Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1984-03-05 to 1985-07-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conduction and documentation of study acceptable. Literature reference and study report available.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
2,4-Pentanedione: 9-Day and 14-Week Vapor Inhalation Studies in Fischer-344 Rats
Author:
Dodd, D.E., Garman, R.H., Pritts, I.M., Troup, C.M., Snellings, W.M., and Ballantyne, B.
Year:
1986
Bibliographic source:
Fundam. Appl. Toxicol. 1986, 7, 329-339.
Reference Type:
publication
Title:
Central Neurotoxicity Induced By Subchronic Exposure To 2,4-Pentanedione
Author:
Garman, R.H., Dodd, D.E., Ballantyne, B.
Year:
1994
Bibliographic source:
Human and Experimental Toxicology, 14/8, 1995, 662-671
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report Date:
1985

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
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
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. (Kingston, NY, USA).
- Age at study initiation: 34 days
- Fasting period before study: not reported
- Housing: two/cage
- Diet (e.g. ad libitum): ad libitum, withheld during exposure
- Water (e.g. ad libitum):ad libitum, withheld during exposure
- Acclimation period:eighteen days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 65-80 °F (ususally 70 to 74 °F)
- Humidity (%): 13-84% (usually 40 to 50 %)
- Air changes (per hr): aprrox. 14
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 1984- 03-05 To: 1984-07-26

