4-phenylbutan-2-one

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2010-10-04 to 2012-07-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to OECD and EPA guidelines

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Reference 2

Reference Type:

other: Abstract

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.6050 (28-Day Oral Toxicity in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Swissmedic

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-phenylbutan-2-one

EC Number:

219-847-4

EC Name:

4-phenylbutan-2-one

Cas Number:

2550-26-7

Molecular formula:

C10H12O

IUPAC Name:

4-phenylbutan-2-one

Test material form:

other: colorless to yellowish clear liquid

Details on test material:

- Name of test material (as cited in study report): benzyl acetone
- Physical state: colorless to yellowish clear liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany GmbH, Sandhoferweg 7, D-97633 Sulzfeld/Germany
- Age at study initiation: ca. 8 weeks
- Weight at study initiation: 215.6-240.1 g (males), 150.3-198.2 g (females)
- Fasting period before study: no
- Housing: in groups of two, three or four in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK).
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2914C (batch nos. 30/10 (B0823) and 68/10 (B0871)) rat / mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland), ad libitum
- Water (e.g. ad libitum): tap-water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Benzyl Acetone was weighed into a glass beaker on a calibrated Mettler balance. A small amount of vehicle was added, shortly stirred and the remaining vehicle was added. The mixtures were stirred using a magnetic stirrer and used at room temperature (20 ± 5 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): sufficient formulation stability, homogeneous
- Concentration in vehicle: 0, 11, 33, 50, 100 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): 6212.1 (Art.-Nr.), 400159216 (Charge)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analysis was performed using a GC method.
After experimental start and during weeks 3 and 13, duplicate samples of the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration. Duplicate samples of about 2 g of each concentration were taken to confirm stability (4 hour and 8 days). The samples were delivered to the analytical department (Harlan Laboratories Ltd., Analytics, Itingen / Switzerland) at room temperature (once on dry ice) and stored there at -20 ± 5 °C until analysis.

Duration of treatment / exposure:

13-week

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (control), 55, 165, 250 or 500 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: no
- Post-exposure recovery period in satellite groups: 28 days
- Section schedule rationale (if not random): no data

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed for clinical signs daily before commencement of administration, twice on treatment day 1 and at least daily on days 2-91 during the treatment period. During days 1 to 28 of the recovery period the animals were observed once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly during acclimatization, treatment and recovery periods and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in the acclimatization period once in all animals, in the treatment period once during week 13 in animals of the control and high dose group (group 5), and in the recovery period once during week 17 in animals of the control and high dose group (group 5).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in weeks 7, 14 and 18
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: in weeks 7 and 14 - all animals, in week 18 - only recovery animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in weeks 7, 14 and 18
- Animals fasted: Yes
- How many animals: in weeks 7 and 14 - all animals, in week 18 - only recovery animals
- Parameters checked in table [No.1] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: in weeks 7, 14 and 18
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters checked in table [No.1] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 13
- Dose groups that were examined: all animals
- Battery of functions tested: grip strength / motor activity

OTHER: VAGINAL SMEAR
- Time schedule for examinations: during treatment weeks 6 and 12
- Dose groups that were examined: all females

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 2)
HISTOPATHOLOGY: Yes (see table 2)

Other examinations:

Vaginal smear for estrus stage

Statistics:

The following statistical methods were used to analyze body weight, food consumption, grip strength, locomotor activity, ophthalmoscopy, sperm analysis, clinical laboratory data, organ weights and ratios as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

One female at 55 mg/kg/day on day 11, one male at 165 mg/kg/day on day 21 and one male at 250 mg/kg/day on day 27 were found dead. One female at 500 mg/kg/day was sacrificed in extremis on day 45 and one female was dead on day 83. This was possibly due to a misgavage because perforation of the esophagus was noted.

Mortality:

no mortality observed

Description (incidence):

One female at 55 mg/kg/day on day 11, one male at 165 mg/kg/day on day 21 and one male at 250 mg/kg/day on day 27 were found dead. One female at 500 mg/kg/day was sacrificed in extremis on day 45 and one female was dead on day 83. This was possibly due to a misgavage because perforation of the esophagus was noted.

