Barium bis[2-chloro-5-[(2-hydroxy-1-naphthyl)azo]toluene-4-sulphonate]

Carcinogenicity

Description of key information

In a longterm 2-year feeding study with Osborne-Mendel rats there was no increase in tumour incidence up to the highest feeding level. ICR mice with dermal exposure for 18 months showed no increased evidence of neoplasms. In the course of a NTP bioassay conducted with rats and mice, no findings were seen in mice or female rats. Fibrosarcomas in spleen were found in high dose male rats. With regard to the absence of genotoxicity and cytotoxicity and the presence of hematotoxicity, fibrosarcoma formation in spleen is considered to be a secondary effect due to methemoglobin formation and excessive erythrocyte damaging. No indication of carcinogenic properties was observed upon drinking water application at 2500 ppm.

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

Study period:

1977-1979

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

GLP compliance:

no

Remarks:

conducted before introduction of GLP

Species:

other: rats and mice

Strain:

other: F344 rats and B6C3F1 mice

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: NCI Frederick Cancer Research Center (Frederick, MD)
- Age at study initiation: 4 weeks
- Weight at study initiation: no data
- Housing: 5 per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 12-16d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 40-60
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12

The start dates were March 10, and March 23, 1977 for male and female rats and April 8, and April 17, 1977 for male and female mice.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Remarks:

mixed with diet

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: formulated by mixing weighed-amounts of Purina® Laboratory Chow and the test chemical for 15 minutes in a Patterson-Kelly® twin-shell blender equipped with an intensifier bar

DIET PREPARATION
- Rate of preparation of diet (frequency): 10d
- Storage temperature of food: 23°C

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulated diets containing 100,000 ppm D & C Red No. 9 were analyzed at Midwest Research Institute and were found to be stable for 2 weeks at temperatures up to 45 C. Every 8 to 10 weeks, analytical concentrations of D & C Red No. 9 were determined in blindly selected batches of formulated diets and were within +10% of the desired concentration.

Duration of treatment / exposure:

2 years / 103 weeks

Frequency of treatment:

daily

Post exposure period:

1 weeks (rats)
2 weeks (mice)

Dose / conc.:

1 000 ppm (nominal)

Remarks:

rats

Dose / conc.:

3 000 ppm (nominal)

Remarks:

rats

Dose / conc.:

1 000 ppm (nominal)

Remarks:

mice

Dose / conc.:

2 000 ppm (nominal)

Remarks:

mice

No. of animals per sex per dose:

50

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: dosel level rationale bases on effects observed in the 91 day study

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: each month

BODY WEIGHT: Yes
- Time schedule for examinations: every 4 weeks

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Animals were statistically censored as of the time that they died of other than natural causes or were found to be missing; animals dying from natural causes were not statistically censored. 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) extensions of Cox's methods for testing for a dose-related trend. One-tailed P values have been reported for all tests except the departure from linearity test, which is reported only when its two-tailed P value is less than 0.05.
The purpose of the statistical analyses of tumor incidence is to determine whether animals receiving the test chemical developed a significantly higher proportion of tumors than did the control animals. As a part of these analyses, the one-tailed Fisher exact test (Cox, 1970) was used to compare the tumor incidence of a control group with that of a group of dosed animals at each dose level. When the results from two dosed groups are compared simultaneously with that for a control group, a correction to ensure an overall significance level of 0.05 may be made. The Bonferroni inequality criterion (Miller, 1966) requires that the P values for any comparison be less than or equal to 0.025. When this correction was used, it is discussed in the narrative section. It is not presented in the tables, where the Fisher exact P values are shown.
Furthermore: Cochran-Armitage test for linear trend in proportions, time-adjusted analysis was applied when numerous early deaths resulted from causes that were not associated with the formation of tumors, life table methods were used to analyze the incidence of tumors,

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

low dose rats had a greater rate of survival than other groups or females

Mortality:

mortality observed, treatment-related

Description (incidence):

low dose rats had a greater rate of survival than other groups or females

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

mean body weight of high-dose female mice was slightly lower than that of the controls

