Results of Long-term Experimental

APPENDIX II-CL: Inert Ingredients-Trimethylbenzene, Ethylbenzene, Benzene (Toluene), Xylene- Cause Cancer: Maltowi, et al, “Results of Long-Term Experimental Carcinogenicity Studies of the Effects of Gasoline, Correlated Fuels, and Major Gasoline Aromatics on Rats, “ Annals New York Academy of Sciences, 1997

This appendix is copied from:

http://www.blackwell-synergy.com/doi/pdf/10.1111/j.1749-6632.1997.tb56863.x

Results of Long-term Experimental

Carcinogenicity Studies of the

Effects of Gasoline, Correlated

Fuels, and Major Gasoline

Aromatics on Rats

CESARE MALTOWI, ADRIAN0 CILIBERTI,

CARMINE PINTO, MORANDO SOFFRITTI,

FIORELLA BELPOGGI, AND LORETTA MENARINI

Cancer Research Center

European Ramazzini Foundation

of Oncology and Environmental Sciences

Bentivoglio Castle

40010 Bentivoglio (BO), Italy

INTRODUCTION

Up to the midseventies the experimental information on the possible carcinogenic

potential of gasolines and their components, with particular regard to aromatic hydrocarbons,

was unsubstantial. In the laboratories of the Cancer Research Center (CRC),

at the Bentivoglio (BT) Castle of the European Ramazzini Foundation of Oncology

and Environmental Sciences, a series of experimental studies was started, aimed

at evaluating the carcinogenic burden of gasolines, correlated fuels, and gasoline

aromatics.

As early as 1977, it was shown for the first time that benzene was a multipotential

carcinogen.' Those results were then confirmed in successive A study

conducted by the American Petroleum Institute in the early eighties showed that a

cross section of significant samples of American gasolines was causing kidney tumors

in rats exposed by inhalation.'

There are no data in the literature on the carcinogenicity of the European type

of gasoline, of diesel oil, and of kerosene. The studies on aromatics other than benzene

are inadequate or missing.

The experimental carcinogenicity studies on toluene, reported up to 1989, have

been considered inadequate.8 This compound was then tested by inhalation on Fischer

344N rats and B6C3F1 mice within the framework of the National Toxicology

Program (NTP) in two year-long bioassays, with negative results.'

The only two experimental carcinogenicity studies on xylenes, performed within

the framework of the NTP, in which the compound was given in corn oil by stomach

tube to Fischer 344N rats and B6C3Fl mice, gave negative results."," Again these

bioassays were truncated after two years.

No experimental carcinogenicity studies have been performed to our knowledge

on ethylbenzene and on 1,2,4-trirnethylbenzene.

16 ANNALS NEW YORK ACADEMY OF SCIENCES

TABLE 1. Weight Percent Composition Analysis of Tested Unleaded Gasolines, as

Evaluated in Two Different Laboratories

Constituents

I analysis

(%I

I1 analysis

(%I

Total saturates

N-paraffins

Isoparaffins

Olefins

Total aromatics

Benzene

2,2,4-TMP

48.0

11.0

28.0

3.3

48.0

5.0

49.0

24.0

25.0

0.2

3.0

48.0

5.6

This lack of proper information on these mixtures and compounds is surprising.

The production, consumption and distribution of these fuels is enormous. Toluene,

xylenes, ethylbenzene, and 1,2,4-trimethylbenzene are present at a high concentration

in gasoline. Toluene and xylenes are widely used in the petrochemical industry, and

for the production of numerous consumer goods.

This report refers the results of our early studies on European unleaded gasoline

with high content of aromatics; leaded gasoline; gasoil (diesel) and kerosene; and

toluene, xylenes, ethylbenzene, and 1,2,4-trimethylbenzene.

Preliminary results on toluene, xylenes and ethylbenzene were already published

in 1985.4

MATERIALS AND METHODS

Gasolines, the other fuels, and the aromatic hydrocarbons were supplied by an

The weight percent composition analysis of the tested unleaded gasolines, evalu-

The analytical characterization of the tested aromatic hydrocarbons resulted in

Italian petrol industry and by an Italian petrochemical industry, respectively.

ated in two different laboratories, was as shown in TABLE 1.

percent, wlw:

toluene: toluene: 98.34; paraffin: 0.83; benzene: 0.28; ethylbenzene: 0.52; mxylenes:

xylenes: - 99.45 (0-xylene: 26.90; m-xylene: 50.3 1; p-xylene: 22.24);

ethylbenzene: ethylbenzene: 99.57; benzene: 0.07; toluene: 0.20; cumene: 0.06;

1,2,4-trimethylbenzene: 1,2,4-trimethyIbenzene: __ >99.

Male (M) and female (F) Sprague-Dawley rats of the colony of the CRC/BT

were used. This colony of rats has been employed for various experiments in the

xylene and p-xylene: 0.029

toluene: 0.29; paraffin: 0.05

paraffin: 0.06; unidentified: 0.04

MALTONI et aL: LONG-TERM CARCINOGENICITY STUDIES 17

BT laboratory for nearly 25 years. Historical data are available on more than 10,000

historical controls, kept under observation for life span and submitted to systematic

necropsies and standardized histopathological examinations. Therefore, data on the

expected incidence of the different types of tumors in the control animals and on its

fluctuations are available.

