Registration Dossier

Toxicological information

Carcinogenicity

Currently viewing:

Administrative data

Description of key information

Based on carcinogenicity studies in animals and lack of evidence of carcinogenicity in humans toluene is considered not to be carcinogenic.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Near guideline, GLP, animal experimental study, available as published report, fully adequate for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
no
GLP compliance:
yes
Species:
rat
Strain:
other: F344/N
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI, USA
- Age at study initiation: approx. 6-7 weeks
- Housing: singly in stainless steel wire mesh cages
- Diet: IH 07 Rat and Mouse Ration (Zeigler Bros., Inc., Gardners, PA, USA) ad libitum (except during exposure)
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 74-80°F during exposures
- Humidity: 45-55 % during exposures
- Air changes (per hr): not reported
- Photoperiod: 12hrs dark / 12hrs light

IN-LIFE DATES: not reported
Route of administration:
inhalation: vapour
Vehicle:
other: air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: no data
- Vapour generation system: toluene vapour was generated by delivering liquid toluene to a heated Spraying Systems atomizer that was operated with nitrogen. Toluene vapour was diluted with chamber ventilation air to produce the desired concentrations in the chamber
- Temperature, humidity: 74-80°F, humidity 45-55 % during exposures

TEST ATMOSPHERE
- Brief description of analytical method used: gas-phase infrared spectophotometry
- Samples taken from breathing zone: not reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of toluene in the chambers was measured in sampled chamber air at 3.3 u by a MIRAN gas-phase infrared spectrophotometer. Air from each chamber was sampled and analyzed about 5 minutes every hour.
Duration of treatment / exposure:
6.5 h/day
Frequency of treatment:
5 days/week for 15 months or 103 weeks
Post exposure period:
None
Remarks:
Doses / Concentrations:

Basis:
nominal conc.
0, 600, 1200 ppm
Remarks:
Doses / Concentrations:

Basis:
analytical conc.
1.3, 593.2, 1179 ppm (time weighted average)
Remarks:
Doses / Concentrations:

Basis:
nominal conc.
0, 2261, 4522 mg/m3
No. of animals per sex per dose:
60
Control animals:
yes, sham-exposed
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes (for dead and moribund animals)
- Time schedule: twice/day

DETAILED CLINICAL OBSERVATIONS: Yes
-Time schedule: every 4 weeks

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for first 13 weeks, once every 4 weeks until week 92, and then once every 2 weeks.

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No
Sacrifice and pathology:
Necropsy and histopathological examinations were performed on all animals and the following tissues were examined: adrenal glands, brain, caecum, colon, duodenum, epididymis/prostate/testes or ovaries/uterus, oesophagus, femur including marrow, gross lesions and tissue masses with regional lymph nodes, heart and aorta, ileum, jejunum, kidneys, liver, lungs and mainstem bronchi, mammary gland, mandibular lymph nodes, nasal cavity and turbinates, pancreas, parathyroid glands, pituitary gland, preputial or clitoral gland, rectum, salivary glands, spleen, stomach, thymus, thyroid gland, trachea, and urinary bladder.
Statistics:
Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups for equality and Tarone’s (1975) life table test for a dose related trend. The primary statistical method for tumour analysis was a logistic regression analysis, which assumed that the diagnosed tumours were discovered as the result of death from an unrelated cause and thus did not affect the risk of death. The dosed and control groups were compared on the basis of the likelihood score test for the regression coefficient of dose. In addition to logistic regression, alternative
methods of statistical analysis were used and include the life table test (Cox, 1972; Tarone, 19751, appropriate for rapidly lethal tumours, and the Fisher exact test and the Cochran-Armitage trend test (Armitage, 1971; Gart et al., 1979), procedures based on the overall proportion of tumour-bearing animals. Tests of significance include pairwise comparisons of each dosed group with controls and a test for an overall dose-response trend. Continuity corrected tests were used in the analysis of tumour incidence. The procedures described above also were used to evaluate selected non-neoplastic lesions.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
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
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Relevance of carcinogenic effects / potential:
Not carcinogenic
Dose descriptor:
NOAEC
Effect level:
1 200 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: no substance related increases in any tumour type
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
Dose descriptor:
NOAEC
Effect level:
4 522 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: no substance related increases in any tumour type
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
Dose descriptor:
LOAEC
Effect level:
600 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: erosion of nasal epithelium
Remarks on result:
other: Effect type: toxicity (migrated information)
Dose descriptor:
LOAEC
Effect level:
2 261 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: erosion of nasal epithelium
Remarks on result:
other: Effect type: toxicity (migrated information)

Clinical signs and mortality

There were no compound related clinical signs and no significant differences in survival between any groups of either sex.

