4-hydroxy-4-methylpentan-2-one

Endpoint summary

Description of key information

Repeated dose studies with Diacetone alcohol (4-hydroxy-4-methylpentan-2-one) indicated that systemic toxicity is minimal. The NOAEL via oral gavage dosing (13-week study) was established at 600 mg/kg/day for male and female rats, excluding the sex- and species-specific hyaline droplet nephropathy in males in the determination of the NOAEL. There were no organ-specific toxic effects for either chemical relevant for human risk assessment. The NOAEC for subacute inhalation exposure was 4685 mg/m3 (6-weeks study with rats).

Oral route

OECD 408 study
The potential toxicity of diacetone alcohol was evaluated following daily oral administration (gavage) to rats for 13 weeks. On completion of the treatment period, designated animalsof the control- and high-dose groups were held for a 6-week treatment-free period in order to evaluate the reversibility of any findings. The study was performed based on the OECD guideline No. 408 and in compliance with the Good Laboratory Practices. Three groups of 10 (low- and mid-dose groups) or 15 (high-dose group) male and female Sprague-Dawley rats each received the test item by daily oral gavage administration for at least 13 weeks. The test item was administered at 25, 150 and 600 mg/kg/day as a solution in the vehicle (corn oil) under a constant dosage-volume of 5 mL/kg/day. A control group of 15 animals per sex received the vehicle alone under the same experimental conditions. On completion of the treatment period, the animals in each group were sacrificed, with the exception of the recovery animals (last 5 animals per sex in the control and high-dose groups) which were kept for a 6-week treatment-free period. The concentration of the dose formulations was checked in Weeks 1, 4, 9 and 13. The animals were checked at least twice daily during the treatment and treatment-free periods for mortality and morbidity, and once daily for clinical signs. In addition, detailed clinical examinations were performed once before the beginning of the treatment period and then weekly. The body weight was recorded once before the beginning of the treatment period, and then at least once a week during the study as well as food consumption. Functional Observation Battery was evaluated on all main animals once in Week 12. Ophthalmological examinations were performed on all main animals before the beginning of the treatment period and once at the end of the treatment period. Estrous cycle stage was determined on all main females daily for 21 consecutive days at the end of the treatment period and at the end of the treatment-free period for recovery animals. Hematology, blood biochemistry and urinalysis were performed towards the end of thetreatment period for main animals and towards the end of treatment-free period for recovery animals. On completion of the treatment period and treatment-free periods, the animals were euthanized and submitted to a full macroscopic post-mortem examination. Sperm investigations were performed at sacrifice of the males. A microscopic examination was performed on selected tissues from animals of the control- and high-dose groups sacrificed at the end of the treatment period, and on macroscopic lesions, kidneys and adrenals (males only) and liver from animals of the low- and mid-dose groups sacrificed at the end of the treatment period. Kidneys and adrenals (males only) and liver of recovery animals were examined microscopically at the end of the treatment-free period. Males kidney slides immunostained with an antibody for Alpha 2u-globulin protein were also microscopically examined at the end of treatment period.
The test item concentrations in the administered dose formulations analyzedin Weeks 1, 4, 9 and 13 were within the acceptance criteria (± 10%) and no test item was detected in control formulations. There were no test item treatment-related deaths or clinical signs.
A dose-related increase in horizontal movement and rearing was observed in males during the recording of motor activity. In absence of correlated clinical signs or other findings at the Functional Observation Battery, this was not considered adverse. Non-adverse slightly lower body weight was recorded from Week 10 in males during the treatment period at 600 mg/kg/day. Body weight gain returned to normal at the end of the treatment-free period, suggesting reversibility. No effects on body weight and body weight gain were noted in females. There were no differences in food consumption between control and test item-treated animals during the treatment and treatment-free periods. At the end of the treatment period, a higher number of females given 600 mg/kg/day remained in diestrous for several consecutive days when compared with controls. This was no longer observed at the end of the treatment-free period and was therefore not considered to be adverse. There were no test item-related ophthalmology findings at the end of the treatment period. At hematology at the end of the treatment period, when compared with controls, a slightly higher neutrophil count was noted in males treated at 600 mg/kg/day (+53%). In females, mean red blood cell count was statistically significantly decreased at 150 and 600 mg/kg/day when compared with controls (up to -6%) and was associated with lower hemoglobin (-5%) and packed cell volume (-7%) at 600 mg/kg/day. Lower total white blood cell (-24%) and lymphocyte counts were also noted (-26%) at the highest dose. These findings were not considered to be adverse in view of their amplitude (remained within the physiological range) and were no longer observed at the end of the treatment-free period, suggesting reversibility. At blood biochemistry in both genders, moderately higher cholesterol concentration was noted at 600 mg/kg/day (+40 % for males and +43% for females). In males, a lower inorganic phosphorus concentration was also noted from 150 mg/kg/day (up to -13%), when compared with controls. These findings were not considered to be adverse and recovery was complete at the end of the treatment-free period. At urinalysis, a slight non-adverse decrease of pH values was noted in males at 600 mg/kg/day. Urinary findings were no longer observed at the end of the treatment-free period. At sperm analyses, when compared to controls, there were no effects attributed to the test item treatment. Test item administration at 600 mg/kg/day induced minimal to slight non-adverse centrilobular hepatocellular hypertrophy that correlated with increases in liver weights and with an increase in the incidence of macroscopically accentuated lobular pattern, in both sexes. In the kidneys, in males only, there were increased incidence and severity of tubular hyaline droplets (consistent with a2u-globulin), tubular basophilia and granular casts, from 25 mg/kg/day, which correlated with increased kidney weights. Given the nature and the severity of the microscopic alterations associated with the hyaline deposits (mostly minimal to slight), hyaline droplet accumulation and related renal lesions were considered to be non-adverse, in this study. Moreover, these findings are specific to male rats and non-relevant for humans. 
Consequently, under the experimental conditions of the study, the No Observed Adverse Effect Level (NOAEL) after the 13-week treatment period was established at 600 mg/kg/day.

OECD 422 study
A repeated dose study according to OECD TG 422 was conducted with Diacetone alcohol (4-hydroxy-4-methyl-2-pentanone) (MHW, 1997a). In this study, 10 rats/sex/group were dosed by gavage with 0, 30, 100, 300, or 1000 mg/kg/day of Diacetone alcohol in distilled water for approximately 45 days. Decreased locomotor activity and stimulation response were observed at 300 and 1000 mg/kg/day for both sexes. Altered blood parameters, dilatation of the distal tubules of the kidneys, hepatocellular hypertrophy, and vacuolization of the zona fasciculata of the adrenals were observed in males from the 1000 mg/kg/day group. Females at 300 mg/kg/day showed dilatation of the distal tubules and fatty degeneration of the proximal tubular epithelium in the kidneys. At 1000 mg/kg/day, female body weight gain was reduced, liver weight was increased, hepatocellular hypertrophy was noted, and kidney lesions similar to those at 300 mg/kg/day were also recorded. In addition, increased incidence and/or severity of hyaline droplets in the tubular epithelium was noted in males at 100 mg/kg/day and greater. The kidney effects observed are suggestive of chronic progressive nephropathy (CPN) and a2u-globulin nephropathy, a condition not relevant for human risk assessment (U. S. EPA. (1991) Alpha2u-globulin: Association with chemically induced renal toxicity and neoplasia in the male rat. Risk Assessment Forum. EPA/625/3-91/019F). Therefore, the NOAEL for this study was 100 mg/kg/day for males and females (excluding the sex- and species-specific hyaline droplet nephropathy observed in the males).

MTD study in rabbits

The potential toxic effects of diacetone alcohol was evaluated following daily oral administration (gavage) in the non-pregnant female rabbit to establish a Maximum Tolerated Dose (MTD) for an OECD 414 study in this species (Bentz, 2018). Three non-pregnant female New Zealand White rabbits received diacetone alcohol by daily oral gavage for 3 weeks. Diacetone alcohol was administered under a volume of 5 ml/kg at 100 mg/kg/day during the first week, 300 mg/kg/day during the second week and 1000 mg/kg/day during the third week, formulated as a solution in drinking water. The animals were observed at least once daily for morbidity, mortality and clinical signs. Body weight was recorded twice during the acclimation period and then every 1 to 3 days. Food consummption was recorded throughout the treatment period for intervals of 2 to 3 days. On completion of the treatment, animals were euthanized and a post-mortem macroscopic examination of the principal thoracic and abdominal organs was performed

No unscheduled death occurred and no clinical signs were observed through the whole treatment period. There were no test item-related effects at 100 and 300 mg/kg/day. During treatment at 1000 mg/kg/day, body weight of the females appeared to be minimally affected (down to -4%), but at the end of the week of treatment 2/3 animals seemed to recover. Food consumption was decreased by -27 to -67%. At necropsy, no findings were observed. 

Under the experimental conditions of this study, the Maximum Tolerated Dose (MTD) was established to be over 1000 mg/kg/day.

