[No public or meaningful name is available]

Carcinogenicity

Description of key information

In the absence of any experimental studies conducted for renewable hydrocarbons of wood origin (diesel type fraction), the CLP mixture rules were applied to evaluate the carcinogenicity of the substance. The generic limit value in CLP regulation triggering the classification of mixtures to carcinogenicity (Cat 2) is 1 %. As the sum of PAH compounds detected in the substance is only 0.0015% (w/w) no classification is warranted.
Two dermal studies have been conducted where carcinogenicity potential of read-across substances (ten middle distillate fuels, marine diesel fuel) have been investigated in mice. These studies showed an evidence of tumorigenic activity. However, the chemical composition of these fuels is different than that of the renewable diesel. Especially, it is expected that the read-across substances contain carcinogenic PAH compounds that are responsible for the tumorigenic effects observed in the studies.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: dermal

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

This study is conducted for a series of read-across substances (i.e. middle distillate fuels). This study is considered as reliable with restrictions since the study was well conducted and acceptable, but no guideline was followed. Furthermore, the analysis for polycyclic aromatic hydrocarbons was not conducted for all test substances.

Justification for type of information:

Read-across justification: Based on the chemical composition, the renewable hydrocarbons produced from raw materials such as fatty acid rich oil like Crude Tall Oil (CTO) or triglyserides, using a hydrotreatment process have similar hydrocarbon fractions and they contain the same critical constituents than fossil diesel fuels. According to the identified hydrocarbon blocks, the typical carbon number ranges and the physicochemical properties, the renewable hydrocarbons with diesel type fractions can be considered as having structural similarities and similar behaviour in contact with water and in the physiological processes than the analogue source substances (fossil diesel fuels). Their irritation properties, skin sensitisation property as well as acute and long-term adverse effects to human health is similar. Therefore, and in order to avoid the unnecessary animal testing, the read-across data from the analogue fossil diesel fuels is used to evaluate skin and eye irritation, the genetic toxicity, carcinogenicity, developmental toxicity and short term and/or long-term toxicological effects of the target substance.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Compound was applied dermally 3 times a week for the lifespan of the animal (only male mice used) and animals were examined for dermal tumours. Animals were examined grossly for internal tumours at the end of the study period.

GLP compliance:

no

Species:

mouse

Strain:

other: C3H/HeJ

Sex:

male

Details on test animals or test system and environmental conditions:

Source: Jackson Laboratories, Bar Harbor, Maine
- Age at study initiation: 6 to 10 weeks old
- Weight at study initiation: Not reported
- Housing: Five per suspended wire mesh cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Not reported

ENVIRONMENTAL CONDITIONS
Only stated to be according to U.S. Department of Health, Education, and Welfare standards.

Route of administration:

dermal

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: Not reported
- % coverage: Not reported
- Type of wrap if used: None reported
- Time intervals for shavings or clippings: Once a week


REMOVAL OF TEST SUBSTANCE
- Washing (if done): None reported

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

lifetime

Frequency of treatment:

three times a week

Post exposure period:

none

Remarks:

Doses / Concentrations:
25 µL
Basis:

No. of animals per sex per dose:

40-50 animals per test group

Control animals:

yes

Details on study design:

The test materials were applied by automatic pipette in 25 µL aliquots. Test materials were applied 3 times/week for the lifetime of each animal or until all the animals in a test group developed grossly diagnosed carcinomas. All test materials were applied undiluted. Animals were observed daily for the appearance of dermal tumours. Each tumour in the treatment area was carefully examined and grossly classified. All grossly diagnosed tumours were examined microscopically following study termination. The final tumor listings were adjusted to reflect the histologic diagnoses, and each mouse was then classified on the basis of the most advanced tumor type in the treatment area. When skin tumors were cannibalized or otherwise lost prior to histologic examination, the gross diagnoses were used for tabulation and statistical analysis.

Positive control:

The positive control was a dilution of a catalytically cracked clarified oil (CCCO) (b.p. > 427°C(800°F)).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: daily observed for tumour development

DETAILED CLINICAL OBSERVATIONS: No data

DERMAL IRRITATION (if dermal study): Yes, but details were not provided

BODY WEIGHT: No data

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Animals were observed daily for the development of skin tumours.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, skin tumours, skin from the treated area, abnormal tissues, brain, heart, lungs, spleen, kidneys. liver, cervical and mesenteric lymph nodes.