After weaning, at 4-5 weeks of age, the experimental animals were identified

by ear punch, randomized in order to have no more than one male and one female

of each litter in the same group, and housed 5 per cage. The animals were 7 weeks

old at the start of the experiments.

The plan of the experiments are shown in T-LE 2. Unleaded and leaded gasoline,

gasoil (diesel), and kerosene were tested at two dose levels within the same experiment.

Toluene, xylenes, and ethylbenzene were tested at two dose levels, with two experiments,

each one with its own control group. 1,2,4-trimethylbenzene was tested only

at one dose.

Every single dose was administered by stomach tube as 1 ml of extra virgin olive

oil solution, 4 days weekly (Monday and Tuesday; Thursday and Friday), for 104

weeks. A 5-6-dose weekly administration of the compound at a higher dose would

not have been tolerated by the rats. The solutions were prepared weekly and maintained

at 4°C. Control animals were given 1 ml of extra virgin olive oil alone.

The animals were kept under observation until death, under highly standardized

housing and diet conditions identical to those used in the CRCBT; the animals were

usually allowed to live until natural death. By doing so, it was possible to develop

all the neoplastic potentialities. Mean daily feed and drinking water consumption

were determined once weekly for the first 13 weeks from the start of the experiments,

then every 2 weeks, until 11 1 weeks of age. Individual animal weight was measured

once weekly from 7 weeks of age for the first 13 weeks, every 2 weeks until 11 1

weeks of age, and then every 8 weeks until the end of the experiments. In order to

detect and register all gross lesions, the animals were examined every week for the

first 13 weeks, and then every 2 weeks until the end of the experiments.

The biophase of the experiments either was truncated after 123 weeks (in the

experiment studying fuels and the higher doses of aromatics), or else lasted for the

life span, up to 145 weeks (in the experiments studying the aromatics at the lower

doses).

Upon death, the animals were submitted to systematic necropsy. Histopathology

was routinely performed on brain, pituitary gland, Zymbal glands, salivary glands,

Harderian glands, head, tongue, thymus and mediastinal lymph nodes, lung, heart,

diaphragm, liver, spleen, pancreas, kidneys and adrenal glands, esophagus, stomach,

intestine (4 levels), bladder, prostate, uterus, vagina, gonads, interscapular fat pad,

subcutaneous and mesenteric lymph nodes, sternum, femur, spinal cord, and any

other organs and tissues with pathological lesions.

All organs and tissues were immediately preserved in 70% ethyl alcohol, except

bones, which were fixed in 10% formalin and then decalcified with 10% formaldehyde

and 20% formic acid in water solution. The normal specimens were trimmed, following

the standard procedures of the BT Laboratory: i.e., parenchymal organs were

dissected through the hilus to expose the widest surface, and hollow organs were

sectioned across the greatest diameter(s). The pathological tissue was trimmed through

the largest surface, including normal adjacent tissues. The trimmed specimens were

18 ANNALS NEW YORK ACADEMY OF SCIENCES

TABLE 2. Long-term Carcinogenicity Bioassays on Unleaded Gasoline with High

Content of Aromatics, Leaded Gasoline, Gasoil (Diesel), Kerosene, Toluene,

Xylenes, Ethylbenzene, and 1,2,4-Trimethylbenzene: Plan of the Experiments

Animals Daily Dose

Test Compound Experiment (mg/kg b.wJ Sex No.

Leaded gasoline BT952

Gasoil (diesel) BT954

Kerosene BT953

Toluene BT910

Unleaded gasoline with high

content of aromatics BT951 800

500

g a b

800

500

p b

800

500

800

500

800

o",'

M + F

100

100

100

MALTONI et al. : LONG-TERM CARCINOGENICITY STUDIES 19

TABLE 2. Long-term Carcinogenicity Bioassays on Unleaded Gasoline with High

Content of Aromatics, Leaded Gasoline, Gasoil (Diesel), Kerosene, Toluene,

Xylenes, Ethylbenzene, and 1,2,4-Trirnethylbenzene: Plan of the Experiments

(Continued)

Animals Daily Dose

Test Compound Experiment (mgkg b.w.) Sex No.

Xylenes

Ethylbenzene

BT903 500

BT911 800

o”,‘

BT904 500

o”,d

BT9 12 800

o”.’

BT905 500

800

o”,‘

BT913

M + F

100

a Olive oil alone.

The same control group.

20 ANNALS NEW YORK ACADEMY OF SCIENCES

TABLE 3. Effects of Treatments on Survival of Male and Female Rats

Daily Dose Survival"

(mgkg b.w.) M F Test Compound

Unleaded gasoline with high 800 + ++

500 + +

Leaded gasoline 800 ++ +

500 -

Gasoil (diesel) 800 + ++

500 +

Kerosene 800 + ++

500 + +

Toluene 800 + +

500

500 +

500 + -

content of aromatics

- -

Xylenes 800 ++ ++

Ethylbenzene 800 +(+) +(+)

1,2,4-Trimethylbenzene 800 +(+) +

a -: unaffected; +: slight reduction; ++: sharp reduction; +(+): intermediate reduction.

processed as paraffin blocks, and 3-5-micron sections of every specimen were obtained.