Inhalation route
A 6-week whole body inhalation study was conducted with Diacetone alcohol (4-hydroxy-4-methylpentan-2-one) in Wistar rats. Animals were exposed to analytical concentrations of 0, 233, 1041 and 4685 mg/m3 of Diacetone alcohol for 6 hours per day for 5 days per week for 6 weeks (Butterworth et al., 1980). This non-GLP study was equivalent to OECD Test Guideline 412. There were no clinical signs of toxicity in the first four weeks of exposure; however, slight lethargy was noted for a few hours in 233 and 4685 mg/m3 exposed animals over the following 2 weeks. The body weights of females exposed to 4685 mg/m3 were significantly lower (-5%) than controls at week 6. After 2 weeks, food consumption was significantly decreased in males compared to controls; however, this was not noted in subsequent weeks. Haemoglobin was significantly increased (+6%) in females exposed to 4685 mg/m3 compared to controls. Lactate dehydrogenase (LDH) was significantly higher (+48%) in females exposed to 4685 mg/m3 compared to controls. In males, plasma protein was increased (+3%) at the 4685 mg/m3 concentration and plasma sodium was reduced (-0.7%) at all concentrations. Liver and kidney weights were increased in males (compared to controls) exposed to 1041 (liver+13%) and 4685 mg/m3 (liver+23%) and 4685 mg/m3 (kidney +17%), respectively. Histologically, the abnormal presence of eosinophilic hyaline droplets in the proximal tubules of males exposed to the high concentration was noted. There was no suggestion of cellular damage and the response may have been due to cellular accumulation of the chemical or a metabolite. A review of the study data suggests that a NOAEC of 4685 mg/m3 and a NOEC of 1041 mg/m3 can be considered for repeated-dose inhalational toxicity, based on liver weight changes not associated with histological alterations and probably secondary to a metabolic over load and based on the male rat-specific eosinophilic hyaline droplets in the proximal tubular cells as hyaline droplet formation in male rats is not considered to be relevant to human health for the purposes of risk assessment (U. S. EPA, 1991). No irritation of the respiratory tract was observed up to the highest concentration tested.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 March 2016 - 18 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Charles River Laboratories Italia, Calco, Italy
- Age at study initiation: 5 to 6 weeks old at the beginning of the treatment period
- Mean body weight at study initiation: the males had a mean body weight of 216 g (range: 191 g to 236 g) and the females had a mean body weight of 168 g (range: 152 g to 193 g)
- Fasting period before study: no
- Housing: the animals were housed by two or three, by sex and group, in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm²) containing autoclaved sawdust
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: the animals were acclimated to the study conditions for a period of 11 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 18 April 2016 to 29 August 2016.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Solution in the vehicle

VEHICLE
- Justification for use and choice of vehicle: suitable formulation in corn oil
- Concentration in vehicle: 5, 30 and 120 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: Gas Chromatography with Flame Ionization Detection (GC-FID)
Once in Weeks 1, 4, 9 and 13
A sample was taken from control and test item dose formulations and analyzed using the validated method.

Duration of treatment / exposure:

13 weeks followed by a 6-week recovery period.

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

600 mg/kg bw/day (nominal)

No. of animals per sex per dose:

15 animals per sex at dose-levels of 0 and 600 mg/kg/day and 10 animals per sex at dose-levels at 25 and 150 mg/kg/day.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected based on the results of a previous study: a repeated dose study according to OECD TG 422 was conducted with 4-hydroxy-4-methyl-2-pentanone (1). In this study, 10 rats/sex/group were dosed by gavage with 0, 30, 100, 300, or 1000 mg/kg/day of 4-hydroxy-4-methyl-2 pentanone in distilled water for approximately 45 days. Decreased locomotor activity and stimulation response were observed at 300 and 1000 mg/kg/day for both sexes. Altered blood parameters, dilation of the distal tubules of the kidneys, hepatocellular hypertrophy, and vacuolization of the zona fasciculata of the adrenals were observed in males at 1000 mg/kg/day. Females at 300 mg/kg/day showed dilation of the distal tubules and fatty degeneration of the proximal tubular epithelium in the kidneys. At 1000 mg/kg/day, female body weight gain was reduced, liver weight was increased, hepatocellular hypertrophy was noted, and kidney lesions similar to those at 300 mg/kg/day were also recorded. In addition, increased incidence and/or severity of hyaline droplets in the tubular epithelium were noted in males at 100 mg/kg/day and greater. The kidney effects observed are suggestive of Chronic Progressive Nephropathy (CPN) and a2u globulin nephropathy.
Therefore, 25, 150 and 600 mg/kg/day were selected.

- Rationale for animal assignment: computerized randomization procedure.

Positive control:

no (not required)

Observations and examinations performed and frequency:

MORTALITY / MORBIDITY: Yes
- Time schedule: each animal was checked for mortality and morbidity once a day during the acclimation period and at least twice a day during the treatment and recovery periods, including weekends and public holidays.

CLINICAL OBSERVATIONS: Yes
- Time schedule: each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations were performed on all animals once before the beginning of the treatment period and then once a week until the end of the study.

BODY WEIGHT: Yes
- Time schedule:
The body weight of each animal was recorded:
- once before the beginning of the treatment period,
- on the first day of treatment,
- at least once a week until the end of the study.

FOOD CONSUMPTION: Yes
- Time schedule: the quantity of food consumed by the animals in each cage was recorded once a week, over a 7-day period, during the study.
Food consumption was calculated per animal and per day.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule:
Ophthalmological examinations were performed on all main animals (recovery animals excluded):
- before the beginning of the treatment period,
- on one occasion at the end of the treatment period (Week 13).

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional Observation Battery (FOB)
Each main animal was evaluated once on Week 12 before the daily treatment.
This evaluation included a detailed clinical examination, the assessment of reactivity to manipulation and different stimuli, and motor activity.
The animals were randomized and observed in the cage, in the hand and in the standard arena.

Motor activity
For each animal, motor activity was measured by automated infra-red sensor equipment over a 60-minute period.

HAEMATOLOGY: Yes
Peripheral blood
The parameters were determined for all main animals at the end of the treatment period.

Bone marrow
Two bone marrow smears were prepared from the femoral bone (at necropsy) of all animals euthanized on completion of the treatment or recovery period.

CLINICAL CHEMISTRY: Yes
The parameters were determined for all main animals at the end of the period.

THYROID HORMONES: Yes
An additional blood sample was taken into K3EDTA tubes from all animals sacrificed at the end of the treatment or recovery period. But, those samples were not analyzed.

URINALYSIS: Yes
The parameters were determined for all main animals at the end of the treatment period.

SEMINOLOGY: Yes
Before euthanasia at the end of the treatment or treatment-free period, each main or recovery male was anesthetized by an intraperitoneal injection of sodium pentobarbital for seminology investigations.

Sacrifice and pathology:

ORGAN WEIGHTS: Yes
see table below
The body weight of each animal was recorded before euthanasia at the end of the treatment or recovery period.

GROSS PATHOLOGY: Yes
Sacrifice
On completion of the treatment or recovery period, after at least 14 hours fasting, all animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital.

Macroscopic post-mortem examination
A complete macroscopic post-mortem examination was performed on all animals.

Preservation of tissues
For all study animals, the tissues specified in the Tissue Procedure Table were preserved in 10% buffered formalin (except for the eyes and Harderian glands and for the right testes and epididymides which were fixed in Modified Davidson's Fixative).

Preparation of histological slides
All tissues required for microscopic examination were trimmed according to the RITA guidelines, when applicable (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004), embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except for the testes and epididymides which were stained with hematoxylin/PAS).
Kidneys from all males (main + recovery) were kept no longer than 96 hours in formalin (tissue intended for immunohistochemistry).
One additional kidney slide of all main males sacrificed at the end of the treatment period was immunostained with an antibody for Alpha-2-microglobulin protein.

HISTOPATHOLOGY: Yes
A microscopic examination was performed on all tissues listed in the Tissue Procedure Table:
- for the control- and high-dose animals (groups 1 and 4) euthanized at the end of the treatment period,
- for all macroscopic lesions, kidneys and adrenals (males only) and liver from all low- and intermediate-dose animals (groups 2 and 3) euthanized on completion of the treatment period,
- immunostained kidneys from all main males sacrificed at the end of the treatment period.

Other examinations:

Monitoring of estrous cycle
The estrous cycle stage was determined daily from a fresh vaginal lavage (stained with methylene blue), daily for all main females for 21 consecutive days before the end of the treatment period.
As treatment-related changes were observed at the end of the treatment period, these examinations were carried out daily on recovery females for 21 consecutive days before the end of the recovery period.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment period
There were no test item treatment-related clinical signs.
From Day 85, piloerection, abdominal breathing, dyspnea and thin appearance were noted on one group 4 male. Respiratory disorders persisted until Day 94 and thin appearance until Day 105. Since it was seen in only one animal and was consecutive to a reflux at dosing, this was considered incidental and linked to the administration procedure.
All other clinical signs recorded in test item-treated groups (chromorhynorrhea, transient piloerection, alopecia/thinning of hair, scabs, abnormal growth of teeth, ptyalism, reflux at dosing, transient hypoactivity) were of comparable incidence of controls, not dose-related and/or common in this strain of rats under laboratory conditions.