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: not reported
Details on inhalation exposure:
Liquid 2,4-pentanedione was metered from a piston pump into a heated-glass evaporator. The temperature in the evaporator was maintained at the lowest level sufficient to vaporize the liquid. The resultant vapor was carried into the chamber by a countercurrent air stream that entered the bottom of the evaporator.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The chamber concentrations of 2,4-pentanedione were analyzed approximately once each hour throughout the 68 days by gas chromatography. The concentration means were 650 (+/- 19.3), 307 (+/-10.3), and 101 (+/- 3.5) ppm for the target concentrations.
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
daily 6 h, 5 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 650 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
20 male and 20 female rats per group, with half being sacrificed  at the end of exposure period and the remaining after a 4 week recovery period for the determination of the reversibility of observable effects, were exposed to nominal concentrations of 0, 100, 300 and 650 ppm test substance, respectively. 10 male rats were added to control and high dose groups for glutaraldehyde perfusion and subsequent ultrastructural examination of sciatic nerves.
- Dose selection rationale: preliminary acute reports
- Rationale for animal assignment (if not random): random
- Post-exposure recovery period in satellite groups: yes
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
Toxicity monitoring: Following parameters were  determined: clinical signs of toxicity (daily), ophtalmoscopy of the eye  (before and after exposure), neurobehavioral screening (monthly before, during  and after exposure), body weight (weekly during the study and before  sacrifice), food and water consumption for 14 h in metabolic cages during the last exposure week (urinalysis), organ weights (liver, kidneys, lungs, brain, heart, thymus and testes), urine chemistry (n=10 each  group), serum chemistry and hematology of blood samples collected at the  end of exposure or the 4-week recovery; gross pathology at termination in  all groups; histopathology in high dose and control group as well as  brains of the medium dose group were processed for histopathology.
Sacrifice and pathology:
Survivors of the 14-week exposure regimen were sacrificed on June 28 (males) and June 29 (females), 1984. The postexposure rats were sacrificed on July 25 (males) and July 26 (females), 1984. The rats were killed by exsanguination via the brachial blood vessels following anesthesia with methoxyflurane.
Additional five male rats from the high concentration and control groups were sacrificed on June 28, 1984 and were perfused with a glutaraldehyde solution for the removal of the sciatic nerve for examination by transmission electron microscopy. Sciatic nerves were also examined by electron microscopy from five recovery high dose and control males although these rats were not perfused with a glutaraldehyde solution prior to sci-atic nerve removal.
Gross necropsies were performed, and selected tissues were fixed in 10% neutral buff-ered formalin. Histologic evaluation was performed on selected tissues from animals in the highest exposure level (males), intermediate exposure level (females), and control groups. This list of tissues is as followsepididymides, spleen, lungs, nasal turbinates, thymus, trachea, urinary bladder, adrenals, brain (5 sections), parathyroids, heart, kid-neys, larynx, testes, thyroids, pituitary, muscle-gastronemius, liver, sciatic nerve and sternal bone.
Other examinations:
A modified Irwin Screen (Irwin, 1968) was performed prioc,r to the first exposure and monthly thereafter (including sacrifice). This examination was also performed on the recovery animals at the time of sacrifice. The following measurements were examined: :tremors, convulsions, tail elevation, impaired gait, paresis, salivation, lacrimation, diar-rhea, piloerection, hypothermia, stereotyphy, surface righting, mid-air righting reflex, wire grasping, body tone, limb rotation, pupil response (pre-exposure only), pupil size, skin color, respiration, locomotor acivlty, corneal response, tail pinch, toe pinch, auditory startle response
Statistics:
Results of quantitative continuous variables (such as body weight changes) were inter-compared among the concentration groups and one control group by use of analysis of variance (ANOVA). 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 hetero-geneous variances, all groups were compared by an ANOVA for unequal variances fol-lowed if necessary by t-tests. Corrected Bonferroni probabilities were used for t-test comparisons. The fiducial limit of 0.05 (two-tailed) was used as the critical level of sig-nificance for all comparisons.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
In the 650 ppm group all females and 10/30 male rats died between the 2nd and 6th week. Rats of this dose group had severe clinical abnormalities (e.g. lacrimation, ataxia, hypoactivity, hypo-thermia). Survivors of the 650 ppm group had decreased body weight gains, decreased organ weights, and minor alterations in hematology (reduced hematocrit and red blood cell counts, increased mean corpuscular hemoglobin and volume), serum chemistry and urinary chemistry. Noteworthy lesions in animals that died after exposure to 650 ppm were acute degenerations in the deep cerebellar nuclei, vestibular nuclei and corpora striata and acute lymphoid degenerations in the thymus. Many of the survivors (7/15, non-recovery and recovery group combined) in this group had gliosis and malacia in the same brain regions but no peripheral neuropathy, minimal squamous metaplasia in the nasal mucosa, and lymphocytosis. Most of the observed alterations in male rats of the 650 ppm group that survived the 14 weeks exposure regimen decreased in frequency and/or severity after the 4 weeks recovery period. There were no substance related mortalities in the 300, 100 and 0 ppm groups. Also there was no evidence of clinical signs or histologic lesions in these rats. However, females of the 300 ppm group had slightly decreased body weight gains and in both sexes minor concentration related alterations in hematology, serum and urine chemistry were observed. Furthermore, these changes were completely reversible following a 4 weeks recovery period. In the 100 ppm group no differences from controls were detectable. In all surviving males the mean testes weights and testes weights expressed as % of organ weight determined on necropsy right at the end of the study were not different from controls in any treatment group. The same observation was made for animals of the recovery group. No histopathological changes were noted in the testes and epididymis in any dose group of surviving males examined immediately after study termination and after a 4 week recovery period, respectively. One/10 control animals of the recovery group was diagnosed with epididymitis. In male animals of the high dose group which died during exposure atrophy of the seminal vesicles were seen in four males and degeneration of the seminiferous tubules in two animals. In the female rats uterus, cervix and ovaries were subject to histopathological examination. No pathological findings were observable after gross and microscopical examination of uterus, cervix and ovaries in any treatment group immediately after study termination. In females of the recovery group ovarial cysts ("cystic ovarian bursa") were found in 2/10 animals of the control group but none in the treated groups. One/10 animals each of the control and intermedi-ate dose group had changes in uterus size ("luminal ectasia") while 1/10 animals of the interme-diate dose group had size changes in the cervix ("luminal ectasia").

