Phenacetin

Carcinogenicity

Description of key information

Carcinogenicity: Supporting study (Nakanishi1982): Test method was realised by long-term feeding study in mice. Based on the results, test item had a dose-related induction relation with renal cell tumors in the mice fed with it. 

Carcinogenicity: Supporting study (Isaka1979): Test item was tested for carcinogenicity using long-term feeding study in rats. Based on the results, the higher the concentration of phenacetin given, higher incidence of tumors was observed.

Carcinogenicity: Supporting study (Johansson1989): Test item was tested for carcinogenicity using feeding study in rats. Based on the results, there is a dose-related association between the labeling index (LI) in rat urinary bladder and renal pelvic urothelium when the rats are treated with up to 1.5% test item.

Carcinogenicity: Supporting study (Bogdanffy1989): The test item was tested for cell proliferative and cytotoxic effects on rat nasal mucosa. Based on the results, test item was considered to produce increase of DNA sytnhesis in respiratory mucosa al lower dose (100 mg/kg) and at high dose (1250 mg/kg) produced necrosis and degeneration of the neuronal cells within the olfactory mucosa.

 

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

GLP compliance:

not specified

Species:

rat

Strain:

other: Crl:CDBR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River Breeding Laboratories (Kingston, NY).
- Age at study initiation: 56 days
- Weight at study initiation: 200 to 250 g
- Fasting period before study: 1 week (stainless-steel wire mesh cages, housed individually)
- Housing: group housed, 5 per cage, i polycarbonate plastic cages on Bed-O-Cobs bedding
- Diet (e.g. ad libitum): provided with Purina certified rodent show
- Water (e.g. ad libitum): ad libitum.

Route of administration:

oral: gavage

Vehicle:

other: methylcellulose suspension

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): methylcellulose
- Concentration in vehicle: 0 mg/ml (control), 17.5 mg/ml (low dose), 109 mg/ml (intermediate dose) or 219 mg/ml (high dose).
- Amount of vehicle (if gavage): 0.25%

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Two-week

Frequency of treatment:

Daily

Dose / conc.:

0 other: mg/kg/day

Dose / conc.:

100 other: mg/kg/day

Dose / conc.:

625 other: mg/kg/day

Dose / conc.:

1 250 other: mg/kg/day

No. of animals per sex per dose:

5 rats/ group

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels chosen for the present study were based on daily phenacetin intake values estimated from the bioassay reported by Isaka et al. (1979). In that study, male Sprague-Dawley rats were fed diets containing phenacetin at concentrations of 1.25 and 2.5%. Average body weight and food consumption
throug

Positive control:

None.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. (by measurement of cell proliferation by scintillation counting and histoautoradiography).

Statistics:

Statistical differences between control and treated groups were evaluated by Student’s t test. A significance level ofp < 0.05 was used in all cases.

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not specified

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:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Phenacetin treatment resulted in a dose-related increase in DNA synthesis in both respiratory and olfactory mucosa.
In contrast to the previous experiment, phenacetin treatment appeared to have
no effect on cell proliferation in the three Incorporation of [3H]thymidine into nasal mucosal DNA following oral phenacetin administration for 1 week. Osmotic minipumps containing [)H]thymidine
were implanted subcutaneously on Day 1 and provided a constant release of [‘H]thymidine through Day 7. Rats were treated by gavage with phenacetin once per day during the 7-day release period. Circles represent respiratory mucosa and triangles represent olfactory mucosa. Each point represents the mean (*SE) for five rats per group. Asterisks denote statistically significant differences from control value (p < 0.05). paired regions of respiratory mucosa during
the first or second week. Although there was a statistically significant increase in the labeling
index in regions 1 and 2 during the first week, this was not observed during the second
week. Therefore, these results were inconsistent with the increased DNA synthesis observed by biochemical analysis.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Key result

Dose descriptor:

LOAEL

Effect level:

100 other: mg/kg/day

Based on:

test mat.