Treatment-free period
There were no test item treatment-related clinical signs.
From Day 130 or Day 131, mydriasis and exophthalmos were recorded in one group 4 male. As they were not seen during the treatment period, and were noted in only 1/5 males, these were not considered as a test item effect.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the treatment and treatment-free periods.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See Table 1.
Treatment period
At 600 mg/kg/day, a statistically significantly lower mean body weight was noted in male animals treated from Week 10 when compared with control animals. A slightly lower body weight gain was also noted all over the treatment period.
A treatment-related effect in males cannot be excluded, and however was not considered as adverse in view of the minimal magnitude of the changes, the clinical conditions of the animals and the partial recovery during the treatment-free period.
No treatment-related effect on body weight or body weight gain was noted in females.

The occasional statistical differences from controls observed in mean body weight and/or mean body weight gain at lower dose-levels in males and females were considered to be incidental (transient, rarely dose-related).

Treatment-free period
Mean body weight of males remained statistically significantly lower during the treatment-free period when compared with controls but the body weight gain of males was higher than in control animals, indicating partial recovery.
There were no differences between control- and test item-treated females during the treatment-free period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no differences between control- and test item-treated animals during the treatment and treatment-free periods.
Very slightly higher mean food consumption was occasionally noted in group 3 females (150 mg/kg/day) during the treatment period and was linked to higher food intake recorded for two females. This was most probably due to spillage frequently observed in the cage. Food consumption was invalidated for these females when spillage was evident.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no test item-related ophthalmology findings at the end of the treatment period.
The chrorioretinopathy observed in 2/10 males treated at 25 mg/kg/day at the end of the treatment period was not considered to be linked to the test item administration since it was not seen at higher doses.
No other ocular findings were noted in any animals.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See Table 2.
There were no adverse findings in any group.

Treatment period
When compared to controls, a statistically significantly higher neutrophil count was noted at the highest dose in males at the end of the treatment period.
In view of the magnitude of this change (values remained within the physiological range), and in absence of effect on the clinical conditions of the animals, this was considered non-adverse.

In females, when compared with controls, mean Red Blood Cell count was statistically significantly lower in animals given 150 and 600 mg/kg/day. This was associated with lower hemoglobin concentration and packed cell volume at the highest dose. At 600 mg/kg/day, a statistically significantly lower White Blood Cell count was also noted, accompanied by a slightly lower lymphocytes count. For all these parameters, individual data remained within the physiological range observed for this type of study and the findings were not considered adverse in view of the clinical conditions of the animals.

The other occasional statistical differences from controls observed at lower dose-levels in males and females were considered to be incidental (not dose-related) and of no toxicologically relevance.

Treatment-free period
No relevant differences were noted at the end of the treatment-free period, suggesting reversibility of the findings.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See Table 3.
There were no adverse findings in any group.

Treatment period
In males, treatment-related changes consisted in statistically significantly lower inorganic phosphorus concentrations at 150 and 600 mg/kg/day, associated with slightly higher total protein and albumin concentrations at the highest dose. These changes were not considered adverse in absence of related clinical signs and since the values remained within the physiological range.
At 600 mg/kg/day, statistically significantly moderately higher cholesterol concentration was also seen. This was not considered adverse in view of the clinical conditions of the animals and in the absence of correlated adverse findings at histopathology.

In females, treatment-related changes consisted in a statistically significantly higher cholesterol concentration at 600 mg/kg/day.
These changes at the end of the treatment period were not considered adverse in view of the clinical conditions of the animals, the absence of correlated adverse findings at histopathology and since the values remained within the physiological range.
The other occasional statistical differences from controls observed in males and females were considered to be incidental or of no toxicologically relevance.

Treatment-free period
Blood biochemistry findings recorded at the end of the treatment period were no longer observed after the 6-week treatment-free period.
A slightly higher potassium concentration was noted in males given 600 mg/kg/day, when compared with controls. This was not considered to be test item-related in view of the low magnitude of the change, not seen at the end of the treatment period.
Slightly higher triglyceride concentration was seen in females when compared with controls. This was attributed to low values in the control group when compared with concentrations measured at the end of the treatment period.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

There were no adverse findings in any group.

Treatment period
A slight decrease in pH was noted in males at the dose 600 mg/kg/day (6.0 vs 6.9 in control group). In view of the magnitude of this change, this was not considered to be adverse.
Lower grades of ammonium phosphate crystals were noted in males, especially at the highest dose where only 1/10 animals presented crystals. In view of the direction of this change, this was not considered biologically relevant.
No relevant changes were noted in females at the end of the treatment period.

Treatment-free period
Urinary findings recorded at the end of the treatment period were no longer observed after the 6-week treatment-free period.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Motor activity and Functional Observation Battery
A dose-related increase in horizontal movements and rearing was observed in males. The severity for horizontal movement increased due to two outliers at 150 and 600 mg/kg/day, leading to a high standard deviation. Even if a relationship with the test item cannot be excluded, this was not considered adverse in the absence of correlation at functional observation battery (no signs of hyperactivity or stereotypy) or related clinical signs during the treatment period.

No relevant differences were noted at Functional Observation Battery between control and test item-treated groups at the end of the treatment period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See Table 4.
End of the treatment period
The main changes in organ weights were seen in the liver, in both sexes, and in the kidneys and adrenals, in males only.

When compared with controls, the mean absolute and relative liver weights were statistically significantly increased in males (+20% and +31%, respectively) and females (+18% and +12%) at 600 mg/kg/day. These weight changes correlated with an increase in the frequency of macroscopically accentuated lobular pattern and, at microscopic examination, with hepatocellular hypertrophy.

Higher statistically significant absolute and relative kidney weights were recorded for males receiving the test item at the highest dose (+16% and +26%, respectively). These changes correlated with microscopic tubular hyaline droplets, tubular basophilia and granular casts, and were considered as test item-related. In females, a statistically significant increase in absolute but not in relative kidney weight was detected in animals given 150 (+13%) or 600 mg/kg/day (+14%). These changes were not considered to be toxicologically significant given their magnitude and the absence of relative to body weight changes or microscopic correlates.

The mean absolute and relative weights of the adrenal glands were statistically significantly increased (+26% and +38%, respectively) in males given Diacetone alcohol at 600 mg/kg/day. These changes were mainly due to higher weights in two individuals. Despite the magnitude and given the absence of histologic correlates and the presence of two outlier animals, these increases were considered to be most likely unrelated to the test item. Furthermore, in females, mean absolute and relative values were also above those of controls (+17% and +11%, respectively) at the highest dose, but these differences were not statistically significant, had no microscopic correlates and therefore were not considered to be toxicologically relevant.
A statistically significantly slight increase in relative (+14%) but not in mean absolute spleen weight was also observed in males given the highest dose. This was considered to be secondary to a decrease in final body weight and, thus, not directly related to the test item.

Other organ weight changes were not considered to be related to the test item as they were small in amplitude, not statistically significant, had no gross or microscopic correlates, and/or were not dose-related in magnitude. Furthermore, they reflected the usual range of individual variations.

End of the treatment-free period
There was complete recovery of the weight increases seen in the liver, in both sexes, at 600 mg/kg/day. In the kidneys and adrenals, in males only (at 600 mg/kg/day), recovery was also considered as complete, since differences between controls and high dose animals were minimal and not statistically significantly.
Other differences in organ weights at the end of the treatment-free period were minor and reflected the usual range of individual variations.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

End of the treatment period
The test item administration induced a slight increase in the incidence of accentuated lobular pattern in the liver in mid or high-dose males (6/10 in both of these groups, as opposed to 4/10 in controls) and in mid- or high-dose females (4/10 in both of these groups, as opposed to 2/10 in controls). At microscopic examination, these changes correlated with minimal to slight hepatocellular centrilobular hypertrophy at the highest dose.
The other macroscopic findings had no significant histologic correlates or correlated with common histologic findings in control rats, and were considered to be incidental.

End of the treatment-free period
There was complete recovery of the accentuated lobular pattern seen in the liver, in both sexes, at high dose.
The few macroscopic findings noted at the end of the treatment-free period were of those commonly recorded in the rat and none were considered to be related to the test item administration.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See Table 5.
End of the treatment and treatment-free periods
The main microscopic findings were seen in the liver, in both sexes, and in the adrenals and kidneys, in males only.

In the liver, minimal to slight centrilobular hypertrophy of hepatocytes was found in all males and in six out of ten females at 600 mg/kg/day. In addition, minimal or slight hepatocellular vacuolation was observed in males at 150 mg/kg/day (1/10) and 600 mg/kg/day (4/10). In females, this finding was also observed in at 150 mg/kg/day (3/10) and 600 mg/kg/day (1/10). Given the low incidence and severity, as well as the lack of dose-correlation in females, this finding was considered as most likely incidental. Of notice, both of these findings (hepatocellular hypertrophy and vacuolation) were completely reversed in both sexes, at the end of treatment-free period.