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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 specified

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

non-neoplastic nodules of the liver in male rats

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

fibrosarcomas in male rats

Details on results:

Rats:
Fibrosarcomas, apparently arising from the red pulp or capsule of the spleen, were found in 17 of 48 high-dose (3,000 ppm) male rats. In high-dose male rats, one animal had a leiomyosarcoma and five had splenic osteosarcoma. A fibroma was found in one low-dose male rat. Eleven of the splenic tumors metastasized to peritoneal tissues. The two sarcomas of multiple organs in high-dose males may have originated in the spleen.
Fourteen of, 48 males had congestion of the splenic parenchyma, 23 had focal or multifocal areas of fibrosis, 3 had diffuse fibrosis, and 13 had areas of fatty metamorphosis in the spleen. Twenty-five high-dose females had multifocal, diffuse, or focal fibrosis. Areas of fibrosis were present in two control male rats.
The splenic lesions in dosed male and female rats ranged from multifocal areas of fibroblastic proliferation in the red pulp to areas of proliferation of pleomorphic spindle cells with an oval to round, open-faced nucleus, and generally, an indistinct nucleolus.
Large areas of pigment were occasionally seen in the fibrous areas in the splenic capsule and parenchyma. The pigment appeared different from the hemosiderin seen in spleens of aging F344 rats.
Hepatic neoplastic nodules were seen in 0/50 control males, 6/50 low-dose males, and 7/49 high dose males. Almost all of these nodules were relatively small and composed of hepatocytes with basophilic or eosinophilic cytoplasm. Hepatocellular carcinoma was seen in 1/50 control males.

Mice:
Histopathologic examination provided no evidence for the carcinogenicity of D & C Red No. 9 in B6C3F1 mice.

Relevance of carcinogenic effects / potential:

Under the conditions of this bioassay, D & C Red No. 9 was carcinogenic for male F344 rats causing an increased incidence of sarcomas of the spleen and a dose-related increase in neoplastic nodules of the liver. D & C Red No. 9 was not considered to be carcinogenic to female F344 rats, although the increased incidence of neoplastic nodules of the liver may have been associated with administration of the test chemical. D & C Red No. 9 was not carcinogenic for B6C3F1 mice of either sex.

Dose descriptor:

NOAEL

Sex:

male

Basis for effect level:

other: carcinogenic for male F344 rats causing an increased incidence of sarcomas of the spleen and a dose-related increase in neoplastic nodules of the liver

Remarks on result:

not determinable

Remarks:

no NOAEL identified. Effect type:carcinogenicity (migrated information)

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)

Table 1: food (mean) and compound intake

rats		0 ppm	1000 ppm	3000 ppm
male	feed (g/day)	20	20.5	20.3
male	dose (mg/kg bw/day)	0	50 - 162	150 - 470
female	feed (g/day	13.9	14.8	14.5
female	dose (mg/kg bw/day)	0	51 - 146	150 - 427
mice		0 ppm	1000 ppm	2000 ppm
male	feed (g/day)	8.3	8.2	8.3
male	dose (mg/kg bw/day)	0	216 - 410	421 - 830
female	feed (g/day)	8.1	8.2	7.8
female	dose (mg/kg bw/day)	0	234 - 432	446 - 821

Table 2: Mean body weight change (relative to controls) of rats fed diets containing D & C Red No. 9 for 103 weeks

Sex	Week No.	Cumulative mean body weight change [g]			Weight change [%] relative to controls
		Controls	Low dose	High dose	Low dose	High dose
Male rats	0 5 26 46 67 88 103	131* 101 240 263 289 277 286	134* 101 236 269 293 288 283	127* 109 245 270 297 285 283	0 -2 +2 +1 +4 -1	+8 +2 +3 +3 +3 -1
Female rats	0 3 24 44 65 86 103	105* 36 103 125 146 168 189	103* 37 104 126 148 166 200	102* 38 105 125 148 162 188	+3 +1 +1 +1 -1 +6	+6 +2 0 +1 -4 -1

