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Carcinogenicity

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Description of key information

The role of irritation needs to be carefully evaluated in assessing the carcinogenic potential of straight run gas oils.  The CONCAWE middle distillate program indicates that in the absence of dermal irritation, tumours are not observed with a straight run hydrotreated gas oil.  The assessment by API indicates that tumours are observed in the presence of moderate to severe dermal irritation.  An initiation/promotion assay with a straight run gas oil sample, indicates that non-genotoxic mechanism is likely involved in the development of tumours.

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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
A number of studies are available. Key information to support a non-genotoxic meachanism for dermal tumpours can be found in Concawe 1996

Justification for classification or non-classification

Prolonged exposure to straight run gas oil can result in severe dermal irritation. This repeated dermal damage can result in the development of dermal tumours. In the absence of any irritation, tumours are not observed. Therefore, straight run gas oils do not meet the requirements for classification as a carcinogen as laid down in the EU CLP Regulation (EC No. 1272/2008).

Additional information

The carcinogenicity of gas oils have been investigated in a number of different studies. The American Petroleum Institute (API) reported on skin cancer studies on crude oil fractions which showed that atmospheric distillates boiling between 49-371°C (light and middle distillates) could produce skin tumours in mice after repeated application (King R. W. et al, 1984; Lewis S. C. et al, 1984). Some of the fractions tested contained polycyclic aromatic compounds (PACs) and it was thought that these constituents might have been the cause of the tumour development, acting via a genotoxic mechanism. Other fractions however e. g. straight-run kerosine and naphtha, had either very low or undetectable amounts of PACs and it was considered unlikely that a genotoxic mechanism was responsible for the tumour development with these samples. Severe skin irritation was noted in the studies reported by the API and it was thought that this was likely to have influenced skin tumour development.  Additional studies have been conducted on gas oils,but in most of the studies interpretation of the results has been confounded by the extent of skin irritation that has occurred during the studies. Therefore studies have been carried out to investigate the influence of skin irritation on the development of tumours in mouse skin painting studies. The results of these studies have been summarised below.

In order to help elucidate the interaction between PAC content, skin irritation and skin carcinogenicity, CONCAWE initiated a middle distillate research program described below.

The middle distillate programme was undertaken to investigate the role of skin irritation in tumour development during long term dermal studies in mice. The programme was conducted in three phases.

Phase 1 involved screening studies on 10 middle distillates. These included the determination of physico-chemical properties and PAC (polycyclic aromatic compound) concentrations, short-term in vitro tests mainly investigating mutagenicity and detailed histopathological examination of mouse skin following repeated dermal application of the distillates. These studies suggest that mutagenic activity is related to PAC content. In particular, relatively high mutagenic activity was evident in a cracked gas oil sample, whereas, all of the kerosine samples which contained low levels of PACs were not mutagenic. Further it was shown that the middle distillates were irritant when applied repeatedly to mouse skin.

In phase 2, five middle distillates were evaluated in dermal studies of up to 90 days duration in mice to establish dilution conditions that would enable repeated application of middle distillates without causing significant skin irritation. It was found that use of a mineral oil diluent enabled daily application of a ‘standard’ amount of middle distillates with no more than slight irritation. Experiments with radiolabelled hydrocarbons showed that skin penetration was not reduced under these conditions. A regime was devised which enabled animals to be tested under conditions producing different degrees of skin irritation but at the same total weekly dose of middle distillate over the course of the study.

Once the preliminary investigations had been conducted to determine the physico-chemical properties of the materials and the doses that could be used while avoiding irritation, three middle distillates were evaluated in a two year mouse dermal carcinogenicity study [Dally et al, 1996]. Selected for the investigation were a straight run kerosine, and two gas oils: a straight run hydrotreated gas oil (from Other Gas Oils) (CAS No. 64742-46-7; MD6) and a catalytically cracked light cycle oil (from Cracked Gas Oils) (CAS No. 64741-59-9; MD7). These were tested alongside a negative control (highly refined, non-irritating mineral oil) and a positive control treatment (heavy clarified oil (HCO), 5% in mineral oil). For the study, groups of 50 male C3H/HeNCrlBR VAF/Plus® mice were administered various test (28.5%, 50%, or 100%) or control materials applied regularly to clipped dorsal skin. The treatment regime established in phase 2 was designed to deliver a constant total weekly dose of each distillate while varying the extent of any local skin irritation by manipulating test material concentration and frequency of application.