Sections were routinely stained with hematoxylin-eosin. Specific stainings

were performed when needed. All slides were examined microscopically by the same

group of pathologists; a senior pathologist reviewed all the tumors and any other lesion

of oncological interest. All pathologists followed the same criteria of histopathological

evaluation and classification.

RESULTS

Survival

Survival was affected by treatment as shown in TABLE 3. The reduction of survival

for several compounds appears clearly correlated to the dose, and seems to be

influenced to some extent by the sex. The different survival rates may have affected

the incidence of tumors.

Tumors

Multiple tumors of the same site (monolateral and bilateral organs) and type were

plotted only once.

MALTONI et aL: LONG-TERM CARCINOGENICITY STUDIES 21

TABLE 4. Results of Long-term Carcinogenicity Bioassays of Unleaded Gasoline

with a High Content of Aromatics, Administered by Gavage to Sprague-Dawley

Rats: Total Tumors

% of Animals

Bearing

Animals Tumors No. of Malignant

Tumors per 100 Group Dose

No. (mgkg b.w.) Sex No. TBMT” MTb Animals

I 800 M 50 52.0 30.0 34.0

F 50 62.0 32.0 40.0

M + F 100 57.0 31.0 37.0

I1 500 M 50 54.0 14.0 16.0

F 50 64.0 36.0 46.0

M + F 100 59.0 25.0 31.0

0 50 54.0 24.0

111 (control) w 50 70.0 22.0 2880

M + F 100 62.0 23.0 24.0

a TBMT: total benign and malignant tumors.

MT: malignant tumors.

TABLE 5. Results of Long-term Carcinogenicity Bioassays of Unleaded Gasoline

with a High Content of Aromatics, Administered by Gavage to Sprague-Dawley

Rats: Mammary Cancers

Group Dose % of Animals Bearing Animals

No. (mgkg b.w.) Sex No. Cancers

I 800 F 50

I1 500 F 50

I11 0 F 50

(control)

16.0

10.0

6.0

Unleaded Gasoline

An increase of total malignant tumors was found in males treated with the higher

dose, and in females treated with both doses (but not dose related) (TABLE 4). A

dose-related increase of mammary cancers was found among treated females (TABLE

5). A slight increase of head cancers was observed in male rats treated with the

higher dose (TABLE 6). A higher onset of malignant tumors of the uterus-vagina was

found in females treated with both doses, although it was not dose related (TABLE

7). Of particular significance is the observation of uterine-vaginal neurinosarcomas

22 ANNALS NEW YORK ACADEMY OF SCIENCES

TABLE 7. Results of Long-term Carcinogenicity Bioassays of Unleaded Gasoline with a High Content of Aromatics, Administered

by Gavage to Sprague-Dawley Rats: Malignant Tumors of Uterus and Vagina

Animals % of Animals Bearing Malignant Tumors

Group Dose Uterus Vagina Uterus-Vagina Uterus:

No. (m@g b.wJ Sex No. CA" CA Neurino SAb Other SA Total

8.0 I 800 F 50 2.0

11 500 F 50 - 4.0 4.0 2.0 10.0

- 2.0 2.0 III 0 F 50

- 6.0 -

- -

(control)

CA cancer.

* SA: sarcoma.

P ..

h) w

24 ANNALS NEW YORK ACADEMY OF SCIENCES

(all grossly detected), which are rarely observed in the historical controls. These

tumors seem to arise from the nerves related to the ganglia cewicalia.

Leaded Gasoline

An increase of total malignant tumors was found among females treated at both

doses, particularly with the lower one; in males an increase was observed only among

the animals treated with the lower dose (TABLE 8). An increase of mammary cancers

was found in the females treated with both doses, but was not dose related (TABLE

9). A slight dose-related increase of head cancers was detected among treated males

(TABLE lo). A sharp increase of malignant uterine-vaginal tumors (including neurinosarcomas)

was found in the females treated with the lower dose (TABLE 11).

Gasoil (Diesel)

A non-dose-related increase of total malignant tumors was found among treated

males and females (TABLE 12). The treatment did not affect the incidence of female

mammary cancers (TABLE 13). An increase in the incidence of head cancer in males

was observed in the group treated with the lower dose (TABLE 14). A sharp, doserelated

increase of malignant uterine-vaginal tumors (including neurinosarcomas)

was found in treated females (TABLE 15).

Kerosene

A dose-related increase of total malignant tumors was observed in treated females

and, to a lesser extent, in males exposed to the higher dose (TABLE 16). Mammary

cancers and head cancers appeared to be increased among females exposed to the

higher dose (TABLES 17 and 18). An increased incidence, not dose-related, of malignant

uterine-vaginal tumors (including neurinosarcomas) was found among treated females

(TABLE 19).

