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Carcinogenicity

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Description of key information

No specific carcinogenicity data are available on any of the streams identified for this category or the C1-C4 alkanes. However there are substantial data on one of the main constituents, propene.The stream studies show negative results. However, there are substantial data on the genotoxicity of some of the specific constituents present in some streams. Streams that contain ≥ 0.1% benzene are considered to be mutagenic and will require classification for this end-point. Benzene and 1,3–butadiene have been identified as potential specific constituents present in some streams and these have been shown to be carcinogenic in animals and man. Presence of >0.1% benzene and 1,3 -butadiene will trigger classification.

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Animal experimental study which predates implementation of GLP and development of study guidelines. Published in peer reviewed literature, limitations in reporting but otherwise adequate for assessment.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
no
Remarks:
Study initiated in 1977 and completed 1980. A third party quality assurance audit of the report was completed by Tracor Jitco, Inc, Rockville, Maryland 20852, USA
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS: Fischer-344 rats [CDF (F-344)/CrlBr]
- Source: Charles River Breeding Labs, Wilmington, Mass., USA
- Age at study initiation: approximately 6-7 weeks
- Housing: individually in stainless steel open-mesh cages (6 x 7.5 x 7.5 inch) in a single layer midlevel in each inhalation chamber
- Diet: Wayne Lab Blox (Allied Mills, Chicago, Ill., USA) ad libitum except during exposure
- Water: tap water ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS IN INHALATION CHAMBERS
- Temperature: 22°C
- Humidity: 50%
- Air changes: approx 15/hr
Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Four 8 m3 glass and stainless-steel chambers
- Chambers were supplied with absolute and charcoal filtered air separate from the room air supply
- Ethylene chambers were maintained at slight subatmospheric pressure (0.1-0.5 in. H2O) to avoid any escape of the gas
- The control chamber was under slight positive pressure (0.1-0.5 in. H2O) to minimize any possibility of ethylene entering
- Air flow rate: approximately 15 air changes per hour
- Temperature, humidity, pressure in air chamber: 22°C and 50%. Ethylene gas was metered via heated pressure regulators and precision rotameters into the air supply duct of each test chamber.

TEST ATMOSPHERE
- Brief description of analytical method used: infrared spectrophotometry
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of ethylene in each chamber was sampled every 24 min. with a Miran 1A infrared spectrophotometer. The analytical concentration during each exposure was interpolated from the standard curve. After 526 exposure periods the time weighted average concentrations of ethylene were 0, 301, 1003 and 3003 ppm.
Duration of treatment / exposure:
106 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Post exposure period:
None
Remarks:
Doses / Concentrations:
0, 300, 1000, 3000 ppm
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
0, 301, 1003, 3003 ppm
Basis:
other: calculated time weighted average concentration
No. of animals per sex per dose:
120
Control animals:
other: yes, air exposed
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once/day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Every 2 weeks

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for 1st 6 months, every 2 weeks thereafter

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to sacrifice
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 6, 12, 18 and 24 months
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 5/sex/group at 6 and 12 months, 10/sex/group at 18 and 24 months.
- Parameters examined: Haemoglobin, haematocrit, total erythrocyte count, total and differential leukocyte counts, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 6, 12, 18 and 24 months
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 5/sex/group at 6 and 12 months, 10/sex/group at 18 and 24 months.
- Parameters examined: Serum urea nitrogen, serum glutamic-pyruvic transaminase (alanine aminotransferase), and serum alkaline phosphatase.

URINALYSIS: Yes
- Time schedule for collection of urine: 6, 12, 18 and 24 months
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Appearance, specific gravity, protein, albumin, pH, ketones, glucose and microscopic appearance.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
TIME OF SCHEDULED TERMINATION: 5/sex/group from each group at 6 and 12 months, 20/sex/group at 18 months, and 63 to 80 animals/sex/group at 106 weeks were killed using carbon dioxide and necropsied. All unscheduled deaths were necropsied as soon as they were found.

GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: Yes. Brain, heart, kidneys, liver, lungs, and gonads.

