Benzene, (1-methylethyl)-, oxidized, polyphenyl residues

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

other: read-across from toxic ingredient

Adequacy of study:

supporting study

Study period:

June 2001 to January 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study: OECD Guideline 422

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

A combined repeated dose toxicity study and reproduction/developmental screening study in Sprague-Dawley rats with acetophenone (OECD guideline No. 422)

Author:

Kapp RW, Thorsrud BA, Moffatt WJ, Lawton L

Year:

2003

Bibliographic source:

Toxicologist 72: 76-77

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Acetophenone

EC Number:

202-708-7

EC Name:

Acetophenone

Cas Number:

98-86-2

Molecular formula:

C8H8O

IUPAC Name:

1-phenylethanone

Test material form:

other: liquid

Details on test material:

- Name of test material: Acetophenone
- Physical state: slight pale yellow liquid
- Analytical purity: 98.80 %
- Impurities (identity and concentrations): no data available
- Lot/batch No.: Lot No. R012-078
- Expiration date of the lot/batch: none provided
- Stability under test conditions: no data available
- Storage condition of test material: ambient conditions

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, Inc., Raleigh, North Carolina
- Age at receipt: ca. 8 wks
- Weight at receipt: males 246 - 285 g, females 164 - 191 g
- Fasting period before study: no
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 36 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: October 3, 2001 To: November 25, 2001

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
GAVAGE SOLUTIONS
For each test article dose group, a specified amount of acetophenone was weighed into a pre-calibrated beaker. A sufficient quantity of corn oil was added to the beaker to achieve the desired concentration and the mixture was stirred for 30 minutes. Each test article solution was prepared fresh weekly, dispensed into daily aliquots and stored in amber glass containers at ambient conditions.

VEHICLE
- Justification for use and choice of vehicle: low solubility in water
- Concentration in vehicle: 37.5, 112.5, 375 mg/mL
- Amount of vehicle (if gavage): 2 mL
- Lot/batch no.: Lot no. QN0035

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration vertification analysis performed on the vehicle and each test article dosing solution prepared for study weeks 0, 2, 4, and 6

Duration of treatment / exposure:

In a reproduction/developmental toxicity screening study gavage treatment of male and female rats starting at a minimum of 14 days before mating, and continued up to lactation day 3

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 75, 225, 750 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

10 males, 5 females (additional 10 females for the reproductive toxicity part of the study)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: high-dose level expected to produce some toxic effects, but no excessive lethality; mid-dose level expected to produce no to minimal observable effects; low-dose level was expected to produce no observable effects
- Rationale for animal assignment: random by computer randomization program

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations within ca. one-half hour following dosing

MORTALITY/GENERAL HEALTH CHECK: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: a minimum of weekly and of the day of scheduled euthanasia

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, 3,7, 12, 16, 20, 23, 27, 30; final body weight on day 33/34

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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 data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day of scheduled euthanasia, day 33 for males, day 34 for females
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia)
- Animals fasted: Yes
- How many animals: 5 males and 5 females per dose group
- Parameters: erythrocyte count, hematocrit, hemaglobin concentration, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, platelet count, total and differential leukocyte counts

COAGULATION PARAMETERS: Yes
- Time schedule for collection of blood: on day of scheduled euthanasia, day 33 for males, day 34 for females
- Anaesthetic used for blood collection: Yes (scheduled anesthesia with carbon dioxide)
- Animals fasted: Yes
- How many animals: 5 males and 5 females per dose group
- Parameters: prothromin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day of scheduled euthanasia, day 33 for males, day 34 for females
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia)
- Animals fasted: Yes
- How many animals: 5 males and 5 females per dose group
- Parameters: alanine aminotransferase, albumin, albumin/globulin ratio, alkaline phosphatase, aspartate aminotransferase, calcium, cholesterol, creatinine, globulin, glucose, electrolytes (sodium, potassium, chloride), phosphorus, total bilirubin, total serum protein, triglycerides, urea nitrogen

