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Toxicological information

Repeated dose toxicity: oral

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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.

Administration / exposure

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
Doses / concentrationsopen allclose all
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)

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

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

1.58a

0.90 - 2.61b

Statistically significant from controls: *: p < 0.05.

aMean

b5th- 95thpercentiles

 

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

8.30a

7.25 - 9.07b

Hemoglobin (g/dL)

% change from control

15.2

-

15.0

-1

14.7

-3

14.5*

-5

15.0a

13.6 - 16.2b

Packed Cell Volume (L/L)

% change from control

0.45

-

0.44

-2

0.43

-4

0.42**

-7

0.45a

0.40 - 0.50b

White Blood Cells (G/L)

% change from control

8.40

-

7.27

-13

7.24

-14

6.41*

-24

6.80a

3.82 - 10.83b

Lymphocytes (G/L)

% change from control

7.26

-

6.01

-17

6.11

-16

5.35*

-26

5.57a

2.89 - 8.75b

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

aMean

b5th- 95thpercentiles

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

2.07a

1.74 - 2.41b

Total protein (g/L)

% change from control

58.4

-

59.2

+1

59.5

+2

62.5**

+7

65a

60 - 71b

Albumin (g/L)

% change from control

37

-

37

0

37

0

39**

+5

38a

33 - 41b

Cholesterol (mmol/L)

% change from control

1.49

-

1.45

-3

1.44

-3

2.09**

+40

1.3a

0.7 - 2.0b

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

aMean

b5th- 95thpercentiles

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

2.0a

1.4 - 2.6b

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

aMean

b5th- 95thpercentiles

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 (106/cauda)a

182

183

190

164

[100-182]

Mean number of epididymal sperm (106/g cauda)a

565

549

579

503

[319-591]

Mean number of testicular sperm heads (106/g testis)a

117

124

116

121

[96-151]

Daily sperm production rate (106/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%].

Applicant's summary and conclusion

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.