HISTOPATHOLOGY: Yes. The following tissues were collected and fixed in 10% neutral buffered Formalin: adipose tissue, adrenal glands, aorta, bladder, bone marrow, brain, ear canal, heart, kidneys, large intestine, liver, lumbar, sacral, and dorsal autonomic ganglia, lungs, lymph nodes, mammary gland, nasal turbinates, oesophagus, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve, pituitary gland, prostate, proximal and distal portions of the hind limb, salivary gland, skeletal muscle, skin, small intestine, spinal cord, spleen, stomach, thymus, tibial and plantar nerves, trachea, and uterus. Eyes and testes were examined and then fixed in Bouin's solution. All fixed tissues from the high dose and control animals were examined histopathologically.
Statistics:
Analysis of variance (Kruskal-Wallis test for ratios) was used to test absolute body weight, total body weight change, organ weights, organ weight/body weight and organ weight/brain weight ratios. Where appropriate, follow-up analysis was performed using multiple comparison techniques including Tukey's for equal-sized groups and Scheffe's for groups of unequal sizes. Haematology data were tested for homogeneity of variance, transformed as needed, and analyzed by analysis of variance.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Relevance of carcinogenic effects / potential:
No evidence of carcinogenicity.
Key result
Dose descriptor:
NOAEC
Effect level:
3 000 ppm
Sex:
male/female
Basis for effect level:
other: no clinical or histopathological effects
Key result
Dose descriptor:
NOAEC
Effect level:
3 445 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: no clinical or histopathological effects

Of the 960 animals used for the study, 151 unscheduled deaths occurred and were distributed evenly throughout the groups. At study termination there were 71 males and 74 females, 68 males and 73 females; 64 males and 80 females and 63 males and 75 females for the 0, 300, 1000 and 3000 ppm groups respectively.

No treatment-related clinical observations were seen. There was no ethylene-induced effect on body weights, organ weights, organ to body weight ratios, or organ to brain weight ratios. The mean body weight for male and female rats exposed to 300 ppm and 3000 ppm for 24 months was significantly increased (p<0.05) as compared to controls; however, this was considered not to be treatment-related. A variety of proliferative, degenerative and inflammatory lesions were observed but occurred with approximately equal frequencies in the control and ethylene-exposed groups and were typical for the F344 rat. Haematology, blood chemistries and urinalysis were all similar to control. There were no statistically significant increases in overall or individual tumour incidences in treated animals relative to controls.

 

Conclusions:
There was no evidence of carcinogenicity in male and female F344/N rats exposed to ethylene by inhalation at concentrations of 0, 301, 1003 and 3003 ppm for 103 weeks. The NOAEC for carcinogenicity was 3003 ppm (equivalent to 3445 mg/m3).
Executive summary:

Groups of 120/sex Fischer 344 rats were exposed to 0, 300, 1000, or 3000 ppm ethylene, 6 h/day, 5 days/week for up to 24 months (average calculated time-weighted concentrations 0, 301, 1003, and 3003 ppm, respectively). Ophthalmology, haematology and clinical chemistry (blood and urine) assessments were made. After 6, 12, and 18 months groups of animals were killed and examined. A complete selection of tissues and organs from all animals in the control and 3000 ppm groups were examined for microscopic lesions.

 

There were 151 unscheduled deaths (15.7% of animals) but no evidence of a treatment-related effect on mortality. There were no gross lesions that were considered treatment-related and histopathological examination revealed a variety of lesions in both the control and 30000 ppm groups, but they were considered typical of those seen in this strain of animal and not related to ethylene exposure. The results provide no evidence that chronic exposure to ethylene at these concentrations, up to 3000 ppm, causes chronic toxicity or is oncogenic in Fischer-344 rats.

The results provide no evidence that repeated exposure to ethylene at these concentrations causes chronic toxicity or is oncogenic in Fischer-344 rats. NOAEC = 3003 ppm (equivalent to 3445 mg/m3).

This was a non-GLP study which predated implementation of study guidelines but it was regularly monitored by independent scientists and the results were subsequently published in peer reviewed literature. Concerns were expressed in the Agrochemical Draft Assessment Report (DAR) in 2008 (fourth stage review under Council Directive 91/414/EEC) specifically over the interpretation of the findings in the study pertaining to mononuclear cell leukaemia (MCL; not detailed in the publication). Subsequent to the publication, the incidence of MCL was discussed in a personal communication to the ACGIH (Swenberg J, Personal Communication July 28 2003). The author recognised the incidence of MCL in males in this study rose from 14% to 22% at the high dose (3000ppm), while in females the respective percentages were 9% (control) and 12% (3000 ppm). However historical data on MCL in inhalation studies suggests the average incidence for MCL in males for the same period was 57% (with a range of 34-70%), while for females was 37% with a range of 24-54%). The author reports that MCL was not significantly increased in this study and that the control groups were unusually low. The personal communication also details re-analysis of the lung pathology slides; this reanalysis confirmed the lung tumours were of spontaneous origin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
3 445 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
Adequate for assessment

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

There is no evidence that ethylene is carcinogenic. Ethylene does not warrant classification for carcinogenicity under CLP.

