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Carcinogenicity

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Description of key information

Based on the available information from animal studies and from long-term observation periods with humans o-toluidine proofed to be a clear carcinogenic substance. Therefore,  IARC has o-toluidine allocated to group 1 = carcinogenic to humans.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions (only 2 doses were employed instead of at least three doses, no clinical pathology, no hematology and no interim kill were performed )
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Deviations:
yes
Remarks:
only 2 doses were employed instead of at least three doses, no clinical pathology, no hematology and no interim kill were performed
GLP compliance:
no
Remarks:
GLP was not mandatory at the time of the study
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
Source: NCI Frederick Cancer Research Center animal farm (Frederick, Md, USA)
- Age at study initiation: 6 week old. Animals were obtained as 4-week-old weanlings, The
animals were housed within the test facility for 2 weeks.

- Weight at study initiation:
Male: 90g-105g (average of 100g), female: 80g-95g (average of 90g)
- Housing: 4 animals per cage
- Diet: ad libitum; autoclaved Wayne® Sterilizable Lab Meal (Allied Mills, Inc., Chicago, 111, USA), replenished at least 3x/week
- Water: ad libitum (pH 2.5)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24°C
- Humidity (%): 45-55%
- Air changes (per hr): 15 times/h
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Test diets containing o-toluidine hydrochloride were prepared fresh every 1 to 1-1/2 weeks in 6- to 12-kg batches at the appropriate doses. A known weight of the chemical was first mixed with an equal weight of autoclaved Wayne® Sterilizable Lab Meal (Allied Mills, Inc., Chicago, 111.), using a mortar and pestle. The mixing was continued with second and third additions of feed, and final mixing was performed with the remaining quantity of feed for a minimum of 15 minutes in a Patterson-Kelly twin-shell blender. The diets were stored at 5°C until used.

Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
104 weeks (females and all control animals) and 101 weeks (males)
Frequency of treatment:
continuous
Remarks:
Doses / Concentrations:
0, 3000 or 6000 ppm (approximately 0, 225 and 450 mg/kg bw/day)
Basis:
nominal in diet
No. of animals per sex per dose:
50 animals/sex were dosed with test substance. Additional 20 animals/sex served as control animals: 104 weeks (females and all control animals) and 101 weeks (males)
Control animals:
yes, plain diet
Details on study design:
50 animals/sex were dosed with test substance. Additional 20 animals/sex served as control animals: 104 weeks (females and all control animals) and 101 weeks (males)
Positive control:
no
Observations and examinations performed and frequency:
All animals were checked twice daily for deaths. Observations for sick, tumor-bearing, and moribund animals were recorded daily. Clinical examination and palpation for masses were performed each month, and the animals were weighed at least once per month. Moribund animals and animals that survived to the end of the bioassay were killed using CO2 and necropsied.
Sacrifice and pathology:
Pathological examination consisted of gross and microscopic analysis of major tissues, major organs, and all gross lesions.
The tissues were preserved in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The following tissues were examined microscopically: skin, lungs and bronchi, trachea, bone marrow (femur), spleen, lymph nodes (mesenteric and submandibular), thymus, heart, salivary glands (parotid, sub lingual, and submaxillary), liver, pancreas, esophagus, stomach (glandular and nonglandular), small and large intestines, kidney, urinary bladder, pituitary, adrenal, thyroid, parathyroid, pancreatic islets, testis, prostate, mammary gland, uterus, ovary, brain (cerebrum and cerebellum), and all tissue masses. Peripheral blood smears also were made for all animals, whenever possible.
Necropsies were also performed on all animals found dead, unless precluded in whole or in part by autolysis or cannibalization.


Statistics:
Probabilities of survival: product-limit procedure of Kaplan and Meier. Animals dying from natural causes were not statistically censored.
Dose-related effect on survival: method of Cox (1972) and Tarone's (1975) extensions of Cox methods for testing for a dose-related trend.
Comparison of tumor incidence: Fisher exact test
Bonferroni inequality (miller 1966)
Cochran-Armitage test (1971)
Details on results:
CLINICAL SIGNS AND MORTALITY
No clinical signs (except body weight reduction) were reported which could clearly be related to administration of the test chemical.
In each sex a significant positive dose-related trend in mortalityrats was reported, but individual animal data were not shown


BODY WEIGHT AND WEIGHT GAIN
Mean body weights of both dosed male and dosed female rats were lower than those of corresponding matched controls and were dose related throughout the bioassay. Some fluctuations in the growth curves may be due to mortality; as the size of a group diminishes, the mean body weight may be subject to variation