In these studies: undiluted straight run kerosine and straight run gas oil caused marked irritation and a number of tumours at the treatment site. The magnitude of response (ranked as slight, moderate or marked on a numerical scale) was negligible-slight in the negative controls (mean 0.06) and also for animals treated with 28.5% or 50% MD6 (0.02 or 0.09, respectively), with moderate irritation present after application of 100% MD6 group (score 2.0). In contrast, concentration-dependent increases in dermal irritation were observed in all of the MD7 treatment groups (0.28, 1.59, 2.40). However, the cracked gas oil (LCO) produced tumours at all three application rates. Undiluted LCO produced marked skin irritation whereas there was moderate irritation with the 50% dilution and slight irritation with the 28.5% dilution. It is concluded that in long-term dermal studies in mice, cracked gas oils containing significant concentrations of 3 to 7 fused ring PACs probably produce skin tumours by a genotoxic mechanism, independent of irritation. Under the same conditions, undiluted straight-run hydrotreated gas oils and kerosines containing undetectable or low concentrations of PACs eventually produce tumours, but only when moderate to severe skin irritation is also present. These tumours are probably the consequence of a continuous cycle of cell damage and repair caused by chronic skin irritation.

For the straight run kerosine, skin tumours only developed in the group of animals in which substantial skin irritation occurred during the study. Since no PACs were detected in the straight run kerosine it is concluded that the occurrence of tumours is likely to have been caused by a non-genotoxic mechanism. This conclusion is consistent with reports by others (Ingram, A. J. and Grasso, P., 1991) that lighter middle distillates are tumour promotors but not initiators and furthermore that skin irritation plays an important role in skin tumour development.

For the straight run hydrotreated gas oil(MD6), which had a low polycyclic aromatic compound content (1.3%), skin tumours only developed in the animals in which substantial skin irritation occurred during the study. This material caused a significant increase in the number of tumours only in the presence of skin irritation, and it is thus concluded that the mechanism is likely to be non-genotoxic. Again, this finding is consistent with results reported previously (Jungen, H. et al, 1993) that gas oils which contain undetectable or low levels of PACs may be tumour promotors but do not possess tumour initiating activity. This is further supported by the results of the supporting IP study. This contrasts with the positive control material, diluted HCO, which had a high polycyclic aromatic compound content and where a relatively short time to tumour formation was recorded.

The catalytically cracked light cycle oil LCO (MD7) contained measurable quantities of PACs and caused the development of skin tumours even at dose levels which did not cause appreciable skin irritation. For this material it is concluded that tumour development was due to a genotoxic mechanism. It was surprising that fewer tumours developed in the group of mice receiving the highest concentration of cracked gas oil. However, this group also showed the most severe skin irritation and it is possible that undiluted cracked gas oil caused a sufficient degree of cytotoxicity to inhibit cell repair and possibly epidermal necrosis destroying initiated cells therefore inhibiting skin tumour formation.

It is concluded that frequent application of middle distillates that contain undetectable or low levels of PACs to mouse skin will produce skin tumours if accompanied by moderate to marked skin irritation. The mechanism is not considered to involve a direct genotoxic process, but rather may result from frequent cell damage and repair. When irritation does not occur, these same middle distillates do not give rise to tumours.

There were no significant treatment-related gross findings at necropsy other than skin irritation and masses (tumours) at the treatment site. Benign and/or malignant skin tumours were diagnosed at the application site in several of the groups, and were frequent in the positive control group where a total of 164 tumours (88 benign, 73 malignant) developed in 47 mice with 217 days to first tumour. This contrasts with the negative control group which was tumour free. Tumours (predominately papillomas or squamous cell carcinomas) occurred in all groups treated with MD7, affecting 1, 17 and 7 mice from the high, intermediate or low dose groups respectively with a time to first tumour of approximately 300-650 days. In contrast, tumours were relatively infrequent in mice treated with MD6 with a total of 6 tumours in 4 mice treated with undiluted distillate, and a single squamous cell carcinoma in the low dose group.

The findings from the above study are further supported by Nessel et al 1999a who state that middle distillate fuels containing limited amounts of PNAs result in tumor formation secondary to chronic skin irritation via a nongenotoxic mechanism (Nessel, 1999a). 

In another study reported by API (1989) and also by Broddle et al (1996), the dermal carcinogenicity of several refinery streams, including one straight run gas oil (straight run middle distillate; CAS 64741-44-2; with a boiling range 186-391 °C), was investigated in mice. No information was provided on PAH levels for this sample. The neat test material (50µl) was applied, twice a week for 24 months, to the shaved skin of the interscapular region of 50 male mice of the C3H/He strain. In addition to test material, sham controls, controls treated with toluene and positive controls treated with 0.01% or 0.05% benzo(a) pyrene in toluene were included. Examinations for skin irritation and tumours were made weekly. At termination animals were subjected to gross pathological examination and the skin of the treatment site was retained for histopathological examination. Any suspected tumours in internal organs were also examined histologically

Significantly reduced survival was seen in all test groups however as there was a clear increase in skin tumour incidences this did not affect the study outcome. Although various tumours were found in non-dermal tissues none of them were reported to be related to treatment.

Virtually no dermal lesions were seen in untreated animals, but 80-100% of animals of the toluene control group showed some signs of skin irritation in the form of mild desquamation with sporadic low incidence observations of alopecia, irritation and scabbing. The dermal lesions with the straight run middle distillate were slightly more severe than those produced by toluene with mild-moderate irritation being seen in a maximum of 60% of animals There was elevated mortality at 18 months compared with the sham control group, which showed a survival at 18 months of 96% or 86% at 24 months. No tumours were found in either the sham control groups or the 24 month toluene control group. Twenty percent of mice treated with the straight run middle distillate showed malignant tumours, while 0% showed benign tumours. 