Toluene

There was a non-dose-related increase of the total malignant tumors among males

and females treated with both doses (TABLE 20); of mammary cancers in femalestreated

with the lower dose (TABLE 21); of head cancers among males treated with

the higher dose and, to a lesser extent, in females treated with the lower dose (TABLE

22); and of lymphomas and leukemias among males treated with the higher dose

and among females treated with both doses (not dose related) (TABLE 23).

Xylenes

A non-dose-related increase of total malignant tumors was found in males and

females treated with both doses (TABLE 24). An increase of mammary cancers was

TABLE 8. Results of Long-term Carcinogenicity Bioassays of Leaded Gasoline with a High Content of Aromatics, Administered by

Gavage to Sprague-Dawley Rats: Total Tumors H

Group Dose Animals

% of Animals Bearing

Tumors

4 CI

No. of Malignant

Tumors per 100 n_ .- .. Animals

No. (mgkg b.w.) Sex No. TBMT" MTb

800

500

M 50 38.0 18.0

F 50 62.0 34.0

M + F 100 50.0 26.0

24.0

42.0

33.0

li E

M 50 70.0 34.0 42.0 E

3- F 50 68.0 44.0 60.0

M + F 100 69.0 39.0 51.0

I1 8 I11 0 M 50 54.0 24.0 26.0

(control) F 50 70.0 22.0 22.0

M + F 100 62.0 23.0 24.0 8

a TBMT total benign and malignant tumors.

* MT: malignant tumors.

26 ANNALS NEW YORK ACADEMY OF SCIENCES

TABLE 9. Results of Long-term Carcinogenicity Bioassays of Leaded Gasoline with

a High Content of Aromatics, Administered by Gavage to Sprague-Dawley Rats:

Mammarv Cancers

Animals Group Dose % of Animals Bearing

No. (mgkg b.w .) Sex No. Cancers

I 800 F 50

I1 500 F 50

111 0 F 50

(control)

12.0

16.0

6.0

observed among females treated with the lower dose (TABLE 25). There was a nondose-

related increase of head cancers and of lymphomas and leukemias among males

and females treated with both doses (TABLES 26 and 27).

Ethylbenzene

A non-dose-related increase of total malignant tumors was found in males and

females treated with both doses (TABLE 28). An increase of head cancers was observed

among males and females treated at the higher dose (TABLE 29).

A slight increase of total malignant tumors was found among treated males and

females (TABLE 30). An increase of the incidence of head cancers was observed

among the treated males (TABLE 3 1).

CONCLUSIONS

The results indicate that, although to a varying extent, all the tested materials

appear to be carcinogenic (TABLE 32). An increase of total malignant tumors and of

head cancers was found among animals treated with all fuels and aromatics, in males

andor in females. An increase of mammary cancers was observed in females treated

with tested gasoline, kerosene, toluene, and xylenes. Malignant tumors of the uterus

and vagina showed an increase in the animals treated with all types of the tested

fuels. Lymphomas and leukemias were increased among males and females treated

with toluene and xylenes.

In several instances no dose-response relationship has been observed. These

results may be due, among other experimental factors (low number of animals per

group; the fact that, in the case of the aromatics, the two doses were tested with two

different experiments; in some cases the different length of the biophase of the

MALTONI ef al. : LONG-TERM CARCINOGENICITY STUDIES 27

TABLE 11. Results of Long-term Carcinogenicity Bioassays of Leaded Gasoline with a High Content of Aromatics, Administered by

Gavage to Sprague-Dawley Rats: Malignant Tumors of Uterus and Vagina

Animals % of Animals Bearing Malignant Tumors

Uterus-

Group Dose Uterus Vagina Vagina Uterus:

No. (mgnCg b.w.) Sex No. CA" CA Neurino SAb Other SA Total

I 800

I1 500

111 0

(control)

- F 50 4.0

F 50 12.0 4.0

F 50 ~ -

4.0

2.0

4.0

20.0

2.0

CA cancer.

* SA: sarcoma.

TABLE 12. Results of Long-term Carcinogenicity Bioassays of Gasoil (Diesel) Administered by Gavage to Sprague-Dawley Rats: 5 Total Tumors r 8

% of Animals Bearing 3 No. of Malignant 2

Group Dose Tumors per 100 p Animals Tumors .. No. (mdk b.w.) Sex No. TBMT" MTb Animals

I 800 M 50 56.0 32.0 38.0 H 2 B F 50 58.0 42.0 56.0

M+F 100 57.0 37.0 47 .O

n 500 M 50 72.0 48.0 56.0

F 50 70.0 38.0 50.0 c1 * M+F 100 71.0 43.0 53.0 56 G

* MT: malignant tumors. cc

M+F 100 62.0 23.0 24.0 $

Iu 0 M 50 54.0 24.0 26.0

(control) F 50 70.0 22.0 22.0

TBMT total benign and malignant tumors.

m 8

m B

30 ANNALS NEW YORK ACADEMY OF SCIENCES

TABLE 13. Results of Long-term Carcinogenicity Bioassays of Gasoil (Diesel)

Administered by Gavage to Sprague-Dawley Rats: Mammary Cancers

Group Dose % of Animals Bearing Animals

No. (mgkg b.w.) Sex No. Cancers

I 800 F 50 4.0

I1 500 F 50 6.0

I11 0 F 50 6.0

(control)

experiments testing two doses of the same compound), mainly to the higher toxicity

of the higher tested doses.