Members and main constituents of this category, including streams that contain <0.1% benzene and 1,3-butadiene, are considered not to be carcinogenic and no labelling is required CLP. However streams that contain ≥0.1% benzene or 1,3-butadiene are likely to be carcinogenic to animals and humans. Some streams are already listed in Annex VI of CLP and are classified Carc 1B:H350 (May cause cancer) (see Section 3). It is proposed that all remaining streams that contain ≥0.1% benzene or 1,3-butadiene are classified Cat1A: H350 (May cause cancer).

Additional information

There are no specific carcinogenicity data are available on any of the streams identified and also no carcinogenicity studies for any of the C1-C4 alkanes in the Other Petroleum Gases category. However, a consideration of their simple chemical structures, which have no reactive groups and carry no alerts for likely genotoxic carcinogenic activity from established Structure Activity Relationship analysis (Tennant RW and Ashby J (1991). Classification according to chemical structure, mutagenicity to Salmonella and level of carcinogenicity of a further 39 chemicals by the US National Toxicology Program.  Mutat Res 257 (3) 209-227), together with the conclusion that C1-C4 alkanes are not genotoxic, provide a strong case for concluding that none will show any significant carcinogenic activity. 

There are data forthe following constituents of this category:

Propene

There was no evidence of carcinogenicity in male and female F344/N rats or in male and female B6C3F1 mice exposed to propene by inhalation at concentrations of 5000 or 10000 ppm (8,600 or 17,200 mg/m3), for 103 weeks (NTP, 1985), or in the supporting study in male and female Swiss mice at concentrations up to 5000 ppm (8,600 mg/m3), following exposure for 78 weeks, or in male and female Sprague-Dawley rats at concentrations up to 5000 ppm (8,600 mg/m3), following exposure for 104 weeks (Ciliberti et al, 1988).

Butene isomers (butenes)

Carcinogenicity studies carried out on 2-methylpropene in rats and mice were reported to produce an increase in thyroid follicular cell tumours (NTP 1998). The thyroid tumours occurred only in male rats at the highest exposure concentration (8000 ppm: 18,359 mg/m3) at an incidence slightly above the laboratory historical incidence in control animals and no tumours were observed in female rats or male and female mice. As the butene isomers are not genotoxic, if 2-methylpropene did cause an increase in thyroid tumors in male rats, a threshold mechanism is likely to be involved and the relevance of tumours of this type to human health is low (IARC, 1999).

Ethylene

The toxicity and oncogenicity of inhaled ethylene was determined in Fischer-344 rats. Groups of 120 of each sex were exposed 6 hr/day, 5 days/week, for up to 24 months to concentrations of 0, 300, 1000 or 3000 ppm ethylene. The calculated time-weighted average concentrations for the 24 months of exposure were 0, 301, 1003, and 3003 ppm, respectively. Animals were investigated for ophthalmological or haematological effects and for clinical chemistry effects on blood and urine. After 6, 12, and 18 months, groups of animals were necropsied and examined. A complete selection of tissues and organs from all animals in the control and 3000 ppm groups were examined for microscopic lesions.

There were 151 unscheduled deaths (15.7% of 960 animals). There was no difference in mortality across groups during the 2-year study. Gross examination of rats dying during the study and those killed as scheduled, did not reveal any lesions attributable to ethylene exposure. Histopathologically, whilst a variety of lesions were observed in both the control and 3000-ppm groups, they were considered typical of those seen in this strain of animal and not related to ethylene exposure.

Overall there was no evidence that repeated exposure to ethylene at concentrations up to 3000 ppm caused chronic toxicity or was carcinogenic in Fischer-344 rats. The NOAEC is 3000 ppm (equivalent to 3445 mg/m3).

Summary:

No specific carcinogenicity data are available on any of the streams identified for this category or the C1-C4 alkanes. However there are data on one of the main constituents, propene, to indicate that members of this category have low potential for carcinogenicity. Propene, like the C1-C4 alkanes, is also considered to be non-genotoxic both in vitro and in vivo, and furthermore, has carcinogenicity data in animals that provides evidence of non-carcinogenicity.

