Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: dermal
Remarks:
other: 9 days
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994-02-22 to 1995-04-06
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Conduction and documentation of study acceptable. Study report available.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995
Reference Type:
publication
Title:
Unnamed
Year:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: stated in report
Deviations:
no
Principles of method if other than guideline:
New Zealand White rabbits were treated by 6 h occluded cutaneous application with undiluted 2,4-pentanedione at dose volumes of 0.25, 1.0 and 1.5 ml/kg body weight. Animals in the control group received occluded applications of Milli-Q filtered water at a volume of 1.5 ml/kg bw. The test or control substance was applied to the clipped dorsal surface of the rabbits. Twelve animals/sex/group were used for the control and high dose groups, 6 animals/sex/group for mid and low dose groups. The original study de-sign included dosing for 5 days the first week and 4 day the second week. The addi-tional 6 animals/sex/group in the controls and the high dose group were used for a 4 week recovery period. Due to mortality and signs of toxicity observable in mid and high dose groups, dosing was discontinued for these groups after day 4. Three surviving males and 2 surviving females from the 1463 mg/kg group were euthanized on day 4 while an additional 4 males and 3 females were retained without further dosing to day 12. Rabbits in the low dose group continued to receive a total of 9 doses (5 in the first week, 4 in the second). On day 12, 6 rabbits/sex from the control group were removed from the study since they were not required for their intended purpose as a recovery group. All other surviving rabbits were euthanized on day 12. Only 3 rabbits/sex from the control group were subjected to necropsy and histopathology. Monitors for toxicity included observations for clinical signs, including skin irritation, food consumption, water consumption, body weight and body weight change, organ weights, gross pathology and histopathology.
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:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HPR Inc denver, PA, USA
- Age at study initiation: 15 to 18 days
- Weight at study initiation: 2.5 to 3 kg
- Fasting period before study: not reported
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 61 - 70 °F
- Humidity (%): 40 - 60 %
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: 4 x 4 inch
- Type of wrap if used: lycra/spandex jacket
- Time intervals for shavings or clipplings: clipping


REMOVAL OF TEST SUBSTANCE
- Washing (if done): not applicable


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.25, 1.0, 1.5 ml/kg


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
not applicable, since substance was administered in neat
Duration of treatment / exposure:
9 days
Frequency of treatment:
5 days first week, 4 days second week
Doses / concentrations
Remarks:
Doses / Concentrations:
0.25, 1.0, 1.5 ml/kg
Basis:
nominal per unit body weight
No. of animals per sex per dose:
6 (mid and low dose group), 12 (high dose and control group)
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: 4 week recovery period
- Dose selection rationale: pretests
- Rationale for animal assignment (if not random): random
- Post-exposure recovery period in satellite groups: 4 weeks (half of control group)
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twiche daily
- Cage side observations checked in table were included.


DETAILED CLINICAL OBSERVATIONS: Yes


DERMAL IRRITATION (if dermal study): Yes / No / No data


BODY WEIGHT: Yes


WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 4 and Day 12
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: No
- How many animals: all remaining animals in high dose and control group



CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 4 and Day 12
- Animals fasted: No
- How many animals: all remaining animals in high dose and control group



URINALYSIS: Yes
- Time schedule for collection of urine: Day 4 and Day 12
- Metabolism cages used for collection of urine: No
- Animals fasted: No


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
none
Statistics:
Results of quantitative continuous variables (such as body weight changes) were intercompared among the treatment groups and one control group by use of analysis of variance (ANOVA)and t-tests. The t-test were used when F value from the ANOVA was significant. When Levene's test indicated similar variances, and the ANOVA was significant, a pooled t-test was used for pairwise comparisons. When Levene's test indicated heterogeneous variances, all groups were compared by a ANOVA for unequal variances followed, when necessary, by a separate

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Dermal irritation:
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):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Occluded cutaneous dosing of rabbits with 2,4-pentanedione for 3 or 4 days resulted in death of 5/12 males and 7/12 females in the 1.5 ml/kg (1463 mg/kg) group and 1/6 males and 3/6 females in the 1.0 ml/kg (975 mg/kg) group. Skin irritation was observed in all dose groups. Time to onset and severity of skin irritation were generally dose-dependent and persistent in all dose groups. Signs of skin irritation included erythema, edema, desquamation/exfoliation, excoriation, fissuring, necrosis and/or ecchymosis. In the mid and high dose groups of rabbits during the first few days of the study, several signs of systemic toxicity were evident. Numerous animals from these dose groups were hypoactive, uncoordinated and/or prostrate, had tremors, salivation, gasping and/or convulsions, and some had blue cutis of the nasal area suggestive of cyanosis. Furthermore, these groups lost mean body weight and had decreased food con-sumption during the first few days of the study. After cessation of dosing in these dose groups, mean food consumption generally returned to control values while mean body weight gains were increased over control values. Excessive vocalization, slow or la-boured breathing, and/or red perioral discharge were also observed in some high dose group animals until cessation of dosing. In the low dose group there were no mortalities, clinical signs of systemic toxicity, or effects on body weight or food consumption. Gross and microscopic evaluation at both day 4 and 12 confirmed dose-related skin irritation in all treatment groups. Microscopic lesions included acanthosis, subcutaneous edema, dermatitis, hemorrhage, congestion and/or necrosis. There were also numerous rabbits with hemorrhaging in various sections of the brain, including the meninges. Additionally, a number of brain sections showed neuronal degeneration, including the hypothalamus, mid brain, piriform cortex, pons and/or hippocampus. At both day 4 and 12, the thymus or thymic region, spleen, and/or lymph nodes of several animals of both sexes from the mid and high dose groups were congested and/or hemorrhaged; some animals also had lymphoid depletion or necrosis. This observation, combined with decreased lymphocyte and eosinophil counts in the high dose group at day 4, suggested possible effects on the immune system. Since the animals from the mid and high dose group had severe skin irritation and many signs of systemic effects a definitive conclusion regarding a treatment related response to the immune system is not possible, as discussed by the study authors. Except clinical pathology changes that may have been related to the skin irritation, no substance related differences from controls were reported in the low dose group. According to the systemic effects observed, 244 mg/kg bw and 975 mg/kg bw correspond to the NOAEL and LOAEL of this dermal study, respectively.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
244 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
LOAEL
Effect level:
975 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: body weight; haematology; clinical chemistry; histopathology;