Sex:

male

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 other: mg/kg/day

System:

respiratory system: lower respiratory tract

Organ:

other: respiratory mucosa

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 250 other: mg/kg/day

System:

respiratory system: upper respiratory tract

Organ:

other: olfactory mucosa

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Test item was considered to produce increase of DNA synthesis in respitarory mucosa at lower dose (100 mg/kg) and at high dose (1250 mg/kg) necrosis and degeneration of the neuronal cells within the olfactory mucosa.

Executive summary:

The test item was tested for cell proliferative and cytotoxic effects on rat nasal mucosa. Animals were daily gavage treated with test item at 100, 625 or 1250 mg/kg during two weeks. The experiment had a control group whom did not recieve test item .Vehicle used was methylcellulose suspension at concentration 0.25%. Test item was considered not to induce effect on the rate of cell replication in respiratory epitelium, but indicated an increase in DNA synthesis in respiratory mucosa. Test item was considered to produce increase of DNA sytnhesis in respiratory mucosa al lower dose (100 mg/kg) and at high dose (1250 mg/kg) produced necrosis and degeneration of the neuronal cells within the olfactory mucosa.

 

 

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Principles of method if other than guideline:

- Principle of test: dose-related effect on the cell proliferation of the urothelium was evaluated in male Sprague-Dawley rats by autoradiography
- Short description of test conditions: . Nine groups of twenty, 6-week old rats were treated with 0.5%, 1.0% or 1.5% of test item in the diet. A tenth group of rats received control diet without test item.
- Parameters analysed / observed: the rats underwent a complete necropsy with careful macroscopical examination of the exterior of the rat and the organs in the thoracic and abdominal cavities.

GLP compliance:

not specified

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, NY
- Females (if applicable) nulliparous and non-pregnant: no
- Age at study initiation: 6 week
- Weight at study initiation: 113 g.
- Fasting period before study: 1 week
- Housing: plastic cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum


ENVIRONMENTAL CONDITIONS

- Air changes (per hr): air conditioned room
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): mixed with powdered laboratory chow, Prolab 3200 (Agway, Inc., St Mary's, OH), at a level of 1.5% by weight, respectively.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

The purity of the compounds was tested by NMR

Duration of treatment / exposure:

6 and 12 weeks

Frequency of treatment:

Daily

Dose / conc.:

0 other: % test item

Remarks:

Control

Dose / conc.:

0.5 other: % test item

Dose / conc.:

1 other: % test item

Dose / conc.:

1.5 other: % test item

No. of animals per sex per dose:

200 animals in total (males) divided randomly into 10 groups

Control animals:

yes, plain diet

Details on study design:

- Toxicokinetic data
- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):

Positive control:

None.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION (if dermal study): No

BODY WEIGHT: Yes
- Time schedule for examinations: biweekly

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 (The food and water consumption was calculated per cage and divided by four or five (the number of rats in each cage).)
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: 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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No


OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. (labeling index (LI) and urothelial cell proliferation)

Statistics:

Statistical analysis was done using Student's /-test and Fisher's exact test between means and portions, respectively

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The higher the dose of the chemicals, the less increase in body weight occurred.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The intake of chemical by kg body weight was markedly diminished throughout the experiment in all groups except the 1.5 % dose A which showed the strongest toxic effect on the body weight.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

The pattern of water consumption was lesser extent in the treated rats

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There is a dose-related association between the LI in rat urinary bladder and renal pelvic urothelium when the rats are treated with up to 1.5%

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Key result

Dose descriptor:

LOAEL

Effect level:

1.5 other: % test item

Based on:

test mat.

Sex:

male

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1.5 other: % test item

System:

urinary

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1. Body weight, water intake and food consumption at 6 and 12 weeks in the different groups 

Group number	Dose (%)	Body weight (g)		Number of rats		Food consumption g/rat/day		Water consumption		Total chemical consumption g/rat
		Week 6	Week 12	Week 6	Week 12	Week 6	Week 12	Week 6	Week 12	Week 6	Week 12
7	0.5	380 e	480 f	20	10	32.6	30.1	47.0	46.8	6.6	12.9
8	1.0	330 f	462 i	20	10	27.8	29.9	48.2	45.9	10.8	23.1
9	1.5	292 g	370 g	19	10	26.9	29.1	42.8	40.2	14.7	31.9
10		415 h	525 j	20	10	31.0	31.5	49.0	50.1	-	-

e'Significantly heavier than groups 2, 3, 5, 6, 8, 9 (P < 0.05, 0.001).
f'Significantly heavier than groups 2, 3, 9 (P < 0.001).
g'Significantly heavier than group 2 (P < 0.02).
h'Significantly heavier than group 2 (P < 0.05, 0.001).
i'Significantly heavier than groups 3, 9.
j'Significantly heavier than groups 2 - 9 (0.05, 0.001). 

