Phenacetin

Endpoint summary

Description of key information

Supporting study: A repeated dose toxicity study of 4 weeks in rats was conducted on the test item and LOEL observed was 500 mg/kg bw/day, based on haematological findings.

Supporting study: A repeated dose toxicity study of 66 weeks in rats was conducted on the test item and MTD observed was 450 mg/kg bw/day. Based on a significant decrease of renal concentrating capacity and this impairment tended to be rapidly reversible after discontinuation administration of the test item.

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:

supporting study

Study period:

1967

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

The test perfomrs rat experiments with long-term feeding with phenacetin to measure the renal concentration capacity, to find out whether and induced decrease in the renal concentrating capacity would be normalized after discontinuing the drug and to examine the kidney morphology.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Anticimex AB, Stockholm
- Weight at study initiation: 190g
- Fasting period before study: no
- Diet: ad libitum.
- Water: tap water ad libitum

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Group 1: receiving powdered food
Group 2: Rats receiving the powdered food to which phenacetin was added in an amount of 5.35 g/kg food (16 rats).
Group 3: Rats receiving the powdered food, to which acetylsalicylic acid was added, in an amount of 2.70 g/kg food (16 rats).

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

66 weeks. The rats had free access to food and tap water. After 40 weeks on this regimen 11 rats in group I, 12 in group II, and 13 in group III were killed and autopsied. In the remaining rats the test was continued for 3 more weeks. The drugs were then discontinued. After 23 additional weeks with control food for all three groups, the remaining 12 rats were killed and autopsied.

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg diet

Dose / conc.:

5 350 mg/kg diet

Dose / conc.:

2 700 mg/kg diet

No. of animals per sex per dose:

Group 1: controls; powdered food with no test item (16 rats)
Group 2: powdered food with phenacetin in an amount of 5.35 g/kg food (16 rats)
Group 3: powdered food with in an amount of 2.70 g/kg food (16 rats)

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

BODY WEIGHT: Yes
- Time schedule for examinations: 2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38 weeks

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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

URINALYSIS: Yes
- Time schedule for collection of urine: various times after withdrawal of the drugs (1,3,7,12,18 weeks)
- Metabolism cages used for collection of urine:No
- Animals fasted: No
- Parameters checked in table [1], Maximale urine osmolality mOsm/kg H2O, were examined.

Clinical signs:

not examined

Mortality:

not examined

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no definite difference in weight gain between the rats in the three groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food intake was greatest for the phenacetin group,

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

After 38 weeks, there was a significantly lowered osmolality in both the phenacetin group. After 41 weeks there was a significantly lower osmolality in both the phenacetin group. The effect is reversible, therefore an MTD= 450 mg/kg is stablished.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

other: MTD (maximum tolerated dose)

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

urinalysis

Key result

Critical effects observed:

no

Table 1. Kidney function and phenacetin 

Group	No. of rats	Maximale urine osmolality mOsm/kg H2O (mean and standard error of mean)
		Period of drug consumption (weeks)		Various times after withdrawal of the drugs (weeks)
		38	41	1	3	7	12	18
I	4	2592±220	2872±76	2752±152	2942±101	3133±408	2592±156	2750±153
II	5	2398±81	2264±125	2466±201	2901±231	2442±236	2354±184	2786±193
III	3	2027±102	2170±112	2624±116	2396±112	2564±104	2249±88	2478±72

Conclusions:

In a repeated dose toxicity test of 66 weeks, the MTD (maximum tolerated dose) of the test item in Sprague-Dawley rats was 450 mg/kg bw/d.

Executive summary:

A 66 weeks repeated dose toxicity study was conducted with the test item. Female rats of Sprague-Dawley strain were used for this study. Control group recieved powdered food with no test item and rats receiving test item was in amount of 5.35 g/kg food. There was a significant decrease of renal concentrating capacity and this impairment tended to be rapidly reversible after discontinuation of the administration of the test item. Therefore, the  MTD (maximum tolerated dose) was 450 mg/kg bw/d.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Qualifier:

no guideline available

Principles of method if other than guideline:

In this paper it is reviewed the methods available fot the detection of hemolytic properties of chemicals.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SIV 50, Ivanovas, Kissleg, F.R.G.
- Weight at study initiation: 180- 200 g
- Housing: Macrolon cages with wood shavings as bedding under controlled conditions of humidity, temperature and light.
- Water: ad libitum.
- Food: ad libitum rat chow.

Route of administration:

oral: gavage

Details on oral exposure:

ADMINISTRATION:
Phenacetin (Ph. Eur., Merck, Darmstadt, F.R.G.) was administered by stomach tube to 5 rats in a dose of 500 mg/kg, 5 times weekly, as a suspension in 0.5 % methyl cellulose in a volume of 2 ml/kg.

Duration of treatment / exposure:

28 days

Frequency of treatment:

5 times weekly

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

- 5 males were given: in a dose of 500 mg/kg, 5 times weekly, as a suspension in 0.5 %
methyl cellulose in a volume of 2 ml/kg.
- 5 males (controls): received the vehicle only
- 10 males were given: phenacetin or 0.5 % methyl cellulose (same treatment as above) for osmotic fragility and surface area/volume ratio tests.