The carcinogenicity of the fuels is also supported by the observation of neurinosarcomas

of the uterus-vagina in treated animals; and the carcinogenicity of ethylbenzene

and 1,2,4-trimethylbenzene is supported by the observation in treated males and

females of neuroesthesioepitheliomas. These two tumors are, in fact, quite rare in

the colony of Sprague-Dawley rats used for these experiments.

The fact that the experimental study performed by NTP on toluene and xylenes

gave negative r e ~ u l t s ~ - ~ ~ may be due to the different experimental conditions in which

these compounds were tested, and particularly to the fact that in those bioassays the

experiments were truncated after 104 weeks.

Since the experiments were performed in a standardized way, our data allow us

to rank the relative carcinogenic potency of toluene, xylenes, and ethylbenzene using

the results obtained with a lower dose, which does not noticeably affect the survival

rate; and also to compare the carcinogenic potential of these aromatics with that of

benzene, which was also tested in the same experimental conditions in our laboratories

(TABLE 33). It must be stressed that benzene causes the onset of a wider range of

different cancers (TABLE 34).

Our results call for further research to better assess the level of carcinogenicity

potential of the tested compounds and also for preventive measures.

SUMMARY

Unleaded gasoline, with high aromatic content, leaded gasoline, gasoil (diesel),

kerosene, toluene, xylenes, ethylbenzene, and 1,2,4-trimethyl-benzene were submitted

to long-term experimental carcinogenicity bioassays. The mixtures and the compounds

were administered by stomach tube, in olive oil, once daily, 4 days weekly, for 104

weeks, to male and female Sprague-Dawley rats. The animals were kept under control

until the end of the experiments. With varying degrees of evidence, all the tested

materials were found to increase the total number of malignant tumors and of some

site-specific tumors. They must therefore be considered carcinogenic. On the basis

of our results the rank of carcinogenic potency of the tested aromatic hydrocarbons

increases in the following order: 1,2,4&methylbenzene, ethylbenzene, xylenes, toluene

(benzene).

TABLE 14. Results of Long-term Carcinogenicity Bioassays of Gasoil (Diesel) Administered by Gavage to Sprague-Dawley Rats:

Head Cancers

Animals % of Animals Bearing Cancers

Group Dose Zymbal Ear Duct Nasal Oral Head

No. (m@g b.w.1 Sex No. Gland CA" CA Cavities CA Cavity CA SAb Total

~ 4.0 I 800 M 50 2.0 2.0

F 50 2.0 4.0 2.0

1 .o 4.0

8.0 4.0

2.0

5.0

2.0

6.0

4.0

- -

~ - -

~ M + F 100 1 .o 2.0 -

I1 500 M 50 4.0 - ~ -

~ F 50 - - 2.0 -

M + F 100 - 2.0 - 3.0 -

~ ~ ~ I11 0 M 50 2.0 -

~ (control) F 50 2.0 2.0 2.0 -

~ - M + F 100 2.0 1 .o 1 .o

P ..

CA: cancer.

SA: sarcoma.

tJ TABLE is. Results of Long-term Carcinogenicity Bioassays of Gasoil (Diesel) Administered by Gavage to Sprague-Dawley Rats:

Malignant Tumors of Uterus and Vagina

Animals % of Animals Bearing Malignant Tumors"

Group Dose Uterus Vagina Uterus-Vagina Uterus:

No. (mgkg b.w .) Sex No. CA" CA Neurino SAh Other SA Total

I 800 F 50 18.0 - 8.0 - 26.0

~ - 12.0 I1 500 F so 6.0 6.0

- - I11 0 F so -

(control)

2.0 2.0

2 5 t:

' CA: cancer.

SA: sarcoma.

TABLE 16. Results of Long-term Carcinogenicity Bioassays of Kerosene Administered by Gavage to Sprague-Dawley Rats: Total

Tumors

Group Dose - - Animals

% of Animals Bearing

Tumors No. of Malignant

Tumors per 100

Animals No. (m&g b.wJ Sex No. TBMT" MTb

111

800

500

M 50 60.0 32.0

F 50 72.0 40.0

M + F 100 66.0 36.0

M 50 56.0 24.0

F 50 68.0 26.0

M + F 100 62.0 25.0

0 M 50 54.0 24.0

M + F 100 62.0 23.0

(control) F 50 70.0 22.0

36.0

50.0

43.0

28.0

40.0

34.0

26.0

22.0

24.0

a TBMT: total benign and malignant tumors.