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: abbreviated FOB on day before start of exposure and on days 7, 14 and 21; full FOB on days 28 to 30
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
Abbreviated functional observation battery (FOB):
Home cage evaluation: body posture, clonic involuntary motor movements, tonic involuntary motor movements, vocalization
Removal from home cage evaluation: ease of removal, reactivity to being handled, ocular discharge, eyelid closure, salivation, piloerection
Open field evaluation: clonic involuntary motor movements, tonic involuntary motor movements, gait score, gait abnormalities, mobility score,
arousal, stereotypy, bizarre behavior, urination, defecation, rearing
Full FOB:
Day 28: home cage, removal from home cage, open field evaluation with all animals
Day 29 manipulative tests: approach, touch and startle response, tail pinch, pupil response, righting ability, grip strengths, landing foot splay,
body temperature, parameters investigated for 10 males each at 0 and 75 mg/kg, for 5 males each at 225 and 750 mg/kg, and for all females
Day 30 motor activity measurements: in open field chamber by measuring the total number of squares entered during the 1-hr test interval,
test performed for 5 males and 5 females per group

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
complete gross necropsy including examination of the external surfaces of the body, of all orifices, and the cranial, thoracic, abdominal and pelvic cavities and their contents

ORGAN WEIGHTS: Yes
adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus

HISTOPATHOLOGY: Yes
Accessory genital organs (epididymides, seminal vescicles and prostate or uterus and vagina), adrenals, all gross lesions, aorta, brain, cecum, colon, duodenum, esophagus, exorbital lachrymal glands, eyes with optic nerve, femur and bone marrow, heart, ileum, jejunum, kidneys, liver, lungs, mammary gland, mandibular lymph node, mediastanal lymph node, mesenteric lymph node, ovaries, pancreas, peripheral nerve, pituitary, rectum, skeletal muscle, skin, spinal cord, spleen, sternum with bone marrow, stomach (glandular, nonglandular), testes, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder (5 rats per dose and sex, gross lesions from all animals)

Other examinations:

Reproduction/Developmental screening study (see Section 7.8.2)

Statistics:

One-way analysis of variance (ANOVA), Tukey-Kramer test, Fisher's exact test, Levene's test for homogeneity of variance, Kruskal-Wallis non-parametric ANOVA, followed by Dunn's test

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

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):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals survived to scheduled euthanasia.
Clinical signs (for details see Table 1): all observed effects were present on a few days only in the single animals
225 mg/kg-group: postdose salivation in 9/10 males and in 3/5 females;
750 mg/kg: predose salivation in 9/10 males and 4/5 females; postdose salivation and wobbly gait observed in all males and females;
postdose urine stain in 2/10 males and 1/5 females; hairloss in 3/5 females
Evaluation: As postdose salivation is not considered to represent an adverse effect, the NOAEL for clinical findings is 225 mg/kg.

BODY WEIGHT AND WEIGHT GAIN (for details see Table 2)
males: 750 mg/kg-group: significant body weight loss during days 0-3; no statistically significant effect on mean body weight (only 5-10 % decrease beginning on day 3 and continuing through day 30).
males and females: all study groups including controls: apparent body weight loss during study days 12-16 followed by a compensatory rebound in weight gain during the following interval (days 16-20).
Evaluation: The definitive reason for the weight loss during study days 12-16 observed in all dosed groups could not be determined, but it may be related to the initiation of mating. The only significant effect on body weight change in high dose males occurred during days 0-3 simultaneously to significantly decreased food consumption. This effect on body weight is considered to be of no toxicological significance.

FOOD CONSUMPTION (for details see Table 2)
males: 750 mg/kg-group: significantly lower during study days 0-3, slightly lower during study days 16-30 (control 25-27, high dose 21-23 g/animal/day)
females: 750 mg/kg-group: significantly lower during study days 0-3
Evaluation: The significant decrease of food consumption on days 0-3 is considered to be of no toxicological significance.