HISTOPATHOLOGY: .
Histopathology revealed in males and in females tumors in different organs (low, high doses versus controls) including
----multiple organs, localization not otherwise specified
(sarcomas, NOS: m: 3/50, 11/49 versus 0/20; f: 1/50, 2/49 versus 0/20;
fibrosarcomas, m: 8/50, 20/50 versus 0/20,
osteosarcomas, m: 3/50, 5/50 versus 0/20, f: 0/50, 18/50 versus 0/20,
malignant mesothelioma, m 4/50, 2/49 versus 0/20, f: 0/50, 0/49 versus 0/20),
-----subcutaneous tissue
(fibromas: m: 28/50, 27/49 versus 0/20, f: 4/50, 2/49 versus 0/20;
fibrosarcomas: m: 1/50, 2/49 versus 0/20),
-----spleen
(sarcomas, NOS: m: 1/49, 3/42 versus 0/20, f: 1/49, 3/49 versus 0/20;
angiosarcomas: m: 7/49, 0/42 versus 0/20, f: 7/49, 9/49 versus 0/20;
fibromas: m: 10/49, 2/49 versus 0/20, f: 4/49, 6/49 versus 0/20),
-----urinary bladder hyperplasia, m: 9/50, 7/44 versus 0/20, f: 21/45, 13/47 versus 0/20),
-----mammary gland
(fibroadenoma: m: 15/50, 9/49 versus 0/20, f: 20/50, 35/49 versus 7/20)
-----mesotheliomas of the tunica vaginalis in the body cavity of males (15/50, 9/49 versus 0/20).

The tumors were regarded to be treatment related, even if there was no clear dose-dependency at all tumor sites






Dose descriptor:
NOAEL
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
not determinable
Remarks:
no NOAEL identified. Effect type:carcinogenicity (migrated information)

In summary the following tumors were related to the administration of o-toluidine hydrochloride:

- Sarcomas of multiple organs in each sex,

- Fibromas of the subcutaneous tissue in male rats

- Sarcomas of the spleen in female rats,

- Transitional-cell carcinomas of the urinary bladder in female rats,

- Fibroadenomas or adenomas of the mammary gland in female rats, and

- Mesotheliomas in multiple organs or the tunica vaginalis in male rats

Table 1 (Histopathological findings, neoplastic lesions)

Male

Female

Control

Low dose

High dose

Control

Low dose

High dose

Subcutaneous tissue

No of tissues examined

20

50

49

20

50

49

Fibroma

0

28

27

0

4

2

Lipoma

0

4

2

0

0

0

Fibrosarcoma

0

1

2

0

0

0

Mammary Gland

Fibroadenoma

0

7

1

6

20

35

Urinary Bladder

No of tissues examined

20

50

44

20

45

47

Transitional-cell Carcinoma

0

3

0

0

9

22

Epithelial Hyperplasia

0

9

7

0

21

13

Spleen

No of tissues examined

20

49

42

20

49

49

Fibroma

0

10

2

0

4

6

Sarcoma, NOS

0

1

3

0

1

3

Angiosarcoma

0

7

0

0

7

9

Multiple Organs

No of tissues examined

20

50

49

20

50

49

Osteosarcoma

0

3

5

0

0

18

Fibrosarcoma

0

8

20

0

1

0

Sarcoma, NOS

0

3

11

0

1

2

Malignant mesothelioma

0

4

3

0

0

0

Body Cavities

No of tissues examined

20

50

49

20

50

49

Tunica Vaginalis

(Malignant mesothelioma)

0

10

6

0

0

0

Executive summary:

In a carcinogenicity bioassay conducted with a method similar to OECD Guideline 451 with deviations (only 2 doses were employed instead of at least three doses, no clinical pathology, no hematology and no interim kill were performed) 50 rats/sex and dose group were fed 0, 3000 or 6000 ppm (approximately 0, 225 and 450 mg/kg bw/day) o-toluidine hydrochloride for a period of 104 weeks (females and all control animals) and 101 weeks (males), respectively. Additional 20 rats/sex served as control animals. No clinical pathology, no hematology and no interim kill were performed.