* MT: malignant tumors.

w w

ANNALS NEW YORK ACADEMY OF SCIENCES

z TABLE is. Results of Long-term Carcinogenicity Bioassays of Kerosene Administered by Gavage to Sprague-Dawley Rats: Head

Cancers 4

Animals % of Animals Bearing Cancers 3

No. ( m a g b.w.) Sex No. Gland CA" CA Cavities CA Cavity CA SAb Total .. R Group Dose Zymbal Ear Duct Nasal Oral Head

I 800 M 50 - - ~ 2.0 ff

I1 500 M 50 - 2.0 - 2.0 2.0' 6.0 E

111 0 M 50 2.0 - - - 2.0 8

2.0 -

F 50 - 2.0 2.0 4.0 2.w 10.0 9

M + F 100 - 2.0 1 .o 2.0 1 .o 6.0 2

~ 4.0 - F 50 - 2.0 2.0

M + F 100 - 2.0 - 2.0 1 .o 5.0 #

6.0 0

- 2.0 - (control) F 50 2.0 2.0

M + F 100 2.0 1 .o - 1 .o ~ 4.0 $

a CA: cancer.

SA: sarcoma.

Osteosarcoma.

TABLE 19. Results of Long-term Carcinogenicity Bioassays of Kerosene Administered by Gavage to Sprague-Dawley Rats:

Malignant Tumors of Uterus and Vagina

Animals % of Animals Bearing Malignant Tumors

Group Dose Uterus Vagina Uterus-Vagina Uterus:

No. (mgflcg b.w.) Sex No. CA" CA Neurino SAb Other SA Total

I 800 F 50 2.0 8.0 - 10.0

I1 500 F 50 4.0 6.0 2.0 2.0 14.0

- 2.0 2.0 rn 0 F 50

- -

(control)

* CA: cancer.

SA: sarcoma.

Tumors cl

Animals Tumors Tumorspex 100 p

% of Animals Bearing 3

8 k

I1 0 M 50 54.0 24.0 26.0 z

c M + F 100 62.0 23 .O 24.0 56 Q

2 Q

TABLE 20. Results of Long-term Carcinogenicity Bioassays of Toluene Administered by Gavage to Sprague-Dawley Rats: Total

No. of Malignant

Group Dose .. No. (mgflcg b.w.) Sex No. TBMT MTb Animals

I 800 M 50 60.0 40.0 42.0

F 50 58.0 30.0 46.0

M + F 100 59.0 35.0 44.0

(control) F 50 70.0 22.0 22.0

I 500 M 40 71.5 45.0 57.5

F 40 90.0 52.5 80.0

M + F 80 83.8 48.8 68.8

I1 0 M 50 58.0 24.0 24.0

M + F 100 59.0 22.0 23.0

(control) F 50 60.0 20.0 22.0 .c

TBMT: total benign and malignant tumors.

MT: malignant tumors.

TABLE 21. Results of Long-term Carcinogenicity Bioassays of Toluene Administered by Gavage to Sprague-Dawley Rats: Mammary

Cancers

’

Group

No.

Animals

Sex No.

% of Animals Bearing

Cancers

I 800 F 50 6.0

(control)

F 50 6.0

I 500 F 40 25.0

(control)

F 50

9 14.0 z

TABLE 22. Results of Long-term Carcinogenicity Bioassays of Toluene Administered by Gavage to Sprague-Dawley Rats: Head

Cancers

Animals % of Animals Bearing Cancers

Group Dose Zymbal Ear Duct Nasal Oral

No. (mgflag b.w.1 Sex No. Gland CA' CA Cavities CA Cavity CA Total

2.0 2.0 14.0 18.0

6.0 6.0

1 .o 1 .o 10.0 12.0

I 800 M 50 -

M + F 100 -

- - - F 50

(control)

500

M 50 2.0

F 50 2.0

M + F 100 2.0

2.0

2.0 6.0

1 .o 4.0

2.5

2.5 2.5 5.0

M + F 80 1.3 1.3 1.3 3.8

- - M 40 2.5 -

F 40 - -

2.0

1 .o

- - - I1 0 M 50 2.0

(control) F 50

M+F 100 1 .o

- - - - -

- - -

CA: cancer.

0 TABLE 23. Results of Long-term Carcinogenicity Bioassays of Toluene Administered by Gavage to Sprague-Dawley Rats:

Lymphomas and Leukemias

Group

No.

Animals

Sex No.

% of Animals Bearing

Lymphomas and

Leukemias

I 800 M 50 16.0

F 50 10.0

M + F 100 13.0

I1 0 M 50 10.0

(control) F 50 6.0

M + F 100 8.0

I 500 M 40 7.5 4

F 40 17.5 G

I1 0 M 50 6 .O <

(control) F 50 2.0 cc

M + F 100 4.0 8

8 5

M + F 80 12.5

4 c,

O'EZ

O'ZZ

0 P Z

0 S S

059

0 SP

0 P Z

0zz

09z

0'9P

0'8P

0.m

O'ZZ 06s 001 d+N

O'PZ 0% 0s w 0

O'OZ 009 OS d (IOJIUO3)

E'9P E'IL 08 d + M

S'LS 0'58 OP d

O'SE S'LS OP M 00s

O'EZ O'Z9 001 d + M

O'PZ O'PS OS M 0

0zz O'OL 0s d (pnuo3)

O'LE 0'09 001 d + M

0 9 E O'Z9 OS d

0'8E 0'8s OS w 008

asoa dnoq

TABLE 2s. Results of Long-term Carcinogenicity Bioassays of Xylenes Administered by Gavage to Sprague-Dawley Rats: Mammary

Cancers

Group

No.

