and vestibular nuclei. These brain lesions were bilaterally symmetrical. Of the surviving males of the 650-ppm group, 7 of 5 had gliosis with or without malacia in the deep cerebellar nuclei, vestibular nuclei, and corpus striatum. Gliosis was most extensive in the deep cerebellar and vestibular nuclei. Many of the cells had irregular or rod-shaped nuclei typical of microglia, but others resembled astrocytes. Multifocal areas of microscopic mineralization, hemosiderosis, and neuronal degeneration were evident. No degenerative changes were seen in the spinal cords of the 650-ppm exposed animals. Transmission electron microscopic evaluation of sciatic nerves from the 650-ppm group did not produce any evidence of a peripheral neuropathy.
In addition, neurobehavioural findings were are follows:


Duration of exposure regimen (months)
Observation 1 2 3 3+1 month recovery

Tremors 1/26* 0/20 0/20 0/5
Impaired gait 4/26 0/20 2/20 0/5
Paresis 4/26 0/20 0/20 0/5
Lacrimation 2/26 1/20 0/20 0/5
Hypothermia 3/26 0/20 0/20 0/5
Abnormal surface righting 2/26 0/20 0/20 0/5
Abnormal midair righting 6/26 3/20 4/20 0/5
Abnormal wire grasping 4/26 0/20 0/20 0/5
Abnormal body tone 6/26 0/20 0/20 0/5
Abnormal limb rotation 3/26 0/20 0/20 0/5
Dilated pupils 6/26 0/20 0/20 0/5
Decreased breathing rate 2/26 0/20 0/20 0/5
Decr. locomotor activity 6/26 0/20 0/20 0/5
Absence of corneal reflex2/26 0/20 0/20 0/5
Abs. of tail pinch resp. 3/26 0/20 0/20 0/5
Abs. of audit. startle resp. 6/26 0/20 0/20 0/5
* No. affected/No. examined.

No effects were observed in the control rats or rats exposed to 101 or 307 ppm.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Light microscopy did not reveal any abnormalities in tissues removed from the animals of the 101- and 307-ppm groups. However, for the 650-ppm animals there were noteworthy histopathologic features in brain, thymus, skin, and nasal mucosa.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The nasal mucosa showed mild multifocal squamous metaplasia at the anterior portion of the nasal cavity, in both the maxillary and nasal turbinates. This change was seen in 9 of the 10 animals exposed to 650 ppm which were sacrificed at the end of the 14-week exposure period, but was present in only one of five and with reduced severity following the 4-week recovery period. The nasal mucosa was normal in female rats exposed to 307 ppm of 2,4-PD vapor.
Other effects:
no effects observed

Effect levels

open allclose all
Dose descriptor:
NOEC
Effect level:
101 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: neoplastic
immunology
mortality
neuropathology
ophthalmological examination
organ weights and organ / body weight ratios
urinalysis
water consumption and compound intake
Dose descriptor:
LOEC
Effect level:
307 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
haematology
urinalysis
Key result
Dose descriptor:
NOEC
Remarks:
calculated from ppm (see 'Overall remarks')
Effect level:
420 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: neoplastic
immunology
mortality
neuropathology
ophthalmological examination
organ weights and organ / body weight ratios
urinalysis
water consumption and compound intake
Key result
Dose descriptor:
LOEC
Remarks:
calculated from ppm (see 'Overall remarks')
Effect level:
1 277.12 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
haematology
urinalysis

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The repeated inhalation exposure of acetylacetonate by vapour atmospheres for a period of 14 consecutive weeks at target concentrations of 100, 300 and 650 ppm produced treatment-related changes at the two higher concentrations. On this basis, a ‘No Observed Effect Concentration’ (NOEC) of 420 mg/m3 could be derived. The lowest effect concentration was 1277.12 mg/m3.

Executive summary:

In this study (similar to OECD Guideline 413), acetylacetonate was administered to assess the toxic effects by inhalation of the acetylacetonate test item as a vapour to rats (20/sex/group) for 6 hours per day, 5 days per week for 14 weeks at target concentrations of 100, 300 and 650 ppm. An additional 10 males were added to the high dose and control groups for glutaraldehyde perfusion and subsequent transmission electron microscopic examination of sciatic and tibial nerves. Control animals (20/sex) received air only. Chamber concentrations of the test item vapor were analyzed approximately once every 33 min during the 6-hr exposure periods using a Perkin-Elmer Model 3920B gas chromatograph equipped with a flame ionization detector.

Animals were observed daily for signs of toxicity. An Irwin screen for the assessment of neurobehavioural effects was performed prior to the first exposure and monthly thereafter, including just before sacrifice of the recovery animals. Body weight measurements were recorded weekly, once before the first exposure, body weight measurements were also obtained just prior to sacrifice.  Animals of the 4-week recovery period were weighed weekly and immediately before sacrifice. Individual animal food and water consumption was measured for approximately 15 hr in metabolism cages with 10 males and 10 females from each exposure concentration; measurements were made during exposure week 14. Urine was collected while animals were in the metabolism cages. Serum chemistry and hematologic evaluations were performed on blood samples collected from survivors at the end of the 14-week exposure or 4-week recovery periods. Animals were sacrificed, selected organs were weighed and organ/body weight ratios were calculated. A complete gross post-mortem examination was conducted on all animals followed by histologic examination of selected tissues.

The mean analytical exposure concentrations were 101, 307 and 650 ppm for the low, mid and high dose groups, respectively. At 650 ppm, all females and 10 of 30 males died between the second and sixth weeks of exposure. These animals had acute degenerative changes in the deep cerebellar nuclei, vestibular nuclei and corpora striata, and acute lymphoid degeneration in the thymus. Seven of 15 male survivors of the 650 ppm group had gliosis and malacia in the same brain regions, minimal squamous metaplasia in the nasal mucosa, decreased body and organ weights, lymphocytosis, and minor alterations in serum and urine chemistries. No ultrastructural evidence of peripheral neuropathy was observed. Except for central neuropathy, many of the adverse effects at 650 ppm were less marked in the 4-week recovery animals. No deaths occurred at 307 ppm, but females had slightly decreased body weight gains, and in both sexes there were minor alterations in hematology, serum chemistry, and urinalysis parameters, which were not present in the 4-week recovery animals. Rats exposed to 101 ppm showed no differences from the control rats. Subchronic exposure to 650 ppm of acetylacetonate vapor causes serious adverse biological effects. Under these study conditions, the minimum-effects concentration was 1277.12 mg/m3, and the no-adverse effects concentration was 420 mg/m3.