Effect levels

open allclose all
Dose descriptor:
NOEC
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEC
Effect level:
300 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight; minor changes in haematology, urinalysis and histopathology; all reversible after 4 weeks
Dose descriptor:
LOAEC
Effect level:
650 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: food consumption; haematology; urinalysis; histopathology; gross pathology

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The most noteworthy observations of the current study were the brain and thymus lesions in animals that died due to inhalation of 650 ppm of 2,4-pentanedione. Because these degenerative lesions were not observed in the 650 ppm male survivors of the 14-week exposure regimen, deaths were attributed to their development. However, half of the survivors of the 650 ppm group did have gliosis or malacia in the brain vestibular nuclei and corpora striata. These central nervous system lesions were accompanied with neurobehavioral abnormalities. Every 2,4-pentanedione-exposed rat exhibiting an abnormality during the modified Irwin Screen Test was subsequently found to have brain lesions. In general, the converse of this statement was true; the exceptions being two 650 ppm males that had normal Irwin Screen Test responses in the presence of brain malacia. Also, several 650 ppm females had acute degeneration of the vestibular nuclei- and corpora striata, but died prior to Irwin Screen testing. Since the electron mi-croscopic findings in the sciatic nerve preparations were negative, the neurotoxic effects of 2,4-pentanedione appear to be central and not peripheral in location. An explanation for the difference in mortality between sexes (30 percent vs. 100 percent for males and females, respectively, of the 650 ppm exposure group) is not known. The sex difference may be related to brain thiamine, folic acid, and/or pyridoxine concentrations since a proposed mechanism of toxicity of 2,4-pentanedione is inactivation of B vitamins or their coenzymes. The concentration-response profile for repeated exposures to 2,4-pentanedione is very steep. A concentration of 300 ppm, which is approximately half of the concentration that was lethal to a majority of exposed rats, did not cause clinical abnormalities or histologic tissue damage. In fact, only minor alterations in body weight and clinical pathology were observed in rats exposed to 300 pppm of 2,4-pentanedione and those alterations were reversible after a 4-week recovery period. Mild squamous metaplasia in the nasal mucosa was observed in the 650 ppm rats. Perhaps inflamma-tion in the nasal mucosa is a transient response at 2,4-pentanedione concentrations of 200 ppm and higher. Rats exposed to 100 ppm of 2,4-pentanedione for 14 weeks showed no signs of irritancy or toxicity. In conclusion, the results of this study would support 100 ppm of 2,4-pentanedione vapor as a no observable effect level in rats.
Executive summary:

Four groups, each consisting of 20 male and 20 female Fischer 344 rats were exposed six hours per day, five days a week, for 14 weeks to 2,4-pentanedione vapor at concentrations of 0 (control), 100, 300, or 650 ppm. Half of the animals were held an additional 4 weeks postexposure to determine the reversibility of any observed toxic effects. Monitors for toxicity were as follows: clinical observations including neurobehavioral signs, body weight, food and water consumption, hematology, serum chemistry, urinalysis, organ weights (liver, lungs, brain, heart, thymus, kidneys and testes); and ophthalmologic, gross pathologic and microscopic evaluations. An additional ten males were added each to the control and high dose group for glutaraldehyde perfusion and removal of sciatic nerve for examination by transmission electron microscopy.

 Rats of the 650 ppm group had closed or partially closed eyes during 2,4-pentanedione exposure throughout the study period. Additional clinical signs observed in the 650 ppm animals were perinasal, periocular, and perioral encrustation, urogenital area wetness, hypoactivity, lack of coordination, paresis, ataxia, irregular gait, slow or negative surface righting reflex, lacrimation, emaciation, hypothermia, and unkempt fur. A majority of the animals exposed to 650 ppm of 2,4-pentanedione died. There were no exposure-related clinical observations in animals of the control, 100, or 300 ppm groups. Additionally, there were no treatment-related clinical observations during the 4-week postexposure period.