In the kidneys, in males, there was a higher incidence of grade 3 or 4 tubular hyaline droplets from 25 mg/kg/day compared to controls. This was characterized by dense eosinophilic droplets in the proximal tubular epithelium. In high-dose males, this finding was frequently associated with tubular basophilia and granular casts. These findings correlated with increased incidence of grade 3/4 positivity for a2u-globulin as detected by immunohistochemistry, using the monoclonal mouse anti-rat a2u-globulin biotinylated antibody (BAM586, R&D Systems), and a semi-automated method (which includes a protease digestion step for antigen retrieval). Hyaline droplet accumulation and granular casts (but not basophilia) were almost completely reversed at the end of treatment free period.
In the adrenals, there was minimal to moderate vacuolation of cortical cells (mostly in the zona fasciculata) in males at 25 mg/kg/day (3/10) and 600 mg/kg/day (4/10), but not at 150 mg/kg/day. This finding was not reversed at the end of treatment-free period. Given the lack of dose correlation and the low amplitude in most of the animals, this finding was considered to be incidental.

Qualitative testis staging did not indicate any abnormalities in the integrity of the various cell types present within the different stages of the spermatogenic cycle.
Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the rat.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Estrous cycles
See Table 6.
At the end of the treatment period, the number of cycles measured during a period of 21 days in the high dose group was slightly lower (not statistically significant) than in control group, due to a higher number of females which remained in diestrous during several consecutive days (6/10 vs. 2/10 in the control group), leading to longer cycles. This was considered treatment-related and non-adverse as this was reversible and in absence of microscopic effects on female reproductive organs.
At the end of the treatment-free period, this was no longer observed. In the high-dose group, 1/5 females remained in estrous for 13 consecutive days before returning to normal. This was not considered adverse since a normal cycle started from Day 18 for this female.

Seminology
See Table 7.
Treatment period
There were no test item treatment-related effects on mean epididymal sperm motility and morphology, mean testicular sperm head and the daily sperm production rate.
At 600 mg/kg/day and when compared with controls, there was a trend towards lower mean epididymal sperm counts (-10 and -11%, as number/cauda and number/g cauda, respectively). A relationship with the test item was considered to be unlikely in view of the low magnitude, the standard deviations, the absence of microscopic finding in the testis and epididymidis and since individual values are comparable to what can be observed in Sprague-Dawley rats in laboratory conditions.

Treatment-free period
At 600 mg/kg/day and when compared with controls, there were still lower mean epididymal sperm counts (-14 and -5%, as number/cauda and number/g cauda, respectively). Changes were of similar magnitude to those observed during the treatment period and were therefore not considered to be related to the previous treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Table 1: Body weight

Treatment period

Sex		Male					Female
	Dose-level (mg/kg/day)		0	25	150	600		0	25	150	600
	Body weight
	. Week 1		217	218	221	212		166	168	171	169
	. Week 14		562	539	549	510**		281	299	305	294
	% change from controls		-	-4	-2	-9		-	+6	+9	+5
	Body weight change
	. Weeks 1-14		+345	+321	+328	+299*		+115	+132	+134*	+125
	% change from controls		-	-7	-5	-13		-	+15	+17	+9

Statistically significant from controls: *: p < 0.05; **: p < 0.01.

-: not applicable

Treatment-free period

Sex	Male					Female
Dose-level (mg/kg/day)		0	25	150	600		0	25	150	600
Body weight
. Week 14		579	-	-	516*		295	-	-	290
. Week 20		598	-	-	554*		306	-	-	303
% change from controls		-	-	-	-7		-	-	-	-1
Body weight change
. Weeks 14-20		+19	-	-	+38		+12	-	-	+13

*: p < 0.05.

-: not applicable.

Table 2: Haematology

Treatment period

Sex		Male			Historical Control Data
Dose-level (mg/kg/day)	0	25	150	600
Neutrophils (G/L) % change from control	1.44 -	1.45 +1	1.67 +16	2.21* +53	1.58^a 0.90 - 2.61^b

Statistically significant from controls: *: p < 0.05.

^aMean

^b5^th- 95^thpercentiles

 

Sex		Female			Historical Control Data
Dose-level (mg/kg/day)	0	25	150	600
Red Blood Cells (T/L) % change from control	8.24 -	8.09 -2	7.85* -5	7.77** -6	8.30^a 7.25 - 9.07^b
Hemoglobin (g/dL) % change from control	15.2 -	15.0 -1	14.7 -3	14.5* -5	15.0^a 13.6 - 16.2^b
Packed Cell Volume (L/L) % change from control	0.45 -	0.44 -2	0.43 -4	0.42** -7	0.45^a 0.40 - 0.50^b
White Blood Cells (G/L) % change from control	8.40 -	7.27 -13	7.24 -14	6.41* -24	6.80^a 3.82 - 10.83^b
Lymphocytes (G/L) % change from control	7.26 -	6.01 -17	6.11 -16	5.35* -26	5.57^a 2.89 - 8.75^b

Statistically significant from controls: *: p < 0.05; **: p < 0.01.

^aMean

^b5^th- 95^thpercentiles

Table 3: Blood biochemistry

Treatment period

Sex		Male			Historical Control Data
Dose-level (mg/kg/day)	0	25	150	600
Inorganic phosphorus (mmol/L) % change from control	2.14 -	2.01 -6	1.95* -9	1.86** -13	2.07^a 1.74 - 2.41^b
Total protein (g/L) % change from control	58.4 -	59.2 +1	59.5 +2	62.5** +7	65^a 60 - 71^b
Albumin (g/L) % change from control	37 -	37 0	37 0	39** +5	38^a 33 - 41^b
Cholesterol (mmol/L) % change from control	1.49 -	1.45 -3	1.44 -3	2.09** +40	1.3^a 0.7 - 2.0^b

Statistically significant from controls: *: p < 0.05; **: p < 0.01.

^aMean

^b5^th- 95^thpercentiles

Sex		Female			Historical Control Data
Dose-level (mg/kg/day)	0	25	150	600
Cholesterol (mmol/L) % change from control	1.58 -	1.65 +4	1.90 +20	2.26** +43	2.0^a 1.4 - 2.6^b

Statistically significant from controls: **: p < 0.01.

^aMean

^b5^th- 95^thpercentiles

Table 4: Organ weights

Treatment period

Sex	Male			Female
Group	2	3	4	2	3	4
Dose-level (mg/kg/day)	25	150	600	25	150	600
Exam. animals / Num. of animals	10/10	10/10	10/10	10/10	10/10	10/10
- Final body weight	-3	-1	-9	+8	+11	+6
- Liver
.absolute	-8	+3	+20*	+8	+10	+18**
.relative	-5	+4	+31**	0	-1	+12**
- Adrenal glands
.absolute	+1	+13	+26**	-1	+10	+17
.relative	+4	+14	+38**	-7	0	+11
- Kidneys
.absolute	-3	+7	+16**	+5	+13*	+14**
.relative	-1	+7	+26**	-3	+3	+8
- Spleen
.absolute	-1	+9	+5	-3	+6	+9
.relative	+1	+10	+14*	-10	-4	+2

Statistically significant from controls: *: p < 0.05; **: p < 0.01.

The significance concerned the organ weights values and not the percentages.

Table 5: Microscopic examination

Incidence and severity of selected microscopic findings at the end of the treatment period

Sex		Male					Female
Group	1		2	3	4	1		2	3	4
Dose-level (mg/kg/day)	0		25	150	600	0		25	150	600
n	10		10	10	10	10		10	10	10
Liver
- Hypertrophy; hepatocyte
Minimal (grade 1)	-		-	-	8	-		-	-	5
Slight (grade 2)	-		-	-	2	-		-	-	1
Kidneys
- Hyaline droplets; tubular epithelium
Minimal (grade 1)	-		1	-	-	-		na	na	-
Slight (grade 2)	-		4	4	-	-		na	na	-
Moderate (grade 3)	1		5	6	4	-		na	na	-
Marked (grade 4)	-		-	-	5	-		na	na	-
- Basophilia; tubule
Minimal (grade 1)	1		6	7	5	-		na	na	-
Slight (grade 2)	-		1	-	1	-		na	na	-
Moderate (grade 3)	-		-	-	3	-		na	na	-
- Granular casts
Minimal (grade 1)	-		4	5	5	-		na	na	-
Slight (grade 2)	-		3	3	1	-		na	na	-
Moderate (grade 3)	1		-	-	1	-		na	na	-
- Immunostaining fora2u-globulin protein*
Minimal (grade 1)	6		1	1	-	na		na	na	na
Slight (grade 2)	3		1	2	-	na		na	na	na
Moderate (grade 3)	1		4	7	7	na		na	na	na
Marked (grade 4)	-		4	-	3	na		na	na	na
Adrenals
- Vacuolation; cortex
Minimal (grade 1)	-		1	-	2	-		na	na	-
Slight (grade 2)	-		2	-	1	-		na	na	-
Moderate (grade 3)	-		-	-	1	-		na	na	-

-: no findings; na: not applicable.