In each sex, a significant positive dose-related trend in mortality rates was reported but individual animal data were not given. According to the authors no clinical signs could clearly be related to the administration of o-toluidine hydrochloride. Mean body weights of both, dosed males and females were lower than those of the corresponding matched controls and were reported to be dose-related throughout the bioassay, but individual animal data were not given. Histopathology revealed in males and in females tumors in different organs (low, high doses versus controls) including multiple organs, localization not otherwise specified (sarcomas, NOS: m: 3/50, 11/49 versus 0/20; f: 1/50, 2/49 versus 0/20; fibrosarcomas, m: 8/50, 20/50 versus 0/20, osteosarcomas, m: 3/50, 5/50 versus 0/20, f: 0/50, 18/50 versus 0/20, malignant mesothelioma, m 4/50, 2/49 versus 0/20, f: 0/50, 0/49 versus 0/20), subcutaneous tissue (fibromas, m: 28/50, 27/49 versus 0/20, f: 4/50, 2/49 versus 0/20; fibrosarcomas, m: 1/50, 2/49 versus 0/20), spleen (sarcomas, NOS, m: 1/49, 3/42 versus 0/20, f: 1/49, 3/49 versus 0/20; angiosarcomas, m: 7/49, 0/42 versus 0/20, f: 7/49, 9/49 versus 0/20; fibromas, m: 10/49, 2/49 versus 0/20, f: 4/49, 6/49 versus 0/20), urinary bladder (transitional cell carcinoma, m: 3/50, 0/44 versus 0/20, f: 9/45, 22/47 versus 0/20; epithelial cell hyperplasia, m: 9/50, 7/44 versus 0/20, f: 21/45, 13/47 versus 0/20), mammary gland (fibroadenoma, m: 15/50, 9/49 versus 0/20, f: 20/50, 35/49 versus 7/20) as well as mesotheliomas of the tunica vaginalis in the body cavity of females (15/50, 9/49 versus 0/20). The tumors were regarded to be treatment related, even if there was no clear dose-dependency at all tumor sites

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Study duration:
chronic
Species:
rat
Quality of whole database:
There is a reliable carcinogenicity study published which evaluated this endpoint adequately although not performed under GLP and only tumor formation is considered. Therefore, based on the given information the study is evaluated with Klimisch score 2 The observed carinogenic effect is confirmed by a reliable mice study which is performed under equal conditions and yielded also tumors in several organs

Justification for classification or non-classification

According to Regulation (EC) 1272/2008 (GHS) o-toluidine has to be allocated to Category 1B of the carcinogenic substances (H350) because o-toluidine is known to have carcinogenic potential in animals and in humans.

Additional information

Exposure to rats and mice

In a carcinogenicity bioassay conducted with a method similar to OECD Guideline 451 with deviations (only 2 doses were employed instead of at least three doses, no clinical pathology, no hematology and no interim kill were performed) 50 rats/sex and dose group were fed 0, 3000 or 6000 ppm (approximately 0, 225 and 450 mg/kg bw/day) o-toluidine hydrochloride for a period of 104 weeks (females and all control animals) and 101 weeks (males), respectively. Additional 20 rats/sex served as control animals. No clinical pathology, no hematology and no interim kill were performed.

In each sex, a significant positive dose-related trend in mortality rates was reported but individual animal data were not given.

According to the authors no clinical signs could clearly be related to the administration of o-toluidine hydrochloride. Mean body weights of both, dosed males and females were lower than those of the corresponding matched controls and were reported to be dose-related throughout the bioassay, but individual animal data were not given. Histopathology revealed in males and in females tumors in different organs (low, high doses versus controls) including multiple organs, localization not otherwise specified (sarcomas, NOS: m: 3/50, 11/49 versus 0/20; f: 1/50, 2/49 versus 0/20; fibrosarcomas, m: 8/50, 20/50 versus 0/20, osteosarcomas, m: 3/50, 5/50 versus 0/20, f: 0/50, 18/50 versus 0/20, malignant mesothelioma, m 4/50, 2/49 versus 0/20, f: 0/50, 0/49 versus 0/20), subcutaneous tissue (fibromas, m: 28/50, 27/49 versus 0/20, f: 4/50, 2/49 versus 0/20; fibrosarcomas, m: 1/50, 2/49 versus 0/20), spleen (sarcomas, NOS, m: 1/49, 3/42 versus 0/20, f: 1/49, 3/49 versus 0/20; angiosarcomas, m: 7/49, 0/42 versus 0/20, f: 7/49, 9/49 versus 0/20; fibromas, m: 10/49, 2/49 versus 0/20, f: 4/49, 6/49 versus 0/20), urinary bladder (transitional cell carcinoma, m: 3/50, 0/44 versus 0/20, f: 9/45, 22/47 versus 0/20; epithelial cell hyperplasia, m: 9/50, 7/44 versus 0/20, f: 21/45, 13/47 versus 0/20), mammary gland (fibroadenoma, m: 15/50, 9/49 versus 0/20, f: 20/50, 35/49 versus 7/20) as well as mesotheliomas of the tunica vaginalis in the body cavity of females (15/50, 9/49 versus 0/20). The tumors were regarded to be treatment related, even if there was no clear dose-dependency at all tumor sites