The pertinent findings following the modified Irwin Screen Test reflect the results of the 650 ppm rats only because there were no treatment-related findings inthecontrol, 100, or 300 ppm groups. Also, due to the excessive mortality in the 650 ppm female group, only two females were available for the first monthly examination. Several rats of the 650 ppm exposure group had abnormal neurobehavioral signs suggesting nervous system toxicity. However, the majority of male rats that survived the first month of exposure to 650 ppm of 2,4-pentanedione did not exhibit neurobehavioral abnormalities. Additionally, there were no treatment-related neurobehavioral signs of abnormality in rats examined following the 4-week recovery period.There were no exposure-related ophthalmologic findings. Incidental lesions were observed in three ratspriorto sacrifice. Deaths were observed in the 650 ppm-exposed animals only. All females died (or were sacrificed moribund) within the first 6 weeks of the exposure regimen. Also, ten of the 30 males died during the first 6 weeks of the study.

The mean body weight changes for males and females and absolute mean body weight values show a significant depression in male body weight gain in the high concentration group throughout the fourteen-week exposure period (days 0 to 94). At the 14-week sacrifice, absolute body weights for the 650 ppm males were decreased compared to the control value. The slight increases in bodyweight gain in the 100 ppm males was not considered treatment-related. The 300 ppm male body weight gain values were similar to the control values. During the 4-week postexposure period, the 650 ppm male body weight mean rose considerably compared to the control male body weight mean (approximately 57 grams compared to 13 grams) indicating recovery. However, the final body weights of the 650 ppm males remained statistically significantly lower compared to the final control male values. Female body weight gains were markedly depressed in the 650 ppm group. This group of females died by study week 6. Females of the 300 ppm group also had mildly depressed bodyweightgains which were statistically significant from study days 45 through 121. Recovery from this mild depression was observed during the 4-week postexposure period. The body weight gains of the 100 ppm females were considered similar to the control female values although occasional instances of an increase in body weight gain in the 100 ppm females were observed.

The food and water consumption results obtained just prior to sacrifice show a statistically significant increase in food consumption in the 300 ppm males. This finding was considered spurious in the absence of an exposure-related response. Therefore, after 14 weeks of 2,4-pentanedione exposures, no treatment-related alterations in food or water consumption were observed in survivors.

Urinalysis results show that only 2,4-pentanedione-exposed males had alterations in urine parameters at the conclusion of the 14-week exposure regimen. For example, in the 650 ppm males, urine pH was low (6 vs. 7), and bilirubin and urobilinogen were slightly increased. Males of the 300 ppm group also had a slight increase in bilirubin and urobilinogen. These alterations were not observed following the 4-week postexposure period.

Hematologic results are show slight differences between 2,4-pentanedione treated and control rats that were observed at the conclusionofthe 14-week exposure regimen. For example, mean red blood cell counts and hematocrits were 4 to 7 percent lower than control values for both the 300 ppm females and the 650 ppm males. A slight increase in mean corpuscular volume and mean corpuscular hemoglobin was also observed in these animals. The lymphocyte count was increased in the 650 ppm males. Following the 4-week postexposure period, hematologic results indicated recovery from the mild changes observed in animals sacrificed after 14 weeks of 2,4-pentanedione exposure. Blood chemistry results show noteworthy alterations related to 2,4-pentanedione exposure in the 650 ppm males. For example, urea nitrogen increased, creatinine decreased, aspartate aminotransferase decreased, alkaline phosphatase increased, and calcium decreased. In the 300 ppm group, males and females had a slight decrease in calcium. The 300 ppm females had an increase in alkaline phosphatase. Most of the blood chemistry values for the 2,4-pentanedione-exposed recovery animals were not different compared to control values indicating reversibility of the aforementioned alterations.

For animals sacrificed after 14-weeks of 2,4-pentanedione exposure, no treatment related gross lesions were found at necropsy. Some gross lesions in animals found dead or sacrificed moribund appeared treatment-related. Specifically, these were ocular and nasal discharges, emaciation, and changes in the skinofthe head region.