Bodyweight

The initial mean body weights of rats exposed at 1200 ppm were 9% greater than those of controls; in the early weeks of the studies, these differences were diminished. Mean body weights of male rats exposed at 1200 ppm were 4%-8% lower than those of controls from week 72 to the end of the study. Mean body weights of female rats exposed at 1200 ppm were 4%-7% lower than those of controls from week 92 to the end of the study.

Pathology

Erosion of the olfactory epithelium and degeneration of the respiratory epithelium were significantly (P < 0.05) increased in exposed rats (erosion of the olfactory epithelium--male: control, 0/50; 600 ppm, 3/50; 1200 ppm, 8/49; female: 2/49; 11/50; 10/50; degeneration of the respiratory epithelium--male: 15/50; 37/50; 31/49; female: 29/49; 45/50; 39/50). Inflammation of the nasal mucosa and respiratory metaplasia of the olfactory epithelium were observed at significantly (P < 0.05) increased incidences in exposed female rats (inflammation of the nasal mucosa: 27/49; 42/50; 41/50; metaplasia of the olfactory epithelium: 0149; 2/50; 6/50). This spectrum of lesions is not unusual in inhalation exposure studies of organic solvents, and the lesions were, for the most part, of mild severity.

Kidney: The severity of nephropathy was increased with exposure concentration in male and female rats. Renal tubule cysts were somewhat increased in male rats at 1200 ppm (control, 1/50; 600 ppm, 2/50; 1200 ppm, 5/50).

Forestomach: Ulcers were marginally increased in exposed male rats (control, 4/50; 600 ppm, 7/50; 1200 ppm, 9/49).

Conclusions:
Survival and tumour incidence were unaltered in male and female rats exposed to toluene vapour for up to two years. Non-neoplastic changes were found in the nasal cavity, forestomach and kidney. A small, not statistically significant, decrease in body weight was noted at 1200 ppm (4522 mg/m3), the highest dose tested.
Executive summary:

Groups of 50 male and 50 female F344N rats were exposed by inhalation to 0, 600, or 1200 ppm toluene (0, 2,261, 4522 mg/m3) 6.5 h/day, 5 days/week for two years.

Mean body weights of male and female rats exposed to 1200 ppm were slightly (4-8%) but not significantly decreased relative to controls at scheduled termination. No compound related clinical signs were recorded, and no significant differences in survival were observed between any groups of either sex. In the nose, erosion of the olfactory epithelium and degeneration of the respiratory epithelium were significantly increased in a dose related manner in exposed rats, while inflammation of the nasal mucosa and respiratory metaplasia of the olfactory epithelium were observed at significantly increased incidences in exposed female rats only. The lesions were for the most part of mild severity. In the stomach ulcers were marginally increased in exposed male rats. The severity of nephropathy was increased with exposure concentration in both sexes and was increased significantly in the high dose group. There were no substance-related increases in any tumour types.

A LOAEC for chronic toxicity of 600 ppm (2261 mg/m3) can be derived from this study. The NOAEC for carcinogenicity was 1200 ppm (4522 mg/m3).

There was no evidence that toluene is a carcinogen in animals and no classification for carcinogenicity is required.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
4 522 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
The available data provide information that is adequate for the purpose of hazard assessment

Carcinogenicity: via dermal route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline and GLP compliant, animal experimental study, published in peer reviewed literature, fully adequate for assessment.
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPP 83-2 (Carcinogenicity)
Deviations:
yes
Remarks:
Toluene was control material, applied twice per week only
Principles of method if other than guideline:
A total of 18 groups were tested, with 3 materials tested using toluene as vehicle. Toluene was included as a vehicle control group.
GLP compliance:
yes
Species:
mouse
Strain:
other: C3H/HeJ
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Jackson Laboratories, Bar Harbor, ME, USA
- Age at study initiation: 7-9 weeks
- Housing: double-housed in suspended stainless steel cages for a quarantine period, and then singly housed prior to study initiation.
- Diet: Purina Certified Rodent Chow 5002 ad libitum
- Water: deionized tap water ad libitum by bottle
- Randomisation: mice were randomized upon receipt

ENVIRONMENTAL CONDITIONS
- Temperature: 24.5 ± 1.7°C
- Humidity: 40 ± 10%
- Air changes (per h): no data
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: no data
Route of administration:
dermal
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: interscapular region of the back
- Coverage: at least 1 cm2
- Time intervals for shavings or clippings: approximately 2 weeks

TEST MATERIAL
- Amount(s) applied: pure toluene was applied topically two times per week in a volume of 50 µL
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Twice weekly
Frequency of treatment:
Lifetime
Post exposure period:
Not applicable.
Remarks:
Doses / Concentrations:
50 µL
Basis:
other: pure toluene
No. of animals per sex per dose:
50 males
Control animals:
yes
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
- A dermal lesion was diagnosed macroscopically as a tumour when the mass reached 1 mm in diameter.