In another carcinogenicity bioassay similiar or equivalent to OECD TG 451, 50 B6C3F1 mice/sex and dose group were fed 0,1000 or 3000 ppm (approximately 0, 150 and 450 mg/kg bw/day) o-toluidine hydrochloride for a period of 103 weeks. Additional 20 mice per sex served as control animals. No clinical pathology, no hematology, and no interim kill were performed.

Mortality occurred in all groups including control groups. Clinical signs could not clearly be attributed to the administration of o-toluidine hydrochloride. Increased incidences of hepatocellular carcinomas or adenomas in female mice and hemangiosarcomas at various sites in males were noted. Therefore, o-toluidine hydrochloride is considered to be carcinogenic in male and female mice.

Exposure to humans

IARC 2012 summarized in the section: "Cancer in Humans"

Several cohort studies have been conducted among workers potentially exposed to o-toluidine. Rubino et al (1982)reported excess bladder-cancer risks in relation to o-toluidine exposure however, other exposure to potential bladder carcinogens also occurred in this work environment. Ward et al (1991) reported an excess in bladder cancer in 1749 US workers employed in the production of rubber additives from o-toluidine and aniline. Risks were greatest for workers with the strongest likelihood of exposure and for those with long-term exposure (>10 years). Further cases of bladder cancer in this facility were reported by Markowith & Levin (2004), but rates were not calculated. Exposure to low-level 4 -aminobiphenyl was suspected, so a protein-adduct biomarker study was carried out (Ward et al 1996), which supported the conclusion that o-toluidine was the most likely cause of the bladder cancer excess, because 4 -aminobiphenyl adducts to haemoglobin were unrelated to work in the facility. Using revised exposure categories, Careon et al (2010) conducted a re-analysis of the data and confirmed that workers in this plant have an increased risk for bladder cancer.

Sorahan et al (2000) and Sorahan (2008) reported an excess in bladder-cancer risk in workers exposed to o-toluidine in the United Kingdom. Sorahan (2008) found increased risks with longer duration of employment in departments where o-toluidine was processed (P<0.05), after adjusting for exposure to other bladder carcinogens in the factory.

Overall, the epidemiological studies show consistent association between exposure to o-toluidine and bladder cancer. Although exposure to other bladder carcinogens occurred for several of the cohorts, the overall evidence is consistent with an association of exposure to o-toluidine and bladder cancer.

Thus, IARC concluded that there is sufficient evidence in humans for the carcinogenicity of o-toluidine. o-Toluidine causes cancer of the urinary bladder.

NTP-DHHS summarizes in the 13th Report on Carcinogens (2014)

o-Toluidine was first listed in the third Annual Report on Carcinogens (1983). o-Toluidine is known to be a human carcinogen based on sufficient evidence of carcinogenicity from studies showing that it causes urinary bladdder in humans together with studies showing that it causes cancer in experimental animals at several tissue sites including the urinary bladder in rats (indicating tumor-site concordance between humans and animals), and studies demonstrating the biological plausibility of mechanisms of its carcinogenicity in humans.

o-Toluidine was first listed in the Third Annual Report on Carcinogens as "reasoneably anticipated to be a human carcinogen" based on sufficient evidence from studies of experimental animals. Since that time , additional cancer studies in humans have been published and the listing was changed to "known to be a human carcinogen" in the Thirteenth Report on Carcinogen (2014).

OVERALL CONCUSION

Based on the available information from animal studies and from long-term observation periods with humans o-toluidine proofed to be a clear carcinogenic substance. Therefore, IARC has allocated o-toluidine to group 1 = carcinogenic to humans and NTP-DHHS listed o-toluidine as "known to be a human carcinogen"


Justification for selection of carcinogenicity via oral route endpoint:
This is a reliable carcinogenicity study published which evaluated this endpoint adequately although not performed under GLP and only tumor formation is considered. Therefore, based on the given information the study is evaluated with Klimisch score 2