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 specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Mean methemoglobin values were slightly reduced in females during week 7 and in all groups of males and females during week 14. There was no statistical significance in the high dose group.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see "Details on results"

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

see "Details on result"

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
One female at 55 mg/kg/day on day 11, one male at 165 mg/kg/day on day 21 and one male at 250 mg/kg/day on day 27 were found dead. One female at 500 mg/kg/day was sacrificed in extremis on day 45 and one female was dead on day 83. This was possibly due to a misgavage because perforation of the esophagus was noted.

BODY WEIGHT AND WEIGHT GAIN
The mean absolute body weights and the mean body weight gain of test item treated males and females were comparable with these of the control animals during the treatment, in males also during the recovery period. In females the mean absolute and relative body weight was statistically significantly lower during recovery when compared with the controls; this was considered to be not test item related.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The mean absolute and the mean relative food consumption of test item treated males and females were comparable with those of the control animals during the course of the treatment and recovery period.

FOOD EFFICIENCY
No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
No data

OPHTHALMOSCOPIC EXAMINATION
No test item related ophthalmoscopic findings were noted in males and females on day 87 during the treatment phase and on day 25 during the recovery period.

HAEMATOLOGY
Mean methemoglobin values were slightly reduced in females during week 7 and in all groups of males and females during week 14. There was no statistical significance in the high dose group. It could not be excluded that this finding was test item related.

CLINICAL CHEMISTRY
No effects

URINALYSIS
No effects

ORGAN WEIGHTS
The mean kidney weight, the mean kidney to body weight ratio and kidney to brain weight ratio were increased with statistical significance in males treated with 500 mg/kg/day of the test item when compared with controls at the end of the treatment. The mean liver to body weight ratios were increased in males and females treated with 165 mg/kg/day, 250 mg/kg/day or 500 mg/kg/day, the mean liver to brain weight ratios were increased in males treated with 165 mg/kg/day or 250 mg/kg/day and in females treated with 250 mg/kg/day or 500 mg/kg/day with different statistical significances compared with controls at the end of treatment. Both changes in organ weights were reversible at the end of the recovery period.

GROSS PATHOLOGY
There were no relevant macroscopic lesions recorded at necropsy.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic Findings at the end of treatment:
• Liver: Minimal to slight centrilobular hepatocellular hypertrophy was present in seven males treated with 250 mg/kg/day of the test item and in all ten males and seven females treated with 500 mg/kg/day. Age-related minimal bile duct hyperplasia was increased in incidence in females treated with 500 mg/kg/day, when compared to controls. The liver cell hypertrophy was considered to represent an adaptive reaction.
• Thyroid gland: Minimal to slight diffuse follicular cell hypertrophy occurred in three males treated with 500 mg/kg/day, consequent to the enhanced liver cell metabolism with accelerated thyroid hormone breakdown in liver cells.
• Kidneys: Minimal to moderate tubular degeneration/regeneration was diagnosed in the outer and inner cortex of three males treated with 250 mg/kg/day and all males treated with 500 mg/kg/day. This lesion was associated with an increase in hyaline droplets in proximal tubules with a moderately increased incidence in males treated with 250 mg/kg/day and a slightly increased severity and moderately increased incidence in the high dose group. A Mallory Heidenhain stain was done on kidneys sections in main and recovery males of controls and group five to confirm and compare the protein droplets (α2u-g) in proximal convoluted tubules.
Microscopic Findings at the end of the treatment free recovery period:
• Kidneys: Minimal to slight tubular degeneration/regeneration was still diagnosed, mainly in the outer cortex, in all five males treated with 500 mg/kg/day, this lesion was associated with a slightly increased incidence of hyaline droplets. This finding is male rat specific and is not considered to be of relevance to humans.

OTHER FINDINGS: VAGINAL SMEAR
No effects

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results of the study, a no-observed-adverse-effect-level (NOAEL) could be established at 500 mg/kg bw/day of Benzyl Acetone.