Table 2. Intakes pf test item expressed as g/kg body weight 

Group	dose (%)	Weeks
		Intake of test item g/kg bw/rat
		2	4	6	8	10	12
7	0.5	0.78	0.52	0.43	0.36	0.34	0.31
8	1.0	1.28	1.04	0.84	0.75	0.68	0.65
9	1.5	1.77	1.66	1.38	1.18	1.24	1.18
10		0.00	0.00	0.00	0.00	0.00	0.00

Table 3. Effects of test item in the LI of the rat renal pelvic urothelium 

	Group	Effective number of rats	dose (%)	Labeling index (% +/- SD)		Group	Effective number of rats	Labeling index (% +/- SD)
Left kidney at 6 weeks	7	7	0.5	0.25±0.17	Right kidney at 6 weeks	7	7	0.31 ± 0.36
	8	9	1.0	1.85 b ± 1.52		8	8	2.78 b ± 1.72
	9	7	1.5	1.22 a ± 0.86		9	7	3.02 ± 6.09
	10	10		0.24 ± 0.23		10	9	0.18 ± 0.08
Left kidney at 12 weeks	7	10	0.5	0.31 ± 0.19	Right kidney at 12 weeks	7	10	0.25 ± 0.21
	8	9	1.0	0.38 ± 0.26		8	9	0.45 ± 0.23
	9	9	1.5	0.52 ± 0.47		9	9	0.77 a ± 0.52
	10	10		0.22 ± 0.10		10	10	0.26 ± 0.12

a'Significantly different from control group (group 10) at P < 0.05 by Student's I test.
b'Significantly different from control (group 10) at P < 0.01 by Student's t test. 

Table 4. the incidence of rats with LI 2-fold or higher than the mean value of control rats 

Group	No. of rats with LI 2-fold or higher than the mean value of control rats
	After 6 weeks		After 12 weeks		After 6 weeks	After 12 weeks
	Left kidney	Right kidney	Left kidney	Right kidney	Bladder
7	1/7	4/4	3/10	1/10	0/7	1/10
8	9/9	9)9	3/9	4/9	2/9	5/9
9	5/7	5/7	2/9	6/9	6/7	9/9
10	2/9	0/9	0/10	0/10	0/10	0/10

Table 5. effects of test item on labeling index of rat urinary bladder urothelium 

	Group	Effective number of rats	Drug (%)	Labeling index % ± SD
At 6 weeks	7	7	0.5	0.23 ± 0.08
	8	9	1.0	0.31 a ± 0.10
	9	7	1.5	5.17 b ± 3.45
	10	10		0.20 ± 0.08
At 12 weeks	7	10	0.5	0.25 ± 0.08
	8	10	1.0	0.37 ± 0.11
	9	9	1.5	2.10 ± 2.12
	10	10		0.21 ± 0.08

a'Significantly different from control (group 10) at P < 0.02 by Student's t test.
b'Significantly different from control (group 10) at P < 0.01 by Student's / test. 

Table 6. The incidence of morphological lesions in the different groeup at 6 and 12 weeks 

	Group number	Number of rats	RPN a	Vaculated pelvic cells			Utothelial hyperplasia
				+	++	+++	Renal pelvis	Papillae	Bladder
6 weeks	7	10		1			-	1	2
	8	10		1			1	3	7
	9	9	1	3			2	3	7 b
	10	10		2			-	-	-
12 weeks	7	10		6			-	3	1
	8	10		6			2	4	3
	9	10	1	7			2	4	5
	10	10		1			-	-	-

b'One rat had papillary and nodular hyperplasia.

Conclusions:

Test item was considered to produce urothelial hyperplasia in 1.5% diet group.