*Post-repetition with the revised IHC protocol:semi-automatic method (Ventana autostainer) + antigen retrieval with protease digestion

Incidence and severity of selected microscopic findings at the end of the treatment-free period

 

Sex	Male
Group	1	4
Dose-level (mg/kg/day)	0	600
n	5	5
Kidneys
- Hyaline droplets; tubular epithelium
Minimal (grade 1)	-	1
- Basophilia; tubule
Minimal (grade 1)	-	4
Slight (grade 2)	-	1
- Granular casts
Minimal (grade 1)	1	-
Moderate (grade 3)	-	1
Adrenals
- Vacuolation; cortex
Minimal (grade 1)	3	1
Slight (grade 2)	-	1

-: no findings.

Table 6:  Estrous cycles

Summary of estrous cycles determined during 21 consecutive days at the end of the treatment and treatment-free periods 

	0	25	150	600
	Treatment period
Number of cycles	3.5	3.1	3.6	2.4
Cycle length (days)	4.7	5.4	4.5	7.8
Rats cycling normally	6	9	5	4
	Treatment-free period
Number of cycles	4.2	-	-	4.0
Cycle length (days)	4.0	-	-	5.2
Rats cycling normally	5	-	-	3

-              : Not performed.

Table 7: Seminology

Treatment period

Mean sperm analysis dataat the end of the treatment period are described in the following table:

Dose-level (mg/kg/day)	0	25	150	600	Reference data
% of motile epididymal sperm	99.8	99.5	99.2	99.1	[86.0-100]
% of morphologically normal epididymal sperm	95.7	-	-	96.5	[90.0-98.1]
Mean number of epididymal sperm (10^6/cauda)^a	182	183	190	164	[100-182]
Mean number of epididymal sperm (10^6/g cauda)^a	565	549	579	503	[319-591]
Mean number of testicular sperm heads (10^6/g testis)^a	117	124	116	121	[96-151]
Daily sperm production rate (10^6/g testis/day)	19.2	20.3	18.9	19.8	[15.8-24.7]

^a:values rounded to three significant digits; -: not performed.

In bold: relevant changes.

Reference control data based onCiToxLAB France/Study Nos. 40814 TCR, 41748 TCR, 38872 TCR, 36834 TCR: individual data percentiles [5%-95%].

Conclusions:

Diacetone alcohol was administered daily for 13 weeks by gavage to male and female Sprague-Dawley rats at the dose-levels of 25, 150 and 600 mg/kg/day in corn oil. No treatment-related clinical signs were noted during the treatment and treatment-free periods. There were no treatment-related or adverse effects on Functional Observation Battery, body weight, estrous cycles, sperm analysis and clinical pathology parameters. There were no effects on food consumption.
At dose-level of 600 mg/kg/day, the test item administration induced non adverse changes in the liver consisting of increased weight associated with hepatocellular hypertrophy, in both sexes. In males only, test item-related non-adverse findings were also found in the kidneys from 25 mg/kg/day and were characterized by increased weights and tubular hyaline droplet accumulation (as confirmed by immunohistochemistry), frequently associated with tubular basophilia and granular casts. These kidney changes are specific to male rats and non-relevant for humans.
Consequently, under the experimental conditions of the study, the No Observed Adverse Effect Level (NOAEL) after the 13-week treatment period was established at 600 mg/kg/day.

Executive summary:

The potential toxicity of Diacetone alcohol was evaluated following daily oral administration (gavage) to rats for 13 weeks. On completion of the treatment period, designated animalsof the control- and high-dose groups were held for a 6-week treatment-free period in order to evaluate the reversibility of any findings. The study was performed based on the OECD guideline No. 408 and in compliance with the Good Laboratory Practices. Three groups of 10 (low- and mid-dose groups) or 15 (high-dose group) male and female Sprague-Dawley rats each received the test item by daily oral gavage administration for at least 13 weeks. The test item was administered at 25, 150 and 600 mg/kg/day as a solution in the vehicle (corn oil) under a constant dosage-volume of 5 mL/kg/day. A control group of 15 animals per sex received the vehicle alone under the same experimental conditions. On completion of the treatment period, the animals in each group were sacrificed, with the exception of the recovery animals (last 5 animals per sex in the control and high-dose groups) which were kept for a 6-week treatment-free period. The concentration of the dose formulations was checked in Weeks 1, 4, 9 and 13. The animals were checked at least twice daily during the treatment and treatment-free periods for mortality and morbidity, and once daily for clinical signs. In addition, detailed clinical examinations were performed once before the beginning of the treatment period and then weekly. The body weight was recorded once before the beginning of the treatment period, and then at least once a week during the study as well as food consumption. Functional Observation Battery was evaluated on all main animals once in Week 12. Ophthalmological examinations were performed on all main animals before the beginning of the treatment period and once at the end of the treatment period. Estrous cycle stage was determined on all main females daily for 21 consecutive days at the end of the treatment period and at the end of the treatment-free period for recovery animals. Hematology, blood biochemistry and urinalysis were performed towards the end of thetreatment period for main animals and towards the end of treatment-free period for recovery animals. On completion of the treatment period and treatment-free periods, the animals were euthanized and submitted to a full macroscopic post-mortem examination. Sperm investigations were performed at sacrifice of the males. A microscopic examination was performed on selected tissues from animals of the control- and high-dose groups sacrificed at the end of the treatment period, and on macroscopic lesions, kidneys and adrenals (males only) and liver from animals of the low- and mid-dose groups sacrificed at the end of the treatment period. Kidneys and adrenals (males only) and liver of recovery animals were examined microscopically at the end of the treatment-free period. Males kidney slides immunostained with an antibody for Alpha 2u-globulin protein were also microscopically examined at the end of treatment period.

The test item concentrations in the administered dose formulations analyzedin Weeks 1, 4, 9 and 13 were within the acceptance criteria (± 10%) and no test item was detected in control formulations. There were no test item treatment-related deaths or clinical signs.

A dose-related increase in horizontal movement and rearing was observed in males during the recording of motor activity. In absence of correlated clinical signs or other findings at the Functional Observation Battery, this was not considered adverse. Non-adverse slightly lower body weight was recorded from Week 10 in males during the treatment period at 600 mg/kg/day. Body weight gain returned to normal at the end of the treatment-free period, suggesting reversibility. No effects on body weight and body weight gain were noted in females. There were no differences in food consumption between control and test item-treated animals during the treatment and treatment-free periods. At the end of the treatment period, a higher number of females given 600 mg/kg/day remained in diestrous for several consecutive days when compared with controls. This was no longer observed at the end of the treatment-free period and was therefore not considered to be adverse. There were no test item-related ophthalmology findings at the end of the treatment period. At hematology at the end of the treatment period, when compared with controls, a slightly higher neutrophil count was noted in males treated at 600 mg/kg/day (+53%). In females, mean red blood cell count was statistically significantly decreased at 150 and 600 mg/kg/day when compared with controls (up to -6%) and was associated with lower hemoglobin (-5%) and packed cell volume (-7%) at 600 mg/kg/day. Lower total white blood cell (-24%) and lymphocyte counts were also noted (-26%) at the highest dose. These findings were not considered to be adverse in view of their amplitude (remained within the physiological range) and were no longer observed at the end of the treatment-free period, suggesting reversibility. At blood biochemistry in both genders, moderately higher cholesterol concentration was noted at 600 mg/kg/day (+40 % for males and +43% for females). In males, a lower inorganic phosphorus concentration was also noted from 150 mg/kg/day (up to -13%), when compared with controls. These findings were not considered to be adverse and recovery was complete at the end of the treatment-free period. At urinalysis, a slight non-adverse decrease of pH values was noted in males at 600 mg/kg/day. Urinary findings were no longer observed at the end of the treatment-free period. At sperm analyses, when compared to controls, there were no effects attributed to the test item treatment. Test item administration at 600 mg/kg/day induced minimal to slight non-adverse centrilobular hepatocellular hypertrophy that correlated with increases in liver weights and with an increase in the incidence of macroscopically accentuated lobular pattern, in both sexes. In the kidneys, in males only, there were increased incidence and severity of tubular hyaline droplets (consistent with a2u-globulin), tubular basophilia and granular casts, from 25 mg/kg/day, which correlated with increased kidney weights. Given the nature and the severity of the microscopic alterations associated with the hyaline deposits (mostly minimal to slight), hyaline droplet accumulation and related renal lesions were considered to be non-adverse, in this study. Moreover, these findings are specific to male rats and non-relevant for humans. 