Statistics:

The Weibull distribution function was used to estimate the median tumor latencies in each of the study groups. The observational parameter which formed
the basis of the median latency estimates was the time to onset of the first squamous cell tumor (either benign or malignant) for each animal. Statistical evaluation was limited to squamous cell carcinomas, squamous cell papillomas, and keratoacanthomas which arose at the site of test material application. Mortality rates were assessed by the product limit method. Tumor yields were compared by the Fisher's Exact Test.

Clinical signs:

no effects observed

Description (incidence and severity):

Mean survival estimates were not different from the concurrent negative controls

Mortality:

no mortality observed

Description (incidence):

Mean survival estimates were not different from the concurrent negative controls

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

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Tumor yields in 8/10 treatment groups were significantly increased from the control.

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Metastatic tumors were occasionally observed.

Details on results:

CLINICAL SIGNS AND MORTALITY: There are no data on the clinical signs. There were no effects on the median survival time in any of the treatment groups. However, treatment with the positive control significantly reduced the median survival time.

GROSS PATHOLOGY: Tumor yields in all treatment groups were statistically significantly increased from the control except in the groups treated withthe virgin heating oil blending oil with 59% catalytically cracked middle distillate and the light catalytic cycle oil (100% catalytically cracked).

The majority of the treated mice had mild to moderate epidermal thickening and hyperplasia, mild to moderate hyperkeratosis, and moderate to markeddermatitis. In addition, many of the animals exhibited epidermal degeneration and necrosis. For most of the treatment groups the incidence and severity of dermal irritation was greater than in the negative controls.

HISTOPATHOLOGY: NEOPLASTIC: Metastatic tumors were occasionally observed. It is evident that tumors of various types were noted, but, in general these appeared sporadically and except for hepatocellular carcinomas, in low frequency.

Relevance of carcinogenic effects / potential:

Compounds were applied dermally 3 times a week for the lifespan of the animal. The tumour yields in 8/10 middle distillate treatment groups were significantly increased from the control.

Dose descriptor:

other:

Effect level:

25 other: µL

Based on:

test mat.

Sex:

male

Basis for effect level:

other: skin tumour development

Remarks on result:

other: Effect type: carcinogenicity

Conclusions:

Middle distillate fuels have a weak carcinogenic potential as demonstrated by the low tumour yield and the long latent periods.

Executive summary:

The carcinogenicity study is conducted for read-across substances (i.e. middle distillate fuels). The study is considered as reliable with restrictions since the study was well conducted and acceptable, but no guideline was followed.

Compounds were applied dermally 3 times a week for the lifespan of the male mice and animals were examined for dermal tumours. Animals were also examined grossly for internal tumours at the end of the study period. It was found that repeated application of middle distillates can produce tumors in mouse skin. The responses were generally weak as characterized by low tumor yields and long median latencies. The tumor responses did not appear to have been directly related to aromatic carbon content in general or, more specifically, to the presence of PAH. It is likely that the irritating properties of the test samples did play a role in the tumorigenic process; however, the actual mechanism of tumor formation is unknown. The positive and negative control materials responded as expected, confirming the validity of the assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Study duration:

chronic

Species:

mouse

Justification for classification or non-classification

In absence of any experimental studies conducted for renewable hydrocarbons of wood origin (diesel type fraction), the CLP mixture rules were applied to evaluate the carcinogenicity. Based on the composition of the substance and the results of the PAH analysis no classification is warranted for the target UVCB substance.

Additional information

In general, the carcinogenic potential of petroleum-derived material is related to the polycyclic aromatic hydrocarbon (PAH) content. PAHs are one of the most widespread organic pollutants being present also in fossil fuels. Several PAHs are known for their carcinogenic, mutagenic and teratogenic properties. The EPA has classified seven PAH compounds as probable human carcinogens: benz(a) anthracene, benzo(a) pyrene, benzo(b) fluoranthene, benzo(k) fluoranthene, chrysene, dibenz(a, h) anthracene, and indeno(1,2,3-cd) pyrene. The carcinogenicity of these PAHs is well established in laboratory animals. Researchers have reported increased incidences of skin, lung, bladder, liver, and stomach cancers, as well as injection-site sarcomas, in animals. Animal studies show that certain PAHs also can affect the hematopoietic and immune systems and can produce reproductive, neurologic, and developmental effects.