Executive summary:

Test item was tested for carcinogenicity using feeding study in Sprague-Dawley (male). Animals were fed with 0.5%, 1% and 1.5% test item diet for 6 and 12 weeks. Control animals were fed with basal diet. Body weight and food consumption were recorded biweekly. The food and water consumption was calculated per cage and divided by four or five. The rats underwent a complete necropsy with careful macroscopical examination of the exterior of the rat and the organs in the thoracic and abdominal cavities. The present study clearly shows that there is a dose-related association between the labeling index (LI) in rat urinary bladder and renal pelvic urothelium when the rats are treated with up to 1.5% phenacetin.

Reference 3

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Principles of method if other than guideline:

- Principle of test: Long-term feeding of test item.
- Short description of test conditions: Two groups of animals containing 50 males and 50 females per group were fed respectively with 2.5% and 1.25% phenacetin diet for 18 months and fed thereafter with basal diet for 6 months. Control animals containing 65 males and 65 females were fed with basal diet for 24 months. Every organ from was fixed in 10% formaldehyde solution and examined histopathologically.
- Parameters analysed / observed: mortality, macroscopic and histopathological examinations

GLP compliance:

not specified

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CLEA Japan Inc., Tokyo.
- Age at study initiation: 9 weeks.
- Housing: in metal cages, 4 rats to each cage.
- Diet (e.g. ad libitum): daily.
- Water (e.g. ad libitum): ad libitum.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23ºC

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Test item was mixed with the powdered basal diet (Charles River Japan Inc., Atsugi) in 2.5% and 1.25%, and made into a cubic diet.

Duration of treatment / exposure:

Animals were fed with test item diet for 18 months and fed thereafter with basal diet for 6 months.

Frequency of treatment:

Daily

Dose / conc.:

2.5 other: % test item

Dose / conc.:

1.25 other: % test item

No. of animals per sex per dose:

Two groups of animals containing 50 males and 50 females per group.
Control animals containing 65 males and 65 females were fed with basal diet.

Control animals:

yes, plain diet

Positive control:

None.

Sacrifice and pathology:

HISTOPATHOLOGY: Yes (see table)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Experimental animals began to die 8 weeks after the feeding, while control rats died 40 weeks after the feeding. The mortality rate of control animals reached 20～40% in thefinal stage. On the other hand, the mortality rate of experimental rats reached more than 65% at the end of experiment.

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:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Neoplasms including sponta-neous tumors were detected in 26 out of 27 males (96.3%) and 21 out of 27 females (77.8%) of 2.5% test item feeding group, while in 1.25% test item feeding group, in 20 out of 22 males (90.9%) and 19 out of 25 females (76.0%). In the control group, 1 out of 19 males (5.3%) and 6 out of 25 females (24.0%) showed spontaneous development of tumors.

Other effects:

no effects observed

Relevance of carcinogenic effects / potential:

Histopathologically, carcinomas of the nasal cavity, such as adenocarcinoma, squamous cell
carcinoma, and transitional cell carcinoma, and the urinary pas-sage, as renal cell carcinoma of the kidney pelvis, and transitional cell carcinoma of the urinary bladder, were most conspicuous, suggesting the target organs of phenacetin carcinogenesis. Males showed higher tumor incidence compared to females. The higher the concentration of phenacetin given, higher incidence of tumors was observed.

Key result

Dose descriptor:

LOAEL

Effect level:

1.25 other: % test item

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1.25 other: % test item

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1.25 other: % test item

System:

urinary

Organ:

bladder

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1.25 other: % test item

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1. Tumors that developed in Sprague-Dawley Rats fed with or without phenacetin 