DERMAL IRRITATION: Yes
- Time schedule for examinations: weekly
- Dermal irritation was evaluated by measuring incidence and estimating severity of erythema, alopecia, and scabbing

BODY WEIGHT: No data

HAEMATOLOGY: No data

CLINICAL CHEMISTRY: No data

URINALYSIS: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All mice were subjected to a limited necropsy upon death or euthanasia

HISTOPATHOLOGY: Yes
- Application site skin was removed and fixed in 10% neutral-buffered formalin. Skin sections were embedded in paraffin, sectioned, stained with haematoxylin and eosin, and examined microscopically
Statistics:
Tumours confirmed by histopathological examination were used in calculating incidence. Statistical methods included: Chi-squared test - test and control tumour incidence; Fisher's Exact Test (one-tailed) - comparing low-tumour incidence groups to the appropriate control. When median time to tumour was under 60 weeks, the number of animals alive at that time was used as the denominator. When median time to tumour was over 60 weeks, the number of animals alive at 60 weeks plus any mice dying with tumours before 60 weeks was used as the denominator. Latency was the time in weeks from start of dosing to appearance of the first tumour.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Relevance of carcinogenic effects / potential:
There is limited, weak evidence that toluene may induce skin irritation and malignant tumours in mice but this is not strong enough to fulfil the EU criteria for classification for carcinogenicity.
Dose descriptor:
NOAEL
Effect level:
50 other: µL applied twice weekly
Sex:
male
Basis for effect level:
other: no increase in tumour frequency
Remarks on result:
other: Effect type: carcinogenicity (migrated information)

Survival at 18 months was 86% in controls and 76% in the toluene-exposed group, and at 24 months the figures were 62% survival for controls and 52% for toluene-exposed mice. A histopathological evaluation was made on untreated and test site skin including dermal and subcutaneous tumours, and on all suspected dermal and systemic neoplasms from animals with life-time exposures.

Skin tumour incidence in untreated controls and toluene-treated group in a skin-painting study

Group

Site

Benign

Malignant

Tumour type

no treatment

test site

0

0

-

non test site

0

0

-

toluene

(life-time exposure)

test site

0

8%

1 fibrosarcoma (2%)

3 squamous cell carcinoma (6%)

non test site

4%

0

2 fibroma (4%)

Conclusions:
Not carcinogenic.
Executive summary:

The carcinogenic potential of toluene following dermal application was investigated in a study of 12 selected petroleum refinery streams where toluene was used as a vehicle. Groups of 50 male mice were dosed with 50 µL pure toluene twice per week for 24 months or entire lifetime.

Although histopathologically highly malignant tumours occurred at the test site in 4 toluene treated mice versus no malignancies in the control group, this finding was not statistically significant. No increase in tumours was apparent in another group given 0.1% catalytic cracked clarified oil in 99.9% toluene. Chronic irritation leading to tumour induction via an epigenetic (non-genotoxic) mechanism may be responsible for the tumours in the toluene vehicle control group.

It is concluded that toluene is not a skin carcinogen and no classification for carcinogenicity is required.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
Dermal application (50 µL, twice per week for up to 2 years) gave no increase in any tumour type.

Justification for classification or non-classification

Toluene is considered not to be carcinogenic and therefore does not warrant classification under GHS / CLP.

Additional information

LOA is currently reviewing the human and animal data supporting Human Health for Toluene. It is expected to be completed by Q4 2020.

Non-human information

Inhalation studies in rats and mice (Huff, 1990; Gibson and Hardisty 1983) and a supporting study in mice using skin application (Broddle et al, 1996), are considered relevant for the risk assessment. Inhalation exposures for 6 or 6.5 h/day, 5 days/week for up to 2 years at concentrations up to 1200 ppm (4522 mg/m3) were used. The dermal application study involved application of 50 µL toluene twice a week for up to 2 years. No statistically significant increase in any tumour type was seen in any study.

Human information

According to the EU RAR (2003), “the only useful epidemiological cancer study which has been found does not show an excess of tumours in toluene-exposed workers".  Toluene has also been evaluated by IARC in 1989 and 1999 and considered to be not classifiable as to carcinogenicity to humans.


Justification for selection of carcinogenicity via inhalation route endpoint:
Inhalation studies in rats and mice of up to 2 years duration, and using exposure concentrations up to 1200 ppm (4522 mg/m3), indicate no increase in tumours.