Animals

Sex No.

% of Animals Bearing

Cancers

800

(control)

500

(control)

2.0

6.0

27.5

14.0

MALTONI et al. : LONG-TERM CARCINOGENICITY STUDIES 43

3 3 CI

P TABLE 2-1. Results of Long-term Carcinogenicity Bioassays of Xylenes Administered by Gavage to Sprague-Dawley Rats:

Lymphomas and Leukemias

Animals % of Animals Bearing

Group Dose Lymphomas and

No. (mgflcg b.w.) Sex No. Leukemias

I 800 M 50 14.0

F 50 14.0

M + F 100 14.0

I1 0 M 50 10.0

(control) F 50 6.0

M + F 100 8.0 *

I 500 M 40 12.5 * 3

F 40 7.5 z

I1 0 M 50 6.0 8

(control) F 50 2.0 4

M + F 100 4.0 8

M + F 80 10.0

R * n *

B m

z n

'SJOUrn] lUeU%JWIl :J,W

'SJOUIII] 1wuSI@m pw U%Naq @lo1 :JJq8L 0 E

0 EZ O'ZZ 065 001 d+PI

OZZ OOZ 009 OS d (104uo3)

OVZ OPZ 08s OS PI 0 I1

S'ZP OSE O'SL ov d

OSE OSE S'L9 op PI 00s I

OPZ O'EZ OZ9 001 d+PI

O9Z O'PZ OPE OS PI 0 I1

OZP O I E OZS 001 d+PI

O8P O8E 009 0s d

E L

zi 8'8E OSE E ' I L 08 d+PI 8

5 OZZ O'ZZ OOL 0s d (IQT1"3)

2 0 9 E OPZ OW OS PI 008 I s

ril c:

TABLE 29. Results of Long-term Carcinogenicity Bioassays of Ethylbenzene Administered by Gavage to Sprague-Dawley Rats:

Head Cancers

Animals % of Animals Bearing Cancers

Nasal Cavities Group Dose Zymbal Ear Duct Oral

No. (mglkg b.w.) Sex No. GlandCA" CA CA NEP Cavity CA Total

I 800 M 50 - 2.0 6.0 2.0 10.0

F 50 2.0 2.0 2.0 2.0 6.0 14.0

M+F 100 1 .o 2.0 1 .o 4.0 4.0 12.0

2.0

2.0 6.0

M+F 100 2.0 1 .o - 1.0 4.0

- - - - I1 0 M 50 2.0

- - (control) F 50 2.0 2.0

~ - - ~ - - I 500 M 40

~ 2.5

1.3

2.0

1 .o

- - - F 40 2.5

M+F 80 1.3

I1 0 M 50 2.0

M+F 100 1 .o

- - - -

~ - - -

- - - - - (control) F 50 ~

~ - - -

a CA: cancer. ' NEP: neuroesthesioepitheliomas.

TABLE 30. Results of Long-term Carcinogenicity Bioassays of 1,2,4-Trimethylbenzene Administered by Gavage to Sprague-Dawley 5 r Rats: Total Tumors 4 s

Group Dose Animals

% of Animals Bearing

Tumors

No. of Malignant

Tumorsper 100

a- TBMT" MTb Animals ..

No. (m&g b.wJ Sex No.

I 800 M 50 62.0 26.0

F 50 66.0 24.0

M+F 100 64.0 25.0

v 34.0 z

32.0

33.0 2 E I1 0 M 50 54.0 24.0 26.0 z

P R

(control) F 50 70.0 22.0 22.0

M+F 100 62.0 23.0 24.0

0 TBMT: total benign and malignant tumors.

MT malignant tumors.

TABLE 31. Results of Long-term Carcinogenicity Bioassays of 1,2,4-Trirnethylbenzene Administered by Gavage to Sprague-Dawley

Rats: Head Cancers

Animals % of Animals Bearing Cancers

Oral Nasal Cavities Group Dose Zymbal Ear Duct

No. (mg/kg b.w.1 Sex No. Gland CA" CA CA NEPb Cavity CA Total

I 800 M 50 4.0 2.0 - 2.0 2.0 10.0

6.0

M + F 100 3.0 1 .o - 3.0 1 .o 8.0

2.0

(control) F 50 2.0 2.0 - 2.0 6.0

M + F 100 2.0 1 .o - 1 .o 4.0

- 4.0 - - F 50 2.0

~ ~ - - I1 0 M 50 2.0

CA cancer.

MP: neuroesthesioepitheliomas.