Tissues	Tumors	2.5 % phenacetin feeding group		1.25% phenacetin feeding group		control group
		males	females	males	females	males	females
Nasal cavity	Adenocarcinoma	7	5	8	1	0	0
	Transitional cell carcinoma	7	1	2	1	0	0
	Squamous cell carcinoma	2	0	3	1	0	0
	Adenoma	0	1	3	3	0	0
Urinary passages	Renal cell carcinoma	1	0	0	0	0	0
	Transitional cell carcinoma	12	4	1	0	0	0
	Papilloma	0	2	0	0	0	0
Myeloid tissue	Myeloid leukemia	7	11	8	6	0	0
Pituitary	Adenoma	0	3	0	0	1	0
Lung	Adenocarcinoma	0	0	0	0	1	0
	Adenoma	0	1	1	0	0	0
Mammary gland	Adenocarcinoma	0	0	0	0	0	1
	Fibroadenoma	1	2	0	6	0	3
	Fibroma	0	0	0	0	1	0
Uterus	Adenocarcinoma	0	2	0	3	0	0
	Myoma	0	0	0	0	0	1
	Hemangioendothelioma	0	0	0	1	0	1
Large intestine	Adenocarcinoma	0	1	0	0	0	0
Others	Neurofibroma	0	1	0	0	0	0
	Myosarcoma	0	0	1	1	0	1
	Total	37	34	27	23	3	7

Table 2. Frequency of tumors arising from the nasal cavity and urinary passage in Sprague-Dawley Rats fed test item 

Sex	Concentration of test item in diet (%)
	2.5	1.25	control
Males	23/27 (29.6)	17/22 (77.3)	0/19 (0)
Females	11/27(40.7)	5/25 (20.0)	0/25 (0)
Total	34/54 (63.0)	22/47 (46.8)	0/44 (0)

The number of animals with tumors/the number of effective animals (%).

Table 3. Frequency of spountaneus tumors in Sprague-Dawley rats fed test item 

Sex	Concentration of test item in diet (%)
	2.5	1.25	control
Males	8/27 (29.6)	11/22 (50.0)	1/19 (5.3)
Females	13/27 (48.1)	14/25 (56.0)	7/25 (28.0)
Total	21/54 (38.9)	35/47 (53.2)	8/44 (18.2)

The number of animals with spontaneous tumors/the number of effective animals (%).

Conclusions:

The test item had a dose-related induction relation with tumors in the rat fed with it.

Executive summary:

Test item was tested for carcinogenicity using long-term feeding study in Sprague-Dawley rats from both sex (male and female). Animals were fed with 2.5% and 1.5% test item diet for 18 months and fed thereafter with basal diet for 6 months. Control animals were fed with basal diet for 24 months. Every organ from the killed and dead animals was fixed in 10% formaldehyde solution and examined histopathologically. Neoplasms including spontaneous tumors were detected in 26 out of 27 males (96.3%) and 21 out of 27 females (77.8%) of 2.5% phenacetin feedding group, and in 20 out of 22 males (90.9%) and 19 out of 25 females (76.0%) of 1.25% phenacetin feeding group. In control group, 1 out of 19 males (5.3%) and 6 out of 25 females (24.0%) showed spontaneous tumor development. Histopathologically, carcinomas of the nasal cavity, such as adenocarcinoma, squamous cell carcinoma, and transitional cell carcinoma, and the urinary passsage, as renal cell carcinoma of the kidney pelvis, and transitional cell carcinoma of the urinary bladder, were most conspicuous, suggesting the target organs of phenacetin carcinogenesis. Males showed higher tumor incidence compared to females. The higher the concentration of phenacetin given, higher incidence of tumors was observed.

Reference 4

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Principles of method if other than guideline:

- Principle of test: Long term feeding study
- Short description of test conditions: Groups of 52 B6C3Fl mice of each sex were maintained on a diet containing I .25 or 0.6 % phenacetin for 96 weeks and then fed a basal diet for 6 weeks. Control groups consisted of 50 mice of each sex and were fed a basal diet for 104 weeks.
- Parameters analysed / observed: Histological examinations and body weight.

