Disodium 4,4'-bis[6-anilino-[4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate

Carcinogenicity

Description of key information

Oral NOAEL: 542 mg/kg bw/day (chronic; rat)

Dermal chronic mouse: the test substance does not contribute to carcinogenicity induced by UV-irradiation.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

other: read-across based on grouping od substances (category approach)

Adequacy of study:

weight of evidence

Study period:

From November 1973 to November 1975.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study carried out on an analogue substance belonging to the category of Stilbene Fluorescent Whitening Agents. The reliability of the source study is 1. Details for category approach are included into the IUCLID section 13.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

no

Remarks:

Pre GLP:

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Dtrainn: Winstar II.
- Source: Winkelmann, Borchen, Germany.
- Age at study initiation: 28 - 32 days.
- Weight at study initiation: 54 g male and 53 g female, average at the start.
- Housing: individually, in Macrolon cages (Type 2).
- Diet: ad libitum, weekly fresh Altromin R-powder feed.
- Water: ad libitum, tap water.

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 1 °C

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
Mixing appropriate amounts with: Altromin R-Pulverfutter (Altromin GmbH, Lage/Lippe, Germany).

Duration of treatment / exposure:

24 months.

Frequency of treatment:

Daily.

Remarks:

Doses / Concentrations:
0, 100, 1000, 10000 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

50 males and 50 females per dose.

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS
The experimental animals were inspected daily and occurring changes and symptoms recorded.

BODY WEIGHT
The body weight of the animals was determined weekly up to the 27th week; after that, the weight determination was carried out at intervals of 14 days.

FOOD CONSUMPTION AND COMPOUND INTAKE
The weekly feed consumption was determined by reweighing.

HAEMATOLOGY
The blood tests included: Erythrocyte and leukocyte count, platelet count, reticulocyte hemoglobin content, haematocrit , MCH, MCV, thromboplastin time.

CLINICAL CHEMISTRY
Clinical laboratory investigations were conducted in 5 male and 5 female rats in each dose at 1, 3, 6 and male 12 months; at the end of the experiment, the analysis were conducted to 10 male and 10 female rats.
Parameters: Alkaline phosphatase (ALP), glutamic-oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), glutam-dehydrogenases (GIDH), creatinin, urea, blood glucose, cholesterin, bilirubin, protein.
To test the liver function following enzymes in heparin plasma were determined: Alkaline phosphatase, glutamic-oxaloacetic transaminase and glutamic pyruvic transaminase.

URINALYSIS
Semiquantitative: glucose, ketone bodies, bilirubin, urobilinogen.
Quantitative: proteins.

Sacrifice and pathology:

SACRIFICE
At the end of the experiment all survivors animals were anesthetized with ether and killed by exsanguination.

GROSS PATHOLOGY
The rats dead during the experiment and the rats sacrified at the end were dissected and examined macroscopically.
The weights of the following organs were determined: thyroid, heart, lungs, liver, spleen, kidneys, adrenal glands, testes and ovaries.

HISTOPATHOLOGY
The following organs were fixed in Bouin solution: Aorta, eyes, intestine (duodenum, jejunum, ileum, colon), femur, brain, bladder, heart, testis, pituitary gland, liver, lung, lymph nodes, stomach, spleen, epididymis, adrenal glands, sciatic nerve, kidney, esophagus, parotid gland , ovaries, pancreas, prostate, seminal vesicles, thyroid, skeletal muscle, sternum, trachea, and uterus, as well as all changes macroscopically found.
From the fixed organs or organ approximately samples of 5 g were prepared and stained with Hamalaun-eosin.
In addition kidney sections were subjected by these rats of the Periodic Acid Schiff (PAS) reaction. The decalcification of the bones was performed in Ethylanadinitrilotetraacetic acid tetrasodium salt (EDTA).

Statistics:

Were calculated: Arithmetic group means, standard deviation s, upper and lower confidence limits on the confidence level α = 95% and 1-α = 99%.

The values ​​of the collective test the investigated doses were compared with the control group with the significance test, U-test according to Mann, Whitney and Wilcoxon on the significance level of α = 5 % and α = 1 %.