Consequently, under the experimental conditions of the study, the No Observed Adverse Effect Level (NOAEL) after the 13-week treatment period was established at 600 mg/kg/day.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study without detailed documentation

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: SD(Crj:CD(SD)) SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Co (955 Kanbayashi, Yatabe-machi, Niihari-gun, Ibaragi-ken)
- Age at study initiation: 8 week old male rats and 7 week old female rats
- Weight at study initiation: 358 g for males and 211 g for females
- Housing: animals were raised in barrier system cages arranged in 5 stainless steel racks
- Diet (e.g. ad libitum): Nihon Nosan Kogyo Co., solid food lab MR, stock, available ad libitum
- Water (e.g. ad libitum): Kanagawa prefecture water supply available ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 40-60
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Vehicle: Japanese Pharmacopeia purified water
- Lot/batch no. (if required): 180889, 180964

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Since the solution was confirmed to be stable for at least 7 days, it was prepared weekly and used within 7 days. The intial and final preparations were analyzed, and confirmation was made that specific concentrations were prepared. Analysis of the raw material of the test substance was conducted.

Duration of treatment / exposure:

44 days for males and 41-45 days for females

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
30, 100, 300, or 1000 mg/kg body weight
Basis:
actual ingested

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes

Details on study design:

Dose levels were selected on the basis of results from a 14-day dose-range finding study.

Positive control:

None used

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on kill date
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on kill date
- Animals fasted: Yes
- How many animals: all

URINALYSIS: Yes
- Time schedule for collection of urine: collected on day 38 and day 41 of administration
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

Reproductive examinations

Statistics:

Statistical significance (danger rate of 5% or less) with the control group on the mean values and frequencies was conducted using the following methods. Parametric data such as body weight, food consumption, hematological and blood chemical data, organ weight, number of corpus luteum, number of implantations, gestation period, and number of pups delivered were subject to Bartlett’s distribution test. If the distribution was normal, an distribution analysis for the normal positions was conducted, and if a significant variance was confirmed from those results, a comparative test was performed on each group compared with the control group using either the Dunnett method or the Scheffe method (if the size of the groups differed). If the distribution was not normal and for non-parametric data such as implantation rate, delivery rate, pregnancy rate, rate of surviving pups, and qualitative data for urine examinations, Kruskal-Wallis analysis of variance was performed, and if a significant variance was confirmed for those results, a comparative test was performed on each group compared with the control group using either the Dunnett method or the Scheffe method (if the size of the groups differed). Categorical data such as the survival rate for newborns, copulation rate, fertilization rate, birth rate, gender ratio of newborns, changes in the overall condition and manifestation of pathological abnormalities were subject to ¿2 tests. Pathological abnormalities were also noted in the control group, and the data for findings where the impact of the test substance exhibited a difference in the distribution for the degree of changes was separated into two categories and tested.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

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):

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:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no deaths among the males. In the females, one animal in the 1000 mg/kg group was weakened during delivery and so was euthanized once reaching a near death state. Other than this one case, no deaths were noted.

BODY WEIGHT AND WEIGHT GAIN
There were no significant changes noted in the weights or weight increases in males. In females, a significant reduction in the amount of weight gain (-31%) during the premating period was noted in the 1000 mg/kg group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Neither males nor females in the groups administered the test substance exhibited significant changes in food consumption.

HAEMATOLOGY
There was a significant increase (+16%) in the number of platelets noted in the 1000 mg/kg group male rats.

CLINICAL CHEMISTRY
In the 1000 mg/kg group in males (females not done), significant increases (50% ) in GOT, choline esterase (x1.8), total protein (10%), total cholesterol (76%), total bilirubin (33%), blood urea nitrogen (19%), creatinine (17%) and calcium (6%) were noted, as well as a significant reduction in glucose (23%). Furthermore, while not a significant difference, an increasing trend was noted in the 300 mg/kg group for choline esterase and total protein, an increasing trend was noted in the 1000 mg/kg group for ¿-GTP and albumin, along with a decreasing trend for the A/G ratio and triglycerides. Significantly low values were exhibited for alkaline phosphatase when compared to the control group but these were not dose dependent changes.

URINALYSIS
There were no significant changes noted for any of the examination items in the groups administered the test substance.

ORGAN WEIGHTS
Significant increases in the absolute (15%) and relative (13%) weights of the kidneys were noted in males in the 300 mg/kg group, and the same increasing trend was noted in the 1000 mg/kg group. Additionally, in the 1000 mg/kg group, significant increases in the absolute (21%) and relative (24%) weights of the adrenals were noted, along with a significant increase in the relative (17%) weight of the liver, and there was an increasing trend exhibited for the absolute weights of the liver. In the females, significant increases in the absolute (26%) and relative (25%) weights of the liver were noted in the 1000 mg/kg group.

GROSS PATHOLOGY, HISTOPATHOLOGY: NON-NEOPLASTIC
Changes thought to be caused by administration of the test substance were noted in the liver, kidney and adrenals.
Five out of ten males in the 1000 mg/kg group exhibited central lobular hepatocellular hypertrophy. Increased deposition of hyaline droplets in the proximal tubular epithelium of the kidney was noted in 8/10 in the 100 mg/kg group, 10/10 in the 300 mg/kg group and 9/10 in the 1000 mg/kg group, exhibiting a strong dose dependency. Additionally, there were 6 animals with increased basophilic tubules in the kidneys in the 300 and 1000 mg/kg group and dilation of the distal tubules noted in 4/10 animals in the 1000 mg/kg group. Vacuolization of the cells of the zona fasciculata in the adrenals was noted in one in the 300 mg/kg group and 5 in the 1000 mg/kg group. Central lobular hepatocellular hypertrophy was noted in 5/10 males in the 1000 mg/kg group.

In the 1000 mg/kg group, central lobular hepatocellular hypertrophy was noted in 6/10 females, vacuolization of the cells of the zona fasciculata in the adrenals in 3/10 females and dilation of the distal tubules of kidneys in 3/10 females.

However, in males and females unsuccessful at impregnation and in females where all of the pups died, there were no changes noted in the pituitary gland and reproductive organs that were thought to be caused by administration of the test substance. In one euthanized female in the 1000 mg/kg group, the fatty degeneration of the proximal tubule epithelium and dilation of distal tubules in the kidneys noted was stronger than the changes in the survivors. Furthermore, central lobular hepatocellular hypertrophic necrosis was noted, along with necrosis of the mucous membranes of the glandular stomach and ileum, hypertrophy of the cells of the zona fasciculata in the adrenals and atrophy of the spleen and thymus. In addition to the findings given above, changes in each of the organs were noted but they were sporadic and not confirmed to be related to administration of the test substance.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant effects at this dose (excluding the sex- and species-specific hyaline droplet nephropathy of the males)

Key result

Dose descriptor:

NOEL

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect

Critical effects observed:

not specified

Conclusions:

Based on the above mentioned results, the non-observable adverse effect levels for repeat dose toxicity of diacetone alcohol in rats are considered to be 100 mg/kg/day in males and females (excluding the sex- and species-specific hyaline droplet nephropathy of the males).

Executive summary:

A repeated dose study according to OECD TG 422 was conducted with 4-hydroxy-4-methyl-2-pentanone (= diacetone alcohol, or HMP). In this study, 10 rats/sex/group were dosed by gavage with 0, 30, 100, 300, or 1000 mg/kg/day of 4-hydroxy-4-methyl-2-pentanonein distilled water for approximately 45 days. Decreased locomotor activity and stimulation response were observed at 300 and 1000 mg/kg/day for both sexes. Altered blood parameters, dilatation of the distal tubules of the kidneys, hepatocellular hypertrophy, and vacuolization of the zona fasciculata of the adrenals were observed in males from the 1000 mg/kg/day group. Females at 300 mg/kg/day showed dilatation of the distal tubules and fatty degeneration of the proximal tubular epithelium in the kidneys. At 1000 mg/kg/day, female body weight gain was reduced, liver weight was increased, hepatocellular hypertrophy was noted, and kidney lesions similar to those at 300 mg/kg/day were also recorded. In addition, increased incidence and/or severity of hyaline droplets in the tubular epithelium was noted in males at 100 mg/kg/day and greater. The kidney effects observed are suggestive of chronic progressive nephropathy (CPN) and a2µ-globulin nephropathy, a condition not relevant for human risk assessment (U. S. EPA. (1991) Alpha2µ-globulin: Association with chemically induced renal toxicity and neoplasia in the male rat. Risk Assessment Forum. EPA/625/3-91/019F). Therefore, the NOAEL for this study was 100 mg/kg/day for males and females (excluding the sex- and species-specific hyaline droplet nephropathy observed in the males).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Two GLP guideline studies

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Shell Toxicology Laboratory (Tunstall)
- Age at study initiation: 10 to 13 weeks
- Weight at study initiation: 413 to 415 g (male) and 245 to 246 g (female)
- Housing: hanging aluminum cages with stainless steel bases
- Diet: Lad 1 (Spillers Spratts Ltd.), ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 31 to 57
- Photoperiod (hrs dark / hrs light):12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

clean air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: aluminum chamber with a volume of 1 m3
- Source and rate of air: laboratory air and 1.8 and 1.9 m3/min
- System of generating particulates/aerosols: by completely evaporating the solvent into the streams of ventilating air entering the chambers using micrometering pumps and vaporisers
- Temperature, humidity, pressure in air chamber: between 19 and 25 °C, humidity and pressure not reported
- Air flow rate: 0.40 to 0.55 meters cubed/min
- Treatment of exhaust air: exhaust ducts from each chamber entered a common exhaust duct through which the air was drawn by a fan situated on the roof of the laboratory.