Taking all these data into account, together with the general lack of toxicity across other endpoints, it is considered that there is no justification for conducting further animal carcinogenicity studies. The above reasoning leads to the conclusion that Other Petroleum Gases category can be considered to have low concern for human carcinogenicity.

However, benzene and 1,3–butadiene have been identified as potential specific constituents present in some streams and these have been shown to be carcinogenic in animals and man:

Benzene

(Classification: CLP - Category 1A, H350): Long term experimental carcinogenicity bioassays have shown that benzene is a carcinogen producing a variety of tumours in animals (including lymphomas and leukaemia). Human epidemiological studies indicate a causal relationship between benzene exposure and acute non-lymphatic leukaemia (Crump, 1994; Glass et al, 2003, 2004, 2006; Rinsky et al, 2002; Schnatter, 2004).

Oral

Oral cancer studies showed increased tumour rates in multiple organs, some of which were also tumour sites in the inhalation studies. The majority of tumour types at sites other than the haematopoietic system are of epithelial origin. In mice benzene produced increased tumour incidences in Zymbal gland, (Cronkite et al,1985; Farris et al, 1993; NTP, 1986; Maltoni et al, 1989), lung (Farris et al, 1993; NTP, 1986; Maltoni et al, 1989), Harderian gland (NTP, 1986), preputial gland (Farris et al,1993; NTP, 1986), forestomach (Farris et al, 1993; NTP, 1996), mammary gland (NTP, 1986; Maltoni et al, 1989), liver (Maltoni et al, 1989) and ovaries (Cronkite et al, 1985; NTP, 1986). In rats, benzene treatment was associated to increased tumour incidences in the Zymbal gland (NTP, 1986; Maltoni et al, 1989), oral cavity (NTP, 1986; Maltoni et al, 1989), forestomach (Maltoni et al, 1989), nasal cavity (Maltoni et al, 1989), and skin (NTP, 1986; Maltoni et al, 1989).

Dermal

No published data are available.

Inhalation

From several animal studies with inhalation and oral exposure there is evidence that benzene is carcinogenic. Target organs were similar in several studies irrespective of the application route and include the haematopoietic system and tissues of epithelial origin. The predominant tumours induced in the inhalation studies were located in the haematopoietic system, particularly lymphomas in mice (Farris et al, 1993; NTP 1986; Cronkite, 1985). In rats, increased frequencies of leukaemia in comparison to controls were found in benzene-exposed Sprague-Dawley rats and Wistar rats (Maltoni et al, 1989) and one case (out of 40 animals) of chronic myelogenous leukaemia was reported in Sprague-Dawley rats exposed to benzene (Goldstein et al, 1982).

Human data

There are substantial uncertainties regarding the shape of the dose response for benzene carcinogenesis below 10 ppm, but in recommending a limit below 1 ppm there can be relatively greater confidence in protection against cancer, based on NOAECs for haematotoxicity and genotoxicity around 0.6 ppm. The analysis by North et al., 2020b using leukemic risk models, concludes that there is no cancer risk to be expected at the proposed OEL of 0.25 ppm.

1,3-Butadiene

(Classification: CLP - Category 1A, H350): In experimental animals, there is a marked species difference in carcinogenicity (EU RAR 2002). In the mouse, 1,3-butadiene is a potent multi-organ carcinogen. Tumours develop after short durations of exposure, at low exposure concentrations and the carcinogenic response includes rare types of tumours (NTP 1993). In the rat, fewer tumour types, mostly benign, develop at exposure concentrations of 100 to1000-times higher (Owen et al 1987). In humans a positive association was demonstrated between workplace exposure to butadiene for men employed in the styrene-butadiene rubber industry and lymphohaematopoietic cancer (leukemia) (Sathiakumar et al 2005, Graff et al 2005, Delzell et al 2006, Cheng et al 2007, Sielken et al 2006, 2007 & 2008).

Non-human information

In the rat an inhalation study was conducted on behalf of the International Institute of Synthetic Rubber Producers (IISRP) (Owen et al 1987). Groups of male and female rats were exposed to 1,3-butadiene at 1000 or 8000 ppm (2212 or 17701 mg/m3) for 6 hr/day, 5 days/week for 2 years. There were increases in the incidences of pancreatic exocrine adenoma (high dose, male); uterine sarcoma (both doses, female); Zymbal gland carcinoma (high dose, female); mammary tumours (both doses, female); thyroid follicular cell tumours (both doses female) and testis Leydig-cell tumours (high dose). These data suggest that 1,3-butadiene is a weak carcinogen to the rat under the conditions of exposure used in this study. The increased incidence of mainly benign tumours, which occur spontaneously in the rat, suggests that 1,3-butadiene may act by a non-genotoxic mechanism, rather than by a direct effect of reactive metabolites.