The only conclusions drawn by the authors with respect to the straight run gas oil was that it was dermatotoxic. The modest tumour incidence with a long latent period suggests that the straight run middle distillate may have produced tumours by a non-genotoxic mechanism.

A dermal initiation/promotion study was conducted on, amongst others middle distillates, a straight run gas oil (MD-57 324) (Jungen et al., 1993; Klimisch score=1).  During the initiation phase tumours were observed in 2 out of 30 animals and in 1 out of 30 animals during the promotion phase. Only information on the straight run gas oil is summarised here (CAS# 64741-87-9; MD 57 324; dodecane content 2.26 g/100g and nitrogen content 22 mg/L).

In the initiation phase, the gas oil sample (50 microlitres) was applied undiluted to clipped dorsal skin (including lumbar and sacral regions) of 30 male CD-1 (ICR) BR mice (Charles River Wiga GmbH; approximately 7 weeks old) for 5 consecutive days during the first week of treatment. Following a two-week rest, TPA (12-0-tetradecanoylphorbol-13-acetate) was applied twice a week (5 micrograms/mouse in 50 microlitres of acetone) from week 4 to week 28. A single application of DMBA (50 microlitres/mouse in 50 microlitres of acetone, given on day 1 of the study) followed by TPA treatment was used as a positive control. Another group, treated during the initiation week with acetone (50 microlitres) (uninitiated mice), followed by application of TPA served as a TPA control.

In the promotion phase, mice were initiated with DMBA (50 microlitres/mouse in 50 microlitres of acetone) on the first day of treatment and, following a rest period, the test site treated with undiluted gas oil (50 microlitres) twice per week from week 4 to week 28. TPA (5 micrograms/mouse in 50 microlitres of acetone) applied to DMBA initiated mice, served as a positive control. An initiation only control group was given DMBA at the start of the study and treated with acetone during the promotion phase.

 

Clinical observations during the initiation study revealed that daily treatment with gas oils was associated with slight to moderate skin irritation in the majority of groups during week 1 of the initiation study. All skin changes were resolved during the 2 week interval between the initiation and promotion phase. Slight to moderate irritation was also present at the test site during the promotion phase of the initiation study. Signs of skin irritation were also seen in animals treated with TPA, however less pronounced reactions were observed with the straight run gas oil.

Various histopathological changes were noted at the application site during both the initiation- and promotion phases of the investigation. Hyperkeratosis was commonly present, most often in the positive control group (DMBA/TPA). Hyperplasia (acanthosis) occurred in the positive control group. Dermal inflammation was the third most prevalent histopathological finding, found in the majority of groups, but occurring at high frequency in mice treated with MD 57324. Parakeratosis, crust formation, ulceration and other changes were also present especially when skin irritation was marked.

In the initiation study, papillomas were observed in all of the positive controls and in 2 out of 30 mice treated with MD 57324. All tumours were benign and showed various intensities of keratinisation on the tumour surface. In the promotion study squamous cell papillomas were diagnosed in animals promoted with MD 57324 (1/30, 3%). All tumours were benign and showed varying degrees of keratinisation.

Based upon the data that was collected the authors concluded that for some of the middle distillates tested a genotoxic mechanism of action is likely, and they also conclude it is likely that there are middle distillates, such as MD 57324 for which other mechanisms may be important. This supports the position that irritation likely plays an important role in determining the mechanism of action and the observed weak initiating activity and borderline promoting activity of the straight run gas oil sample is of lesser importance. 

Additional data supports that straight run gas oils are not carcinogenic (Biles et al., 1998). This information is presented in the dossier. The dermal carcinogenicity of 10 middle distillate samples, including a straight run gas oil with a boiling range of 142-307 °C, was tested in this study. The results obtained with the test material are: median survival of 75 weeks, 7/50 animals bearing tumours (14%), 58 days to first tumour, and median time to first tumour of 110 days. Clear evidence of skin irritation/skin injury was seen with all the test materials including hyperplasia, hyperkeratosis, dermatitis, epidermal degeneration and epidermal necrosis. Tumour incidences in internal organs were reported to be sporadic and, except for hepatocellular carcinoma, to be of low frequency. No treatment related increase in tumours in internal organs was reported. It was noted by the authors that there was a significant increase in skin tumour yield with the majority of samples tested, but that non-neoplastic dermal changes including hyperplasia may have contributed to the tumourigenic response. 

Justification for selection of carcinogenicity via dermal route endpoint:

This is one of a number of studies available that investigate the dermal carcinogenicity of straight run gas oils.The weight of evidence suggests that  straight-run gas oils, with low levels of PACs, are not genotoxic carcinogens. Tumours are not formed in the absence of significant dermal irritation.

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