*Initial weight

Table 3: Numbers of rats with neoplastic and non-neoplastic lesions in the spleen

	Males			Females
	Control	Low dose	High dose	Control	Low dose	High dose
No. of spleens examined	50	50	48	50	50	50
Spleen lesions: Fibroma Fibrosarcoma Leimyosarcoma Osteosarcoma Congestion, NOS or passive Fibrosis, focal or multifocal Fibrosis, diffuse Necrosis, focal Fatty metamorphosis Hemosiderosis Splenic capsule: Sarcoma Fibrosarcoma Splenic red pulp: Fibrosarcoma	0 0 0 0 1 1 1 0 0 2   0 0   0	1 0 0 0 0 0 0 0 0 1   0 0   0	0 17 1 5 14 23 3 2 13 2   1 1   1	0 0 0 0 0 0 0 0 0 1   0 0   0	0 0 0 0 6 2 0 0 0 0   0 0   0	0 0 0 0 26 15 10 0 0 0   0 0   0

Table 4: Mean body weight change (relative to controls) of mice fed diets containing D and C Red No. 9

Sex	Week No.	Cumulative mean body weight change [g]			Weight change [%] relative to controls
		Controls	Low dose	High dose	Low dose	High dose
Male rats	0 5 27 46 66 88 100	21* 7 14 18 18 17 15	20* 10 15 19 18 17 17	21* 7 14 19 19 17 17	+43 +7 +6 0 0 +13	0 0 +6 +6 0 +13
Female rats	0 4 26 45 65 87 99	19* 3 9 14 16 18 16	19* 3 9 13 15 17 17	19* 4 9 13 15 16 15	0 0 -7 -6 -6 +6	+33 0 -7 -6 -11 -6

*Initial weight

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

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:

experimental study

Adequacy of study:

supporting study

Study period:

1961 -1969

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: protocol agreed between the U.S. Food and Drug Agency and CTFA

Principles of method if other than guideline:

Dermal application onto shaved mouse skin twice weekly for 18 months.

GLP compliance:

no

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: no data
- Fasting period before study: not applicable
- Housing: single
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
no data

Route of administration:

dermal

Vehicle:

water

Details on exposure:

Application of 0.1 ml of a 1% suspension in water
application site: 6 cm2

Initially. the hair on the dorsal area of each animal was clipped with an animal clipper free of lubricatirig oil. Subsequent periodic clipping was performed according to the rate of hair growth.

The test item suspension was prepared fresh weekly

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

483 days (ca 18 months)

Frequency of treatment:

twice per week

Post exposure period:

none

Dose / conc.:

1 other: mg (total paint on mouse)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

0.166 other: mg/cm² (nominal)

No. of animals per sex per dose:

50 for test item
150 for control
50 for positive control

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on human exposure from use in lipstick

Positive control:

3,4-benzpyrene, dissolved in acetone

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations not included

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily
DERMAL IRRITATION (if dermal study): No data
BODY WEIGHT: No data
OPHTHALMOSCOPIC EXAMINATION: No data
HAEMATOLOGY: No data
CLINICAL CHEMISTRY: No data
URINALYSIS: No data
NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (preparation of: brain, pituitary, thyroid, thymus, liver, spleen, kidney, adrenal, stomach, small and large intestines, urinary bladder, axillary lymph nodes, testes, ovary, skin from area of treatment, any tissue massess, grossly abnormal organs; complete pathology only for each five males and females; skin and grossly abnormal organs for all animals)

Statistics:

no data

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

>= 0.17 other: mg/cm2

Sex:

male/female

Basis for effect level:

other: corresponds to 1 mg per mouse or roughly 50 mg/kg bw; skin histopathology performed for all animals, complete histopathology only performed for 5 of 50 animals.

In the mid-1960s a number of cosmetic color additives originally identified by US Food and Drug Administration and the Cosmetic, Toiletry and Fragrance Association (CTFA) (formerly the Toilet Goods Association) were evaluated as part of an overall program to determine safety for human usage in a series of coal tar derived colors which were then in wide use by the cosmetic, drug and food industries.