The US National Toxicology Program has conducted two carcinogenicity studies in mice. In the first (NTP, 1984), male and female B6C3F1mice were exposed to 1,3-butadiene by inhalation at 625 or 1250 ppm (1382 or 2765 mg/m3), 6 hrs day, 5 days per week for 61 weeks. The study was scheduled for 2 years but was stopped earlier because of high mortality in both treated groups. There was clear evidence of multiple organ carcinogenicity for 1,3-butadiene in both sexes, as shown by increased incidences and early induction of haemangiosarcomas of the heart, malignant lymphomas, alveolar/bronchiolar adenomas and carcinomas, and papillomas of the stomach in males and females; and of acinar cell carcinomas of the mammary gland, granulosa cell tumours of the ovary, and hepatocellular adenomas and carcinomas in females. This study demonstrated that 1,3-butadiene is a potent carcinogen in mice causing multi-organ tumours that develop after only 1 year.

The second study extended the dose range of the first and also included a “Stop-Exposure” study where mice were exposed for a period then left untreated (NTP 1993). Survival in treated groups was reduced in both standard and “Stop-Exposure” studies due to the presence of malignant neoplasms. The standard and “Stop-Exposure” studies confirmed the clear evidence of carcinogenicity of 1,3-butadiene in both sexes. In the standard study, male and female B6C3F1mice were exposed to 1,3-butadiene by inhalation at 6.25, 20, 62.5, 200 or 625 ppm (13, 44, 138, 442 or 1382 mg/m3), 6 hrs day, 5 days per week for up to 2 years. Tumours arose at all exposure levels. In males there were increased incidences of neoplasms in the haematopoietic system, heart, lung, forestomach, liver, harderian gland, preputial gland, brain and kidney. In females there were increased incidences of neoplasms in the haematopoietic system, heart, lung, forestomach, liver, harderian gland, ovary and mammary gland. Low incidences of intestinal carcinomas in male mice, Zymbal's gland carcinomas in male and female mice, and renal tubule adenomas and skin sarcomas in female mice may also have been related to administration of 1,3-butadiene. In the “Stop-Exposure” study male B6C3F1 mice were exposed to 1,3-butadiene by inhalation at 200 ppm (443 mg/m3) for 40 weeks, 312 ppm (690 mg/m3) for 52 weeks, 625 ppm (1383 mg/m3) for 13 weeks, or 625 ppm (1383 mg/m3) for 26 weeks. After exposure the mice were then left untreated for the remainder of the 2-year study. Tumours at multiple sites were observed at all dose levels with the first tumours appearing after only 13 weeks of exposure to 650 ppm.

These NTP studies (standard and "Stop Exposure") also show that 1,3-butadiene causes multi-site carcinogenicity in mice. Tumours arose at all exposure levels. These data indicate that 1,3-butadiene is a genotoxic carcinogen and the risk of carcinogenicity in mice is high even at low exposure levels (NTP 1984, 1993).

 

Category streams are considered to be carcinogens if they contain ≥0.1% benzene or 1,3-butadiene.

Additional Reference:

EU RAR (2002). European Union Risk Assessment Report for 1,3-butadiene. Vol. 20. European Chemicals Bureau (http: //ecb. jrc. ec. europa. eu/DOCUMENTS/Existing-Chemicals/RISK_ASSESSMENT/REPORT/butadienereport019. pdf)


Justification for selection of carcinogenicity via oral route endpoint:
These streams are gases at room temperature, hence carcinogenicity toxicity testing via the oral route is not technically feasible.

Justification for selection of carcinogenicity via inhalation route endpoint:
There was no evidence of carcinogenicity in male and female rats or mice exposed to propene by inhalation at concentrations of 8,600 or 17,200 mg/m3 for 103 weeks, or in a supporting studies in mice (up to 8,600 mg/m3, 78 weeks) or rats (up to 8,600 mg/m3, 104 weeks). However streams containing >0.1 % butadiene or benzene should be considered potentially carcinogenic.

Justification for selection of carcinogenicity via dermal route endpoint:

These streams are gases at room temperature, hence carcinogenicity toxicity testing via the oral route is not technically feasible.

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