The PAH analysis was conducted for renewable hydrocarbons of wood origin (diesel type fraction) to investigate whether the substance contains carcinogenic PAH compounds. The following PAHs were analysed: naphthalene, 1-methylnaphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, dibenzo(a,h)anthracene, benzo(g,h,i)perylene and indeno(1,2,3,c,d)pyrene. The results show that no other PAHs than naphthalene (0.0015% (w/w)) and pyrene (0.00034% (w/w)), were detected in the substance. Naphthalene is identified as carcinogenic PAH whereas pyrene is not. The detection limit of the analysis for an individual PAH compound was 0.00024 % (w/w). AS naphthalene has a harmonized classification for Carcinogenicity Category 2 with general concentration limit of c ≥ 1 % set in CLP Annex VI no classification for carcinogenicity is warranted.

Furthermore, benzo(a) pyrene, that is the first chemical carcinogen to be discovered, is found generally in both fossil gasoline and diesel fuel. The substance has been shown to be carcinogenic to experimental animals and is presumed to have carcinogenic potential for humans. The substance has harmonized classification as Carc. Category 1B with specific concentration limit of c ≥ 0.01 % set in CLP Annex VI. However, in the PAH analysis conducted for renewable diesel no benzo(a)pyrene was detected.

There is available one carcinogenicity study by Biles et al. (1988) conducted for ten read-across fossil fuels (i. e. middle distillate fuels) showed weak carcinogenicity. The study is considered as reliable with restrictions since the study was well conducted and acceptable, but no guideline was followed. The test compounds were applied dermally 3 times a week for the lifespan of the male mice and animals were examined for dermal tumours. Animals were also examined grossly for internal tumours at the end of the study period. It was found that repeated application of middle distillates can produce tumours in mouse skin. The responses were generally weak as characterized by low tumour yields and long median latencies. It is likely that the irritating properties of the test samples did play a role in the tumorigenic process; however, the actual mechanism of tumour formation is unknown. The positive and negative control materials responded as expected, confirming the validity of the assay.

Carcinogenic evaluation has been performed for another read-across substance, i.e. marine diesel fuel (NTP/NIH, 1986 and cited by ATDSR, 1995). In this study an increased incidence of squamous cell papilloma and/or carcinoma was observed in mice exposed dermally to 250 or 500 mg/kg/day marine diesel fuel. Furthermore, hepatocellular adenoma and carcinoma were noted in male, but not in female mice exposed to 250 or 500 mg/kg/day marine diesel fuel. This study is reported in the section 5.6.1.3. Repeated dose toxicity: dermal.

Weak tumorigenic responses observed in both studies conducted for fossil diesel fuels and summarised above may be due to the different composition compared to the renewable diesel. Especially, it is expected that PAH content is higher in fossil diesel fuels than in renewable diesel.

Justification for selection of carcinogenicity via oral route endpoint:
No oral studies conducted for the substance itself and no studies available from the read-across substances with respect to carcinogenicity. Evaluation of carcinogenicity endpoint was based on the negative genoxicity test results and composition of the substance and the concentrations of the carcinogenic PAH-compounds in the substance.

Justification for selection of carcinogenicity via inhalation route endpoint:
No inhalation studies conducted for the substance itself and no studies available from the read-across substances with respect to carcinogenicity.Inhalation is not considered as relevant route of exposure. Evaluation of carcinogenicity endpoint was based on the negative genoxicity test results and composition of the substance and the concentrations of the carcinogenic PAH-compounds in the substance.

Justification for selection of carcinogenicity via dermal route endpoint:
No carcinogenicity study conducted for renewable hydrocarbons of wood origin (diesel type fraction). The selected study is reliable study that evaluated carcinogenic potential of read-across substances (i. e. middle distillate fuels) similar to the target substance. The dermal route is considered the most relevant route of human exposure.