Conclusions:

Dermal application of 1 mg per mouse twice per week for 18 months did not cause skin cancer. Full histopathology of a low number of randomly chosen animals did not give an indication of systemic toxicity or carcinogenicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.

Additional information

Procedure and observations

100 ICR mice received pigment red 53:1 two times per week for 18 months at the shaven back (Carson et al 1974). Dosage levels were based on lipstick use determinations made in a group of human female volunteers. Twice each week a 0.1 ml dose containing 1 mg of the dye was applied to the dorsal skin of each mouse. All animals were necropsied; after termination of the study, tissues were selected for histopathology, sectioned, stained and examined by a pathologist. The repeated application of 0.1 ml containing 1 % dye did not increase the incidence of neoplasms when compared to the vehicle controls.

Groups of 50 B6C3F1 mice received diets containing 1,000 or 2,000 ppm of test substance for 103 weeks (NTP 1982). After week 50, the mean body weight of high-dose female mice was lower than that of the controls. No compound-related effect on survival or clinical signs were observed for mice of either sex. Under the conditions of this bioassay, D & C Red No. 9 was not carcinogenic for B6C3F1 mice of either sex.

Fisher 344 rats received 0, 1000 or 3000 ppm pigment red 53:1/kg bw for 103 weeks with the daily feed (NTP 1982). In males and females of the 3000 mg group non-neoplastic damage to the spleen (focal and diffuse fibrosis, lesions and splenic capsule) occured. There were no splenic sarcomas in low dose males or any of the female groups. There were small increases in neoplastic nodules of the liver in male rats.

Osborne-Mendel rats received 0, 0.01, 0.05, 0.25 and 1% pigment red 53:1 of the daily feed for two years (David and Fitzhugh 1962). Growth and mortality was unaffected. In the two highest dosage groups depressed haemoglobin levels, abnormal shape of erythrocytes and slight hyperplasia of bone marrow occurred. In the highest dosage group moderately to strong splenomegaly as well as haemosiderosis and partial infarction of the spleen was seen. Compared to the untreated control animals there was no increase in tumour incidence.

In a chronic drinking water study Barium chloride dihydrate was administrated for 104 weeks to rats and mice at concentrations of 500, 1250 or 2500 ppm (NTP 1994). Based on drinking water consumption, the average uptake of the highest dose was 60 and 75 mg/kg bw for males and female rats, respectively and 160 and 200 mg/kg bw for male and female mice, respectively. Systemic availability of Barium was confirmed by monitoring of serum concentrations. For mice, a reduction in survival rate was recorded for the highest dose group, most likely due to renal toxicity. There was no increased evidence for tumor formation in rats or mice.

Discussion

The substance was tested for its carcinogenic potential in several studies. In one study with rats, high dose male animals developed splenic sarcomas and neoplastic nodules of the liver. Whereas, female animals and mice had no tumors. The neoplastic nodules in male high dose rats were not malignant and represent an early stage in the development of liver tumors in rats. It is not clear that any of them would have progressed to hepatocellular carinomas even if the rats had been allowed to live longer. Oral or dermal application of the test item to mice for 2 years gave no hints for a carcinogenic potential. Also oral administration to Osborne-Mendel rats for 2 years did not result in tumor formation. However, splenomegaly, hemosiderosis and splenic infarcts were seen at the high dose group.

Taken together, the substance has no genotoxic potential and does not interact with DNA or genetic material. Most likely, metabolites of the test article (1-amino-2-naphtol) cause methemoglobinamia leading to disturbances of iron metabolism and subsequently increased iron deposition in liver and spleen (hemosiderosis). Moreover, the substance or metabolites are attached at methemoglobin and transported via red blood cells into the spleen which acts as a filter for old or damaged erythrocytes. During degradation of methemoglobin, the substance or metabolite is released and affects spleenic mesenchymal tissue leading to fibrosis and promotion of tumor formation (sarcomas). Finally, the substance acts as a secondary carcinogen after oral administration of high dose level. The substance was not considered to be carcinogenic after dermal application.