F v1

TABLE 32. Tumors in Which an Increase Was Observed Following Treatment with the Tested Fuels and Aromatics

Increased Tumors

Total Uterus-Vagina

Malignant Mammary Head Malignant Lymphomas

Tumors Cancers Cancers Tumors and Leukemias Daily Dose

Test Compound (mgkg b.w.) M F F M F F M F

Unleaded gasoline with high

content of aromatics

Leaded gasoline

Gasoil (diesel)

Kerosene

Toluene

Xylenes

Ethylbenzene

800

500

800

500

800

500

800

500

800

500

800

500

800

500

800

+ +

+ +DR

+ +

+DR" +

+ +DR

+ +

+DR

DR: dose related.

50 ANNALS NEW YORK ACADEMY OF SCIENCES

TABLE 33. Long-term Carcinogenicity Bioassays of Benzene, Toluene, Xylenes,

and Ethylbenzene Administered at 500 mgkg b.w.: Comparative Results

Total Malignant Tumors/ Carcinogenicity

Test Compound 100 Animals (M + F) Index"

Benzene" 160.8 6.56

Toluene 68.8 2.81

Xylenes 56.4 2.30

Ethylbenzene 40.3 1.64

Olive oil alone (control) 24.5 1 .oo

Ratio between observed and expected total malignant tumors.

" See Reference 4.

TABLE 34. Tumors Associated with Benzene Exposure on the Basis of the Experimental Project of the Cancer Research Center of

the European Ramazzini Foundation of Oncology and Environmental Sciences"

5 r 2 w

AnimalsRoute of Administration 3

Sprague-Dawle y Wistar Swiss RF/J I

Rat Rat Mouse Mouse 8 Tumors Ingestion Inhalation Ingestion Ingestion Ingestion

Total malignant tumors

Carcinomas of Zymbal gland

Carcinomas of oral cavity

Carcinomas of nasal cavities

Carcinomas of the skin

Carcinomas of the forestomach

(+)

Carcinomas of the mammary gland (+I (+) + +

He pa tom as (+) (+)

Angiosarcomas of the liver +

Hemolymphoreticular neoplasias (+) +

Tumors of the lung + + 9

a See Reference 4. 4

52 ANNALS NEW YORK ACADEMY OF SCIENCES

10.

11.

REFERENCES

MALTONI, C. & C. SCARNATO. 1977. Le prime prove sperimentali dell’azione cancerogena

del benzene. Osp. Vita 4: 111-113.

MALTONI, C. & C. SCARNATO. 1979. First experimental demonstration of the carcinogenic

effects of benzene. Long-term bioassays on Sprague-Dawley rats by oral administration.

Med. Lav. 70: 352-357.

MALTONI, C., B. CONTI & G. COTTI. 1983. Benzene: A multipotential carcinogen. Results

of long-term bioassays performed at the Bologna Institute of Oncology. Am. J. Ind.

Med. 4: 589-630.

MALTONI, C., B. CONTI, G. COTTI ef al. 1985. Experimental studies on benzene carcinogenicity

at the Bologna Institute of Oncology: Current results and ongoing research. Am.

J. Ind. Med. 7: 415-446.

HUFF, J. E. 1986. Toxicology and carcinogenesis studies of benzene (CAS No. 71-43-2)

in F344/N rats and B6C3F1 mice (gavage studies). Technical Report No. 289. National

Toxicology Program. US Department of Health and Human Services. Washington, DC.

MALTONI, C., A. CILIBERTI, G. Con1 et al. 1989. Benzene, an experimental multipotential

carcinogen: Results of the long-term bioassays performed at the Bologna Institute of

Oncology. Environ. Health Perspect. 82: 109- 124.

KITCHEN, D. N. 1984. Neoplastic renal effects of unleaded gasoline in Fischer 344

rats. In Advances in Modem Environmental Toxicology, Renal Effects of Petroleum

Hydrocarbons, Vol. VII. M. A. Mehlman, G. P. Hemstreet III, J. J. Thrope & N. K.

Weaver, Eds.: 65-72. Princeton Scientific Publishers. Princeton, NJ.

INTERNATIONAL AGENCY FOR RESEARCH ON CANCER. 1989. Monographs on the evaluation

of carcinogenic risks to humans, Vol. 47. Some organic solvents, resin monomers

and related compounds, pigments and occupational exposure in paint manufacture.

IARC. Lyon.

NATIONAL TOXICOLOGY PROGRAM. 1990. Toxicology and carcinogenesis studies of toluene

(CAS No. 108-88-3) in F344/N rats and B6C3F1 mice (inhalation studies). NTP TR

371, NIH Publ. No. 90-2826. US Department of Health and Human Services. Research

Triangle Park, NC.

NATIONAL TOXICOLOGY PROGRAM. 1986. Toxicology and carcinogenesis studies of xylenes

(mixed) (60% m-xylene, 14% p-xylene, 9% o-xylene, and 17% ethylbenzene) (CAS

No. 1330-20-7) in F344/N rats and B6C3F1 mice (gavage studies). NTP TR 327, NIH

Publ. No. 87-2583. US Department of Health and Human Services. Research Triangle

Park, NC.

HUFF, J. E., W. EASTIN, J. ROYCROFT et al. 1988. Carcinogenesis studies of benzene,

methylbenzene, and dimethylbenzenes. Ann. N.Y. Acad. Sci. 534: 427 -440.