HAEMATOLOGY (for details see Table 3)
males: significant decrease of mean prothrombin time at 75 mg/kg and significant increase in mean MCV value at 750 mg/kg
faamles: significant decrease of mean erythrocyte number and hematocrit at 750 mg/kg
Evaluation: No toxicological meaningful differences since statistically significant differences did not follow a consistent pattern and the mean values remained well within the range of the laboratories historical control data.

CLINICAL CHEMISTRY (for details see Table 4)
males: 750 mg/kg: significant increase of mean total protein, calcium and cholesterol (all outside historical control data), significant increase of mean total albumin, globulin, glucose, sodium, potassium and phosphorus (all inside historical control data)
225 mg/kg: significant increase of mean total protein, albumin, calcium and phosphorus (all inside historical control data)
females: 750 mg/kg: significant increase of cholesterol (outside historical control data); significant increase of mean total protein, albumin, potassium and ALT (all inside historical control data)
225 mg/kg: significant increase of mean total protein, albumin, globulin (all inside historical control data)
75 mg/kg: significant increase of mean total bilirubin ( inside historical control data)
Evaluation: The dose-related increase in total protein, albumin and globulin appeared to correlate with a dose-related increase in liver weight. These changes may be related to a compensatory reaction to the test article, possibly due to its metabolism. Most values of parameters that showed signficant changes to the concurrent control values were within the historical control range of animals of comparable age (13-24 weeks). The elevated values of the cholesterin level in both sexes, of total protein in females and of calcium in males were within the historical control range of animals of up to 12 weeks of age. Consequently, the toxicological significance of the significant changes of clinical chemistry parameters is questionable.

NEUROBEHAVIOUR (Functional Observation Battery)
males: 750 mg/kg: statistically significant lowering of mean forelimb grip strength and mean motor activity, p<0.05; values for grip strength were 1.880, 1.817, 1.684, and 1.572 and for motor activity 2796.4, 3222.4, 2675.6, and 1632.8 for the control, low, mid and high-dose group
75 mg/kg: significant changes of body posture on day 21, p<0.05
females: 750 mg/kg: significantly different body posture on day 21, p<0.05
Evaluation: The significant changes of body posture were not considered as toxicologically meaningful finding as there was no dose-response for males and as there was no consistent pattern in females. Thestatistically significant lowering of mean forelimb grip strength and mean motor activity was considered as toxicologically meaningful finding.

ORGAN WEIGHTS
males: 750 mg/kg: significant decrease of absolute weight of heart and epididymides, signifcant increase of relative liver weight
females: 225 and 750 mg/kg: significant increase of absolute and relative liver weight, and of relative kidney weight
Evaluation: Changes were not considered to represent toxicological meaningful differences as the statistically significant differences of organ weights did not correlate with any abnormal biochemistry or histopathology. The increases of liver weights may be related to a compensatory reaction to the test article, possibly due to its metabolism.

GROSS PATHOLOGY
No remarkable findings

HISTOPATHOLOGY: NON-NEOPLASTIC
Males: all dosed groups: minimal to mild haline droplet formation sometimes accompanied by minimal tubular epithelial
degeneration and regeneration.
750 mg/kg: vacuolar change in hepatocytes: minimal effect in 4/5 males, mild effect in 1/5 males
Evaluation: The vacuolar change in hepatocytes was interpreted as a background lesion based on the nature and the distribution of the changes. Additionally, similar changes have been seen in some control animals from other toxicity studies. In summary, no toxicologically significant test-article related lesions were found as hyaline droplet formation is not considered toxicologically significant for humans.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

2

Dose descriptor:

LOAEL

Effect level:

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

Sex:

male

Basis for effect level:

other: neurobehavioural effects: reduced forelimb grip strength and motor activity; clinical signs

3

Dose descriptor:

LOAEL

Effect level:

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

Sex:

female

Basis for effect level:

other: clinical signs

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Summary of changes of clinical signs (frequency of observation / number of affected animals)

Timepoint of observation	Clinical parameter	Males (N=10)				Females (N=5)
		Dose groups (mg/kg bw/d				Dose groups (mg/kg bw/d
		0	75	225	750	0	75	225	750
Predose	Salivation	0/0	0/0	3/1	24/9	0/0	0/0	0/0	18/4
Postdose	Salivation	0/0	0/0	37/9	69/10	0/0	1/1	4/3	36/5
	Wobbly gait	0/0	0/0	0/0	34/10	0/0	0/0	0/0	27/5
	Urine stain	0/0	0/0	0/0	3/2	0/0	0/0	0/0	5/1
General observation	Hairloss	0/0	0/0	1/1	2/1	0/0	0/0	0/0	20/3

Table 2: Summary of body weight development and food consumption

Timepoint of observation	Parameter		Males (N=10)				Females (N=5)
			Dose groups (mg/kg bw/d				Dose groups (mg/kg bw/d
			0	75	225	750	0	75	225	750
Days 0-3	Body weight change (g)	mean	2	-1	-4	-21^a	-2	-1	-7	-7
		SD	5.2	5.5	5.4	7.7	8.0	4.0	4.7	6.7
	Food consumption (g/animal/day)	mean	22	22	22	13^a	16	16	13	8^a
		SD	2.5	2.2	1.9	2.0	1.0	3.5	2.0	2.6
Days 3-7	Body weight change (g)	mean	7	9	11	10	4	8	6	11
		SD	5.9	4.3	6.6	4.3	1.9	3.5	4.0	6.9
	Food consumption (g/animal/day)	mean	23	24	25	23	16	18	16	15
		SD	2.1	1.5	3.0	1.2	0.9	0.9	1.6	1.6
Day 12-16	Body weight change (g)	mean	-22	-26	-23	-19	-8	-5	-14	-17
		SD	10.4	11.9	10.5	11.1	3.8	3.4	9.0	13.9
	Food consumption (g/animal/day)	mean	27	27	25	22	16	17	16	16
		SD	2.5	3.0	3.1	4.6	1.2	1.3	1.1	2.2

Level of significance: ^a p<0.01

Table 3: Summary of significant changes of hematological parameters and of historical control data

Hematological parameter (mean values)	Males (N=10)					Females (N=5)
	Dose groups (mg/kg bw/d					Dose groups (mg/kg bw/d
	0	75	225	750	Range of historical controls^c	0	75	225	750	Range of historical controls^c
MCV	46.8	48.2	47.0	49.9^b	42.7-62.8
Prothrombin time (sec)	12.64	11.80^a	12.28	12.68	13.9-20.7
Erythrocytes (10^6/mL)						8.06	7.66	7.83	7.42^b	7.07-8.55
Hematocrit (%)						41.7	40.6	38.8	38.4^b	37.3-47.3

Level of significance: ^a p<0.01; ^b p<0.05

Historical control data of the test laboratory presented in the study report: ^c data from age range 13-24 weeks (N=36 males and 34 females)

Table 4: Summary of significant changes of clinical chemistry parameters and of historical control data