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions used the systemic NOAEL of the substance is 244 mg/Kg bw/d.
Executive summary:

New Zealand White rabbits were treated by 6 h occluded cutaneous application with undiluted 2,4-pentanedione at dose volumes of 0.25, 1.0 and 1.5 ml/kg body weight for 9 days. Animals in the control group received occluded applications of Milli-Q filtered water at a volume of 1.5 ml/kg bw. The test or control substance was applied to the clipped dorsal surface of the rabbits. Twelve animals/sex/group were used for the control and high dose groups, 6 animals/sex/group for mid and low dose groups. The original study design included dosing for 5 days the first week and 4 day the second week. The additional 6 animals/sex/group in the controls and the high dose group were used for a 4 week recovery period. Due to mortality and signs of toxicity observable in mid and high dose groups, dosing was discontinued for these groups after day 4. Three surviving males and 2 surviving females from the 1463 mg/kg group were euthanized on day 4 while an additional 4 males and 3 females were retained without further dosing to day 12. Rabbits in the low dose group continued to receive a total of 9 doses (5 in the first week, 4 in the second). On day 12, 6 rabbits/sex from the control group were removed from the study since they were not required for their intended purpose as a recovery group. All other surviving rabbits were euthanized on day 12. 3 rabbits/sex from the control group were subjected to necropsy and histopathology. Monitors for toxicity included observations for clinical signs, including skin irritation, food consumption, water consumption, body weight and body weight change, organ weights, gross pathology and histopathology. Used doses of 0.25, 1.0 and 1.5 ml/kg bw as given by the authors equivalent to 244, 975 and 1463 mg/kg bw, respectively.

Occluded cutaneous dosing of rabbits with 2,4-pentanedione for 3 or 4 days resulted in death of 5/12 males and 7/12 females in the 1.5 ml/kg (1463 mg/kg) group and 1/6 males and 3/6 females in the 1.0 ml/kg (975 mg/kg) group. Skin irritation was observed in all dose groups. Time to onset and severity of skin irritation were generally dose-dependent and persistent in all dose groups. Signs of skin irritation included erythema, edema, desquamation/exfoliation, excoriation, fissuring, necrosis and/or ecchymosis. In the mid and high dose groups of rabbits during the first few days of the study, several signs of systemic toxicity were evident. Numerous animals from these dose groups were hypoactive, uncoordinated and/or prostrate, had tremors, salivation, gasping and/or convulsions, and some had blue cutis of the nasal area suggestive of cyanosis. Furthermore, these groups lost mean body weight and had decreased food consumption during the first few days of the study. After cessation of dosing in these dose groups, mean food consumption generally returned to control values while mean body weight gains were increased over control values. Excessive vocalization, slow or laboured breathing, and/or red perioral discharge were also observed in some high dose group animals until cessation of dosing. In the low dose group there were no mortalities, clinical signs of systemic toxicity, or effects on body weight or food consumption. Gross and microscopic evaluation at both day 4 and 12 confirmed dose-related skin irritation in all treatment groups. Microscopic lesions included acanthosis, subcutaneous edema, dermatitis, hemorrhage, congestion and/or necrosis. There were also numerous rabbits with hemorrhaging in various sections of the brain, including the meninges. Additionally, a number of brain sections showed neuronal degeneration, including the hypothalamus, mid brain, piriform cortex, pons and/or hippocampus. At both day 4 and 12, the thymus or thymic region, spleen, and/or lymph nodes of several animals of both sexes from the mid and high dose groups were congested and/or hemorrhaged; some animals also had lymphoid depletion or necrosis. This observation, combined with decreased lymphocyte and eosinophil counts in the high dose group at day 4, suggested possible effects on the immune system. Since the animals from the mid and high dose group had severe skin irritation and many signs of systemic effects a definitive conclusion regarding a treatment related response to the immune system is not possible, as discussed by the study authors. Except clinical pathology changes that may have been related to the skin irritation, no substance related differences from controls were reported in the low dose group. According to the systemic effects observed, 244 mg/kg bw and 975 mg/kg bw correspond to the NOAEL and LOAEL of this dermal study, respectively.

The severity of the skin irritation, as well as systemic toxicity, observed in the study was considered to be at least partially related to the occlusive conditions used in this study.