Executive summary:

In this subchronic toxicity study, Benzyl Acetone was administered daily by oral gavage to Sprague Dawley rats of both sexes at dose levels of 55, 165, 250 and 500 mg/kg body weight/day for a period of 13 weeks. A control group was treated similarly with the vehicle, Corn oil, only. The groups comprised 10 animals per sex which were sacrificed after 13 weeks of treatment. Additional five rats per sex and group were used at 0 and 500 mg/kg. These animals were treated for 13 weeks and then allowed a four-week treatment-free recovery period after which they were sacrificed. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 13. Ophthalmoscopic investigations were performed during acclimation, and at the end of the treatment and recovery periods. At week 7 and the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. A vaginal smear was taken during treatment weeks six and 12 from all females, and the stage of estrus was evaluated, if possible. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, as well as selected organs from group two to four and all gross lesions from all animals. The liver, spleen, thyroid, kidneys and ovaries of animals of the low and two middle dose groups were examined to establish a no-effect level. At necropsy of the males, an epididymal sperm sample was obtained from the left cauda epididymidis for motility investigation, a sperm sample from the left vas deferens was used for morphological assessment and the left caudal epididymidis and the left testis were taken for determination of homogenization resistant spermatids and caudal epididymal sperm reserve.

Mortality / Viability

All control animals, all males treated with 55 mg/kg/day or 500 mg/kg/day and all females treated with 165 mg/kg/day or 250 mg/kg/day survived the scheduled treatment or recovery periods. One female treated with 55 mg/kg/day was found dead on day 11 (before the administration on this day). One male treated with 165 mg/kg/day was found dead on treatment day 21. The reason

for these two deaths could not be established. One male treated with 250 mg/kg/day, was found dead on day 27. It could not be excluded that this was due to a misgavage. One female treated with 500 mg/kg/day was sacrificed in extremis on day 45. This was possibly due to a misgavage because perforation of the esophagus was noted. Another female of this group was found dead on day 83. The reason for the death was not clear.

Clinical Signs (Daily and Weekly)

The one female treated with 55 mg/kg/day which was found dead on day 11 showed decreased activity, hunched posture, abnormal gait and tachypnea on day 10. It could be not excluded that this was test item related because the reason of the death could not be established. Slight up to marked salivation was noted in some males treated with 500 mg/kg/day of the test item from treatment week three until the end of treatment, red salivation was noted on four days in week 6. In a few females of this group slight salivation was noted on three days in week five and reddish nasal secretion was noted on two days in week seven. No test item related findings were noted during the recovery period.

Functional Observational Battery

No test item related clinical signs were noted at any dose tested.

Grip Strength

No relevant changes in the mean grip strength of test item treated males and females were noted at the end of the treatment period when compared with the control animals.

Locomotor Activity

The locomotor activity of test item treated males and females was comparable to the control animals during the one hour observation period (six 10-minutes intervals) at the end of the treatment period.

Food Consumption

The mean absolute and the mean relative food consumption of test item treated males and females were comparable with those of the control animals during the course of the treatment and recovery period.

Body Weights

The mean absolute body weights and the mean body weight gain of test item treated males and females were comparable with these of the control animals during the treatment, in males also during the recovery period. In females the mean absolute and relative body weight was statistically significantly lower during recovery when compared with the controls; this was considered to be not test item related.

Ophthalmoscopic Examinations

No test item related ophthalmoscopic findings were noted in males and females on day 87 during the treatment phase and on day 25 during the recovery period.

Vaginal Smear for Estrus Stage

No changes in the estrus cycle of test item treated females were noted when compared with controls on treatment days 37 to 40 and 78 to 81.

Clinical Laboratory Investigations

Hematology

The mean methemoglobin values of test item treated males and females were slightly decreased in all groups with statistical significance compared with controls in treatment week 14. In treatment week seven these values were slightly decreased in females only. There was no statistical significance in the high dose group. It could not be excluded that this finding was test item related.

Clinical Biochemistry and Urinalysis

No test item related differences in parameters of clinical biochemistry and urinalysis were noted when comparing test item treated animals with controls in treatment week seven and 14 and at the end of the treatment free recovery period.