TEST ATMOSPHERE
- Brief description of analytical method used: hydrocarbon analysers fitted with flame-ionisation detectors

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

hydrocarbon analysers fitted with flame-ionisation detectors.

Duration of treatment / exposure:

6 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
50, 225 and 1000 ppm
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
233, 1041 and 4685 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

12 animals/sex/dose

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment (if not random): no data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:Yes
-Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the end of the experiment

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the experiment
- Anaesthetic used for blood collection: No data
- Animals fasted: No data

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the experiment
- Animals fasted: No data

URINALYSIS: Yes
- Time schedule for collection of urine: overnight following the last exposure
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

ORGAN WEIGHT: Yes
After post-mortem examinations the following organe were weighed:
Brain Liver
Heart Spleen
Kidneys Testes

HISTOPATHOLOGY: Yes
Tissues taken for histological examination were: *
Mammary gland (posterior site with skin)
Mesenteric lymph node
Pancreas
Stomach
Intestine at 5 levels
Caecum*
Spleen
Liver (middle, left and trianguler lobes)
Adrenals
Kidneys
Ovaries or testes
Uterus or prostate
Seminal vesicles*
Urinary bladder
Thyroid with oesophagus and trachea
Trachea (mid course and bifurcation)
Heart
Lungs
Nasal cavity
Thymus
Eye and lacrimal glands
Salivary gland (submaxillary)
Brain
Spinal cord (thoracic)*
Pituitary
Tongue*
Sciatic nerves*
Muscle (femoral)*
Knee joint and femur*
Plus any other macroscopic lesions in any tissues

The samples marked * were held in 4% neutral formalin and only processed for histological examination if indicated by clinical or other pathological findings.

Statistics:

Body and organ weights were analysed by covariance analysis using initial body weight as the covariate. Reported means were adjusted for initial body weight if a significant covariance relationship existed: where no significant covariance relationship was found, unadjusted means were reported.Organ weights were further examined by covariance analysis using the terminal body weight as the covariate. The organ weight means are reported as adjusted for terminal body weight if a significant covariance relationship existed. Although not a true covariance analysis (because the terminal body weights are dependent upon treatment), the analysis does provide an aid to the interpretation of organ weights when there are differences in terminal body weights. The analysis attempts to predict what the organ weights would have been, had all the animals had the same terminal body weight. Clinical chemical and haemlatological parameters were examined using analysis of variance. The analysis allowed for the fact that animals were multi-housed.Differences in response can be affected by cage environment as well as by treatment (this is indicated by 'C' - inflated standard deviation due to significant cage effect), but this effect is minimal in a study of this duration. The significance of any difference between treated and control group means was tested using the Williams t –test (1971, 1972). However, if a monotonic dose response could not be assumed Dunnett's test (1964) was used.

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):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY: No clinical signs of toxicity were noted during the first four weeks of exposure, but over the next two weeks slight lethargy was noted in several of the animals exposed to the high and medium concentrations when they were examined 30 minutes after cessation of exposure. These signs persisted for a few hours but all the rats were fully recovered before the next day's exposure.

BODY WEIGHT AND WEIGHT GAIN: The body weights of females exposed to the high concentration to be significantly lower (-5%) than controls at week 6. No significant differences were noted in any other group.

FOOD CONSUMPTION: The results show no significant differences in the female food intakes but a significant reduction from control in the males exposed to the high concentration (-14%) after 2 weeks, but not at subsequent weeks.

HAEMATOLOGY: The results of the haematological analysis of the blood taken from each rat 18 hours after the last exposure to DAA show no significant differences in the males. Haemoglobin was significantly higher (+6%) than control at the high concentration exposure level in females.
Differential leucocytes count of blood taken 18 hours after the last exposure to the high concentration did not show any significant differences from control.

CLINICAL CHEMISTRY: In females only one significant difference was seen, LDH being significantly higher (+48%) than control at the high concentration. In males plasma protein was increased (+3%) at the high concentration, plasma chloride was reduced (-1%) at the medium and high concentration, and plasma sodium was reduced (-0.7%) at the low, medium and high concentrations.

URINALYSIS: The results show blood to be present in the urines of two rats but this could be attributed to slight damage to the claws from the mesh floor of the urine collector. Glucose was also present in the urine of many of the rats , but since the urine was collected overnight between exposures and the animals had to be fed during the collection period, the presence of glucose is not unexpected. What was unexpected was the very low incidence of rats with glycosuria at the high dose compared with controls and low dose rats .

ORGAN WEIGHTS: The organ weights of the rats showed male liver weights to be significantly higher than control at the medium (+13%) and high (+23%) concentrations, and male kidney weights to be significantly higher ( +17%) than control at the high concentration. When adjustment was made for terminal body weight, not only was there a significant increase in the males in these variates but the females also were significantly higher than controls .

HISTOPATHOLOGY: With the exception of one animal, the kidneys of all males exposed to the high concentration showed eosinophilic hyaline droplets in the proximal tubular cells. They were not easily identified without the use of the X100 objective. The cells containing the droplets were of limited distribution, being mainly close to the surface around the superficial glomeruli. These eosinophilic hyaline droplets were not seen in the females exposed to the high concentration or in the males and females exposed to the medium and low concentrations. In other organs, abnormalities included alveolar wall thickening and minor inflammatory infiltrates in the lungs, and similar infiltrates in the nasal cavities and trachea. Inflammatory changes in the lacrimal and salivary glands were found in a minority of animals. None of these changes appeared to be compound related.

Key result

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

4 685 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: increased kidney and liver weights in males and eosinophilic hyaline droplets in the proximal tubular cells in males

Key result

Dose descriptor:

NOEC

Remarks:

systemic toxicity

Effect level:

1 041 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect

Key result

Dose descriptor:

NOAEC

Remarks:

local irritation of the respiratory tract

Effect level:

>= 4 685 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: no effect

Critical effects observed:

not specified

Executive summary:

A 6-week whole body inhalation study was conducted with 4-hydroxy-4-methylpentan-2-one in Wistar rats. Animals were exposed to analytical concentrations of 0, 233, 1041 and 4685 mg/m³of 4-hydroxy-4-methylpentan-2-one for 6 hours per day for 5 days per week for 6 weeks (Butterworth et al., 1980). This non-GLP study was equivalent to OECD Test Guideline 412. There were no clinical signs of toxicity in the first four weeks of exposure; however, slight lethargy was noted for a few hours in 233 and 4685 mg/m³4-hydroxy-4-methylpentan-2-one-exposed animals over the following 2 weeks. The body weights of females exposed to 4685 mg/m³were significantly lower (-5%) than controls at week 6. After 2 weeks, food consumption was significantly decreased in males compared to controls; however, this was not noted in subsequent weeks. Haemoglobin was significantly increased (+6%) in females exposed to 4685 mg/m³4-hydroxy-4-methylpentan-2-one compared to controls. Lactate dehydrogenase (LDH) was significantly higher (+48%) in females exposed to 4685 mg/m³4-hydroxy-4-methylpentan-2-one compared to controls. In males, plasma protein was increased (+3%) at the 4685 mg/m³concentration and plasma sodium was reduced (-0.7%) at all concentrations. Liver and kidney weights were increased in males (compared to controls) exposed to 1041 (liver+13%) and 4685 mg/m³(liver+23%) and 4685 mg/m³(kidney +17%) 4-hydroxy-4-methylpentan-2-one, respectively. Histologically, the abnormal presence of eosinophilic hyaline droplets in the proximal tubules of males exposed to the high concentration was noted. There was no suggestion of cellular damage and the response may have been due to cellular accumulation of the chemical or a metabolite. A review of the study data suggests that a NOAEC of 4685 mg/m³and a NOEC of 1041 mg/m³can be considered for repeated-dose inhalational toxicity, based on liver weight changes not associated with histological alterations and probably secondary to a metabolic over load and based on the male rat-specific eosinophilic hyaline droplets in the proximal tubular cells as hyaline droplet formation in male rats is not considered to be relevant to human health for the purposes of risk assessment (U. S. EPA, 1991). No irritation of the respiratory tract was observed up to the highest concentration tested.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

4 685 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

A study comparable to guideline study with acceptable restrictions

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Shell Toxicology Laboratory (Tunstall)
- Age at study initiation: 10 to 13 weeks
- Weight at study initiation: 413 to 415 g (male) and 245 to 246 g (female)
- Housing: hanging aluminum cages with stainless steel bases
- Diet: Lad 1 (Spillers Spratts Ltd.), ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 31 to 57
- Photoperiod (hrs dark / hrs light):12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

clean air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: aluminum chamber with a volume of 1 m3
- Source and rate of air: laboratory air and 1.8 and 1.9 m3/min
- System of generating particulates/aerosols: by completely evaporating the solvent into the streams of ventilating air entering the chambers using micrometering pumps and vaporisers
- Temperature, humidity, pressure in air chamber: between 19 and 25 °C, humidity and pressure not reported
- Air flow rate: 0.40 to 0.55 meters cubed/min
- Treatment of exhaust air: exhaust ducts from each chamber entered a common exhaust duct through which the air was drawn by a fan situated on the roof of the laboratory.