Clinical chemistry parameter (mean values)	Males (N=5)					Females (N=5)
	Dose groups (mg/kg bw/d					Dose groups (mg/kg bw/d
	0	75	225	750	Range of historical controls^c	0	75	225	750	Range of historical controls^c
Total protein (g/dL)	6.09	6.36	6.72^b	7.31^a	5.11 -7.21	6.48	6.65	7.18^a	7.11^a	5.77 -7.11
Albumin (g/dL)	3.03	3.21	3.37^a	3.62^a	2.68-3.87	3.16	3.29	3.53^b	3.57^a	2.99-4.04
Globulin (g/dL)	3.06	3.15	3.35	3.69^a	2.34-3.82	3.32	3.36	3.66^a	3.54	2.50-3.72
Glucose (m/dL)	152	160	151	127^b	74-212
Sodium (mmol/L)	139	139	140	141^a	137-145
Potassium (mmol/L)	4.91	5.27	5.43	5.66^a	4.15-5.83	4.14	4.38	4.40	4.62^b	3.77-5.87
Calcium (mg/dL)	9.44	9.97	10.41^a	10.84^a	8.61-10.68 (9.26 -11.19)^d
Cholesterol (mg/dL)	34	37	42	60^a	17-54 (18-69)^d	47	42	48	71^a	16-58 (12-88)^d
Phosphorus (mg/dL)	5.7	6.0	6.6^b	7.1^a	5.2-8.6
ALT (IU/L)						23	26	27	43^a	18-78
Total bilirubin (mg/dL)						0.50	0.65^b	0.58	0.61	0.27-0.77

Level of significance: ^ap<0.01; ^b p<0.05

Historical control data of the test laboratory presented in the study report: ^cdata from age range 13-24 weeks (N=84 males and 83 females); ^d data from age range 5-12 weeks (N=273 males and 253 females);  age of test animals at sacrifice ca. 13 weeks

Table 5: Overview on organ weights with significant changes

Organ	Weight parameter	Males (N=10)				Females (N=5)
		Dose groups (mg/kg bw/d)				Dose groups (mg/kg bw/d)
		0	75	225	750	0	75	225	750
Heart	Absolute	1.49	1.44	1.42	1.31^a
	Relative	0.306	0.294	0.299	0.296
Liver	Absolute	17.20	17.00	18.09	19.84	7.40	7.69	9.12^b	10.63^a
	Relative	3.470	3.487	3.779	4.457^a	2.784	2.884	3.345^a	4.006^a
Kidney	Absolute					1.82	2.08	2.12	2.08
	Relative					0.687	0.779	0.779^b	0.784^b
Epididymides	Absolute	1.36	1.31	1.29	1.24^b
	Relative	0.280	0.268	0.272	0.283

Level of significance: ^ap<0.01; ^b p<0.05

Applicant's summary and conclusion

Conclusions:

For a oral exposure by gavage (>46d) the overall LOAEL for male and female rats is 750 mg/kg bw/day based on neurobehavioral findings and clinical signs.

Executive summary:

Groups of 10 male and 10 female Sprague-Dawley rats were exposed to 0, 75, 225 and 750 mg acetophenone/kg bw/day for >46 days by daily gavage application. The test protocol was a combination of OECD Guidelines 421 and 407 and included the investigation of signs of overt toxicity and clinical signs, body weight development and food consumption, and a functional observation battery. Blood samples were collected on the day of scheduled euthanasia (study day 33 or 34) for evaluation of selected hematology, coagulation and clinical chemistry parameters. Animals were subjected to a complete gross necropsy examination and recording of organ weights (all rats). Microscopic examination was performed on organs and tissues from 5 males and 5 females per group.

No mortality occurred. Effects were restricted to the 750 mg/kg-dose group. Postdose wobbly gait and urine stain appeared in males and females, and hairloss in females. Mean forelimb grip strength and mean motor activity of males were statistically lower than in controls on day 29. During days 0 -3, males lost weight, while food consumption was depressed in both sexes. This initial weight loss as well as changes of hematological and biochemical parameters (significant difference to actual control group for both sexes, but values within historical controls) were considered to be of no toxicological significance. A dose-related increase in total protein, albumin and globulin appeared to correlate with a dose-related increase in liver weight. These changes may be related to a compensatory reaction to the test substance, possibly due to its metabolism. A minimal to mild hyaline droplet nephropathy was observed for males of all dose groups; however, this finding is not considered toxicologically significant for humans.

The NOAEL was 225 mg/kg bw/d and, based on clinical and neurobehavioral findings, the LOAEL for male and female rats was 750 mg/kg bw/day for a oral exposure by gavage (>46d).