Organ Weights

The mean kidney weight, the mean kidney to body weight ratio and kidney to brain weight ratio were increased with statistical significance in males treated with 500 mg/kg/day of the test item when compared with controls at the end of the treatment. The mean liver to body weight ratios were increased in males and females treated with 165 mg/kg/day, 250 mg/kg/day or 500 mg/kg/day, the mean liver to brain weight ratios were increased in males treated with 165 mg/kg/day or 250 mg/kg/day and in females treated with

250 mg/kg/day or 500 mg/kg/day with different statistical significances compared with controls at the end of treatment. Both changes in organ weights were reversible at the end of the recovery period. No other statistically significant changes in mean organ weights, mean organ to body weight ratios and mean organ to brain weight ratios were noted when compared with controls at the end

of the treatment period or recovery period.

Macroscopic / Microscopic Findings

There were no relevant macroscopic lesions recorded at necropsy.

Microscopic Findings at the end of treatment:

Liver: Minimal to slight centrilobular hepatocellular hypertrophy was present in seven males treated with 250 mg/kg/day of the test item and in all ten males and seven females treated with 500 mg/kg/day. Age-related minimal bile duct hyperplasia was increased in incidence in females treated with 500 mg/kg/day, when compared to controls. The liver cell hypertrophy was considered to represent an adaptive reaction.

Thyroid gland: Minimal to slight diffuse follicular cell hypertrophy occurred in three males treated with 500 mg/kg/day, consequent to the enhanced liver cell metabolism with accelerated thyroid hormone breakdown in liver cells.

Kidneys: Minimal to moderate tubular degeneration/regeneration was diagnosed in the outer and inner cortex of three males treated with 250 mg/kg/day and all males treated with 500 mg/kg/day. This lesion was associated with an increase in hyaline droplets in

proximal tubules with a moderately increased incidence in males treated with 250 mg/kg/day and a slightly increased severity and moderately increased incidence in the high dose group. A Mallory Heidenhain stain was done on kidneys sections in main and recovery males of controls and group five to confirm and compare the protein droplets (α2u-g) in proximal convoluted tubules Microscopic Findings at the end of the treatment free recovery period:

Kidneys: Minimal to slight tubular degeneration/regeneration was still diagnosed, mainly in the outer cortex, in all five males treated with 500 mg/kg/day, this lesion was associated with a slightly increased incidence of hyaline droplets. This finding is male rat specific and is not considered to be of relevance to human.

The type, incidence, and/or severity of remaining histopathologic findings observed were considered not to distinguish treated rats from control rats.

Seminology and Spermatid Count

No test item related changes in the parameters of motility or morphology and in the sperm count were noted in test item treated males when compared with controls at the end of the treatment period and at the end of the recovery period.

Conclusion

Under the conditions of this study, Benzyl Acetone induced some histopathological findings that were considered to be test item-related.

The reversible hepatocellular hypertrophy in male and female liver was considered to be an adaptive change. The hypertrophy with enhanced liver cell metabolism led to a consequent acceleration of thyroid hormone breakdown resulting in reversible thyroid follicular cell hypertrophy in males.

The tubular lesions in male kidneys are considered to be an effect representing hyaline droplet nephropathy which was partly reversible within 4 weeks of recovery period. The increase in hyaline droplets is due to an increase in the renal content of alpha-2μ-globulin (α2u-g) in proximal convoluted renal tubules. This globulin is of hepatic origin and the rate of synthesis declines during aging. Therefore, the animal age is an important determinant in the development of hyaline droplet nephropathy and only younger male rats which produce large amounts of α2u-g are susceptible to development of such kidney pathology. However, it is a known specific effect in male rat only, which is not relevant in human.

A no-observed-effect-level (NOEL) of 165 mg/kg bw/day was proposed in the original study report, but from a regulatory point of view the adaptive processes in liver and thyroid gland may be interpreted as a non-adverse effect. Consequently, these effects were not considered in the derivation of a no-observed-adverse-effect-level NOAEL and the NOAEL is 500 mg/kg bw/day of Benzyl Acetone.