The mean absolute and relative (a percentage of body weight) organ weights for males and females sacrificed after 14 weeks of 2,4-pentanedione exposure show that after 14 weeks of 2,4-pentanedione exposures, a statistically significant decrease in most of the absolute organ weights of the 650 ppm males was observed. However, relative weights of these same organs were increased, which suggests that the alterations in absolute organ weights were simply a reflection of decreased body weights. Organ weights of the 300 and 100 ppm males were indistinguishable from control values similar organ weights of the 300 and 100 ppm females were comparable to control values, although spurious statistically significant differences were observed in absolute lung weights of 2,4-pentanedione-exposed females. Organ weight values for the 4-week recovery animals followed a similar pattern to that observed in the 14-week sacrifice. However, due to the substantial body weight gain of the 650 ppm males during this interval, the magnitude of the decreases in absolute organ weights (and concomitant increases in relative organ weights) compared to respective control values was much less in the recovery animals versus the 14-week sacrificed animals. This observation further supports the hypothesis that altered organ weights in this study were reflective of depressed body weights.

The results of histopathologic examination of tissues show that there were no 2,4-pentanedione-related lesions in rats exposed to 100 or 300 ppm. However, in male and female rats of the 650 ppm group, noteworthy lesions were observed in 4 tissues; the brain, the thymus, the skin, and thenasalcavity. Specifically, the brain lesions consisted of acute degeneration in the deep cerebellar nuclei, vestibular nuclei, and the corpora striata of animals that died during the study. For survivors of the 650 ppm exposure regimen, about half of the rats had gliosis or malacia in the same brain regions. Thymic and skin lesions were restricted to rats that died during the study and consisted of acute lymphoid degeneration and dermal necrosis (head region), respectively. In the nasal mucosa, mild squamous metaplasia located in the anterior portion of the nose was observed in the14-week 650 ppm survivors. This lesion lessened in frequency and severity following the 4-week postexposure interval, indicating recovery.

The most noteworthy observations of the current study were the brain and thymus lesions in animals that died due to inhalation of 650 ppm of 2,4-pentanedione. Because these degenerative lesions were not observed in the 650 ppm male survivors of the 14-week exposure regimen, deaths were attributed to their development. However, half of the survivors of the 650 ppm group did have gliosis or malacia in the brain vestibular nuclei and corpora striata. These central nervous system lesions were accompanied with neurobehavioral abnormalities. Every 2,4-pentanedione-exposed ratexhibitingan abnormality during the modified Irwin Screen Test was subsequently found to have brain lesions. In general, the converse of this statement was true; the exceptions being two 650 ppm males that had normal Irwin ScreenTestresponses in the presence of brain malacia. Also, several 650 ppm females had acute degeneration of the vestibular nuclei-and corpora striata, but died prior to Irwin Screen testing. Since the electron microscopic findings in the sciatic nerve preparations were negative, the neurotoxic effects of 2,4-pentanedione appear to be central and not peripheral in location. An explanation for the difference in mortality betweensexes(30 percent vs. 100 percent for males and females, respectively, of the 650 ppm exposure group) is not known. The difference may be related to brain thiamine, folic acid, and/or pyridoxine concentrations since a proposed mechanism of toxicity of 2,4-pentanedione is inactivation of B vitamins or their coenzymes. The concentration-response profile for repeated exposures to 2,4-pentanedione is very steep. A concentration of 300 ppm, which is approximately half of the concentration that was lethal to a majority of exposed rats, did not cause clinical abnormalities or histologic tissue damage. In fact, only minor alterations in body weight and clinical pathology were observed in rats exposed to 300 ppm of 2,4-pentanedione and those alterations were reversible after a 4-week recovery period. Mild squamous metaplasia in the nasal mucosa was observed in the 650 ppm rats. Perhaps inflammation in the nasal mucosa is a transient response at 2,4-pentanedione concentrations of 200 ppm and higher. Rats exposed to 100 ppm of 2,4-pentanedione for 14 weeks showed no signs of irritancy or toxicity. In conclusion, the results of this study would support 100 ppm of 2,4-pentanedione vapor as a no observable effect level in rats.