GLP compliance:

not specified

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc., Kanagawa.
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: 5 week old.
- Housing: plastic cages according to sex and diet, with 5 per cage using wood chips for bedding
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24ºC
- Humidity (%): 50 %

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Charles River basal diet (Charles River Japan, Inc., Kanagawa). Test item was incorporated into the diet at concentrations of 1.25% and 0.6% and made into pellets.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:

Duration of treatment / exposure:

Mice were maintened on a diet containing test item from the age of 6 weeks to 102 weeks and then given a nasal diet for 8 weeks (total of 104 weeks)

Frequency of treatment:

Daily for 96 weeks

Dose / conc.:

0.6 other: % phenacetin

Dose / conc.:

1.25 other: %phenacetin

No. of animals per sex per dose:

52 mice/sex were maintened on tets item diet

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food intake was measured at regular intervals

Sacrifice and pathology:

GROSS PATHOLOGY: Not specified

HISTOPATHOLOGY: Yes. After 104 weeks on these diets, all mice were anesthetized with ether and killed. All tissues were fixed in
10 % phosphate-buffered formalin and embedded in paraffin. Sections were stained.with hematoxylin and eosin for histological examination.

Statistics:

The incidences of tumors and other lesions were compared by the x2 test.

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Although the average body weight gain was similar through the 24th week for males and 36th week for females, the weights in both sexes were subsequently higher in rats fed 0.6 % and lower in rats fed 1.25 % phenacetin compared to controls.

Food consumption and compound intake (if feeding study):

no effects observed

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:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Renal cell carcinomas and adenomas developed in mle mice fed 1.25% test item, and renal cell adenomaas developed in male mice fed 0.6% test item at statistically significantly higher incidences than in controls.
In addition, small foci of dilated and hyperplastic or dyplastic tubules were observed and considered to be preneoplastic lesions which occured alone or coincidentally with adenomas or carcinomas in the same kidney.
Papillary or nodular hyperplasia which are considered to be preneoplastic lesions of the urinary bladder occurred in male and female mice fed 1.25% test item at statistically significantly high& incidences than those in controls.
63 adenomas and 20 carcinomas of the kidney were obtained in 36 male mice fed 1.25% test item and 13 adenomas and 1 carcinoma developed in 12 males mice fed 0.6% test item.

Key result

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: Test item at dose 0.6% induced only significantly increased incidence of renal cell adenoma, but a dose of 1.25% was carcinogenic in kidney

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.6 other: % test item

System:

urinary

Organ:

other: Renal cell adenoma

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1.25 other: % test item

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1. Neoplastic lesions of the kidney and urinary bladder in B6C3F mice treated with phenacetin. 

Sex and dose of phenacetin	Effective No. of mice	Kidney (%)				Urinary bladder (%)
		dyplastic lesion	Renal cell adenoma	Renal cell carcinoma	Pelvic tumor	Papillary or modular hyperplasia	Papilloma	Transitional cell carcinoma
Male 1.25 %	48	46 (96)**	32 (67) **	14 (29) **	2 (4)	7 (15)*	0 -	0 -
Male 0.6 %	48	27 (56)**	11 (23) **	1 (2)	0 -	1 (2)	0 -	0 -
Male control	48	0 -	0 -	0 -	1 (2)	0 -	0 -	0 -
Female 1.25%	49	0 -	0 -	1 (2)	0 -	7 (14)*	2 (4)	2 (4)
Female 0.6 %	50	0 -	0 -	0 -	0 -	0 -	0 -	0 -
Female control	48	0 -	0 -	0 -	0 -	0 -	0 -	0 -

* p <0.05; ** p 10.01, compared with control.

Conclusions:

The test item had a dose-related induction relation with renal cell tumors in the mice fed with it.

Executive summary:

Test item was tested for carcinogenicity using long-term feeding study in B6C3F1 mice from both sex (male and female). Animals were fed with 1.25 or 0.6% test item for 96 weeks and then fed a basal diet for 8 weeks. Control groups were fed a basal diet for 104 weeks. animals were killed at the end of the experiment and organs were examined histopatholigically. Dose-related induction of renal cell tumors in the male mice fed with test item was clearly dmeonstrated. Urinary bladder lesions that developed in mice of either sex fed 1.25% test item were also considered to be due to the tumorigenicity of test item. tumors of other organs in either the test item treated or the control group were regarded as strain-related spontaneous tumors of mice. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 2

System:

respiratory system: lower respiratory tract

Organ:

other: respiratory mucosa

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Carcinogenicity. Based on the available data, the substance is considered to be carcinogenic Category 2 according to CLP Regulation no. 1272/2008.