TEST ATMOSPHERE
- Brief description of analytical method used: hydrocarbon analysers fitted with flame-ionisation detectors

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

hydrocarbon analysers fitted with flame-ionisation detectors.

Duration of treatment / exposure:

6 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
50, 225 and 1000 ppm
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
233, 1041 and 4685 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

12 animals/sex/dose

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment (if not random): no data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:Yes
-Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the end of the experiment

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the experiment
- Anaesthetic used for blood collection: No data
- Animals fasted: No data

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the experiment
- Animals fasted: No data

URINALYSIS: Yes
- Time schedule for collection of urine: overnight following the last exposure
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

ORGAN WEIGHT: Yes
After post-mortem examinations the following organe were weighed:
Brain Liver
Heart Spleen
Kidneys Testes

HISTOPATHOLOGY: Yes
Tissues taken for histological examination were: *
Mammary gland (posterior site with skin)
Mesenteric lymph node
Pancreas
Stomach
Intestine at 5 levels
Caecum*
Spleen
Liver (middle, left and trianguler lobes)
Adrenals
Kidneys
Ovaries or testes
Uterus or prostate
Seminal vesicles*
Urinary bladder
Thyroid with oesophagus and trachea
Trachea (mid course and bifurcation)
Heart
Lungs
Nasal cavity
Thymus
Eye and lacrimal glands
Salivary gland (submaxillary)
Brain
Spinal cord (thoracic)*
Pituitary
Tongue*
Sciatic nerves*
Muscle (femoral)*
Knee joint and femur*
Plus any other macroscopic lesions in any tissues

The samples marked * were held in 4% neutral formalin and only processed for histological examination if indicated by clinical or other pathological findings.

Statistics:

Body and organ weights were analysed by covariance analysis using initial body weight as the covariate. Reported means were adjusted for initial body weight if a significant covariance relationship existed: where no significant covariance relationship was found, unadjusted means were reported.Organ weights were further examined by covariance analysis using the terminal body weight as the covariate. The organ weight means are reported as adjusted for terminal body weight if a significant covariance relationship existed. Although not a true covariance analysis (because the terminal body weights are dependent upon treatment), the analysis does provide an aid to the interpretation of organ weights when there are differences in terminal body weights. The analysis attempts to predict what the organ weights would have been, had all the animals had the same terminal body weight. Clinical chemical and haemlatological parameters were examined using analysis of variance. The analysis allowed for the fact that animals were multi-housed.Differences in response can be affected by cage environment as well as by treatment (this is indicated by 'C' - inflated standard deviation due to significant cage effect), but this effect is minimal in a study of this duration. The significance of any difference between treated and control group means was tested using the Williams t –test (1971, 1972). However, if a monotonic dose response could not be assumed Dunnett's test (1964) was used.

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):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY: No clinical signs of toxicity were noted during the first four weeks of exposure, but over the next two weeks slight lethargy was noted in several of the animals exposed to the high and medium concentrations when they were examined 30 minutes after cessation of exposure. These signs persisted for a few hours but all the rats were fully recovered before the next day's exposure.

BODY WEIGHT AND WEIGHT GAIN: The body weights of females exposed to the high concentration to be significantly lower (-5%) than controls at week 6. No significant differences were noted in any other group.

FOOD CONSUMPTION: The results show no significant differences in the female food intakes but a significant reduction from control in the males exposed to the high concentration (-14%) after 2 weeks, but not at subsequent weeks.

HAEMATOLOGY: The results of the haematological analysis of the blood taken from each rat 18 hours after the last exposure to DAA show no significant differences in the males. Haemoglobin was significantly higher (+6%) than control at the high concentration exposure level in females.
Differential leucocytes count of blood taken 18 hours after the last exposure to the high concentration did not show any significant differences from control.

CLINICAL CHEMISTRY: In females only one significant difference was seen, LDH being significantly higher (+48%) than control at the high concentration. In males plasma protein was increased (+3%) at the high concentration, plasma chloride was reduced (-1%) at the medium and high concentration, and plasma sodium was reduced (-0.7%) at the low, medium and high concentrations.

URINALYSIS: The results show blood to be present in the urines of two rats but this could be attributed to slight damage to the claws from the mesh floor of the urine collector. Glucose was also present in the urine of many of the rats , but since the urine was collected overnight between exposures and the animals had to be fed during the collection period, the presence of glucose is not unexpected. What was unexpected was the very low incidence of rats with glycosuria at the high dose compared with controls and low dose rats .

ORGAN WEIGHTS: The organ weights of the rats showed male liver weights to be significantly higher than control at the medium (+13%) and high (+23%) concentrations, and male kidney weights to be significantly higher ( +17%) than control at the high concentration. When adjustment was made for terminal body weight, not only was there a significant increase in the males in these variates but the females also were significantly higher than controls .

HISTOPATHOLOGY: With the exception of one animal, the kidneys of all males exposed to the high concentration showed eosinophilic hyaline droplets in the proximal tubular cells. They were not easily identified without the use of the X100 objective. The cells containing the droplets were of limited distribution, being mainly close to the surface around the superficial glomeruli. These eosinophilic hyaline droplets were not seen in the females exposed to the high concentration or in the males and females exposed to the medium and low concentrations. In other organs, abnormalities included alveolar wall thickening and minor inflammatory infiltrates in the lungs, and similar infiltrates in the nasal cavities and trachea. Inflammatory changes in the lacrimal and salivary glands were found in a minority of animals. None of these changes appeared to be compound related.

Key result

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

4 685 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: increased kidney and liver weights in males and eosinophilic hyaline droplets in the proximal tubular cells in males

Key result

Dose descriptor:

NOEC

Remarks:

systemic toxicity

Effect level:

1 041 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect

Key result

Dose descriptor:

NOAEC

Remarks:

local irritation of the respiratory tract

Effect level:

>= 4 685 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: no effect

Critical effects observed:

not specified

Executive summary:

A 6-week whole body inhalation study was conducted with 4-hydroxy-4-methylpentan-2-one in Wistar rats. Animals were exposed to analytical concentrations of 0, 233, 1041 and 4685 mg/m³of 4-hydroxy-4-methylpentan-2-one for 6 hours per day for 5 days per week for 6 weeks (Butterworth et al., 1980). This non-GLP study was equivalent to OECD Test Guideline 412. There were no clinical signs of toxicity in the first four weeks of exposure; however, slight lethargy was noted for a few hours in 233 and 4685 mg/m³4-hydroxy-4-methylpentan-2-one-exposed animals over the following 2 weeks. The body weights of females exposed to 4685 mg/m³were significantly lower (-5%) than controls at week 6. After 2 weeks, food consumption was significantly decreased in males compared to controls; however, this was not noted in subsequent weeks. Haemoglobin was significantly increased (+6%) in females exposed to 4685 mg/m³4-hydroxy-4-methylpentan-2-one compared to controls. Lactate dehydrogenase (LDH) was significantly higher (+48%) in females exposed to 4685 mg/m³4-hydroxy-4-methylpentan-2-one compared to controls. In males, plasma protein was increased (+3%) at the 4685 mg/m³concentration and plasma sodium was reduced (-0.7%) at all concentrations. Liver and kidney weights were increased in males (compared to controls) exposed to 1041 (liver+13%) and 4685 mg/m³(liver+23%) and 4685 mg/m³(kidney +17%) 4-hydroxy-4-methylpentan-2-one, respectively. Histologically, the abnormal presence of eosinophilic hyaline droplets in the proximal tubules of males exposed to the high concentration was noted. There was no suggestion of cellular damage and the response may have been due to cellular accumulation of the chemical or a metabolite. A review of the study data suggests that a NOAEC of 4685 mg/m³and a NOEC of 1041 mg/m³can be considered for repeated-dose inhalational toxicity, based on liver weight changes not associated with histological alterations and probably secondary to a metabolic over load and based on the male rat-specific eosinophilic hyaline droplets in the proximal tubular cells as hyaline droplet formation in male rats is not considered to be relevant to human health for the purposes of risk assessment (U. S. EPA, 1991). No irritation of the respiratory tract was observed up to the highest concentration tested.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

4 685 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

A study comparable to guideline study with acceptable restrictions

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance does not meet the criteria for classification and labelling for this endpoint, as set out in Regulation (EC) NO. 1272/2008.