Control animals:

yes, concurrent vehicle

Details on study design:

Twenty rats were randomly divided into three treatment groups and a control group, and each group had 5 rats being all males. Phenacetin (Ph. Eur., Merck, Darmstadt, F.R.G.) was administered by stomach tube to 5 rats in a dose of 500 mg/kg, 5 times weekly, as a suspension in 0.5 % methyl cellulose in a volume of 2 ml/kg. Controls received the vehicle only (n = 5). Drug treatment was discontinued after 4 weeks. In a separate experiment, two groups of 5 rats were given phenacetin or 0.5 % methyl cellulose (same treatment as above) for osmotic fragility and surface area/volume ratio tests.

Observations and examinations performed and frequency:

HEMATOLOGIC PARAMETERS:
- Time: at 2 and 4 weeks of phenacetin treatment and 2 weeks after discontinuation of the drug.
- RBC count, Hkt ratio, and MCV were measured with a Coulter Counter, Model S.
- The number of Ret and Heinz body formation were determined in freshly prepared blood smears.
- MHb was assayed spectrophotometrically as the difference in absorbance of cyan-MHb with and without previous addition of ferricyanide.

OSMOTIC FRAGILITY AND SURFACE/VOLUME RATIO OF ERYTHROCYTE:
- Osmotic fragility tests as described by Beutler for human RBC were adapted for rat RBC. 1.5 ml blood was drawn percutaneously into a heparinized 5-ml syringe from the V. subclavia in light ether anesthesia.
- For estimating the surface/volume ratio of RBC, the maximal volume of globular red cell ghosts was determined.

Clinical signs:

effects observed, non-treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Test item caused reversible formation of methemoglobin and Heinz bodies and an increase in peripheral reticulocytes after 2 and 4 weeks of treatment. Furthermore, an increase in the mean corpuscular volume of red blood cells (RBC) and the volume of RBC ghosts in hypotonic solutions, and a decrease of the mean corpuscular fragility was observed. The latter changes are considered to be a consequence of regenerative RBC compensation rather than due to structural membrane alteration caused by the test item.
The results suggest that only a combination of several hematological tests can provide comprehensive information about the hemolytic potential of chemical substances, and that for screening purposes small numbers of animals are often sufficient.

Clinical biochemistry findings:

effects observed, non-treatment-related

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Dose descriptor:

LOEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

haematology

Critical effects observed:

no

Table 1: METHEMOGLOBIN (MHb), RETICIJLOCYTE RATIO (Ret), HEINZ BODY RATIO (Heinz), RED
BLOOD CELL (RBC) COUNT, AND HEMATOCRIT (Hkt) OF FRESHLY OBTAINED ERYTHROCYTES

Duration of treatment	MHb (%)	Ret (per 10^3 RBC)	Heinz (per 10^3 RBC)	RBC (10^12/1)	Hkt
2 weeks
Phenacetin	7.2±7.9^a	156±19^a	118±16^a	5.930.09^a	0.40±0.004^a
Controls	1.2±0.4	28±3	0	7.74±0.16	0.47±0.009
4 weeks
Phenacetin	12.0±0.8^a	41±4^a	7±3^a	7.09±0.12^a	0.44±0.009
Controls	7.6±1.7	29±3	0	7.55±0.13	0.42±0.004
2 weeks after drug
Phenacetin	2.7±0.3	29±1	0	7.19±0.16	0.40±0.009
Controls	2.9±0.2	24±1	0	7.66±0.12	0.40±0.004

 

Table 2: MEAN CORPUSCULAR VOLUME (MCV) OF RED BLOOD CELLS (RBC) IN ISOTONIC PBS,
AND VOLUME AND SURFACE AREA OF RBC GHOSTS IN HYPOTONIC (0.3%) PBS 

Duration of treatment	MCV ( μm^3)	Volume (V) of RBC ghosts ( μm^3)	Surface area (S) of RBC ghosts ( μm^2)	S/V ratio
2 weeks
Phenacetin	68±0.7^a	124±1.8	120±1.2	0.968
Controls	60±0.2	122±1.8	119±1.1	0.975
4 weeks
Phenacetin	62±0.4^a	134±2.2^a	127±1.4^a	0.945^a
Controls	55±0.3	118±1.6	116±1.1	0.986
2 weeks after drug continuation
Phenacetin	56±0,2^a	nd	nd	nd
Controls	53±0.4

Conclusions:

In a 4-week study and that the LOEL is based on haematological effects. The low observed effect level (LOEL) of the test item in rats was 500 mg/kg bw/day

Executive summary:

A repeated dose toxicity study in rats was conducted with the test item. The administration of the test item was by oral gavage and the dose used was of 500 mg/kg. Also, a control group was monitored. Haematological and clinical-chemistry examinations were performed and haematological effects on MHb and Heinz bodies were observed. Therefore, the low observed effect level (LOEL) of the test item in rats was 500 mg/kg bw/day. 

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

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

Based on the available data, for repeated dose toxicity, the substance is not classified