The mortality rates were compared using Fisher's exact de-tests on the significance level of α = 5 % and α = 1 % with the control group.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

dosed and control goups showed a temporarily low but significant weight depression

Food consumption and compound intake (if feeding study):

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

Description (incidence and severity):

no treatment-related histomorphological alterations

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

no treatment-related histomorphological alterations

Details on results:

CLINICAL SIGNS AND MORTALITY
During the experimental period no differeces in appearance and behavior between treated groups and control were noted. Furthermore no differences in the vividness and coat condition were observed.
Mortality of the dosed groups is comparable to that recorded into the control group.

BODY WEIGHT AND WEIGHT GAIN
The rats of the dosed groups at 100, 1000 and 10000 ppm as well as the rats of the control group, showed a temporarily low but significant weight depression. No significant difference between dosed and control groups has been found.

FOOD CONSUMPTION AND COMPOUND INTAKE
No difference from the control group were recorded in food and drink consumption in all dosed groups.

CLINICAL CHEMESTRY
At 1 month from the start of the test no significant difference between dosed goups and control were recorded. The treated rats did not differ significantly and in dose-dependent manner at 5, 6, and 12 months test period and at end of test from the control animals.
At the end of the experiment the values ​​of alkaline phosphatase (ALP) in the male dosed goups at 1000 and 10000 ppm was higher than in the control ( P < 0.05 and P < 0.01, respectively) and the protein contents (GPT) resulted higer in the female groups dosed at 1000 and 10000 ppm (both P < 0.01), than in the control.
Blood sugar and cholesterol levels were determined to not lying in the pathological range in the dose groups up to 10000 ppm.

URINALYSIS
In all of the investigated rats, glucose, ketone bodies or bilirubin found in the urine, protein and blood-positive urine findings were present in approximately the same abundance.
Urobilinogen content and pH-value of the treated animals did not differ significantly from that of control animals.
The examination of the sediment revealed no treatment-related effect.
At three months after beginning the test creatinine conted resulted in all the females dosed groups significantly and dose-dependent higher than in the control group. Significant and dose-dependent lower protein contents are also recorded at 12 months in the female animals after doses of 1000 and 10000 ppm at end of test.

ORGAN WEIGHTS
In comparison to the control group significantly different organ weights were recorded.
In comparison to the values ​​of the control group, the liver weights of the male rats were not significantly increased up to the administered dose of 10000 ppm.
The kidney weights of female animals doses at 1000 and 10000 increased respect to thde control group.
The remaining organ weight differences are distributed low and independent of dose.

GROSS PATHOLOGY
_RATS DEAD during the experiment: no pathological changes which could be attributed to treatment were found in all the animals.
_RATS KILLED at the end of the experiment: no evidence of specific injury in the experimental groups to 10000 ppm.

HISTOPATHOLOGY
In summary, it was found that all the investigated organ exhibited no treatment-related histomorphological alterations.

HISTOPATHOLOGY - Neoplastic
In the In the male rats were observed adenomas, essentially benign tumors, of the pituitary gland, mammary and thyroidhe endocrine system; of the thyroid adenoma and pheochromocytoma and adenoma of the Hodeninterstitiums and in female rats in all groups at approximately the same frequency. The malignant neoplasms of various types are distributed randomly on all experimental groups. Type and location of all observed tumors are fir the rat strain used typical.

Relevance of carcinogenic effects / potential:

Non carcinogen

Dose descriptor:

NOAEL

Effect level:

542

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Dose descriptor:

NOAEL

Effect level:

779

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Food and active ingredient intake

Dosis [ppm]	mean food intake		mean active ingredient intake
	kg/animal	g/animal/day	g/kg bw	mg/kg bw /day
	male
0	14.85	20.21	-	-
100	15.82	21.52	3.92	5.33
1000	15.62	21.25	39.75	54.08
10000	15.28	20.79	398.96	542.80
	female
0	13.25	18.03	-	-
100	13.48	18.33	5.74	7.80
1000	13.40	18.23	58.78	79.97
10000	13.40	18.24	572.84	779.37

Mortality

Dose ppm	N. dead / N rat Male	%	N. dead / N rat Female	%
1 year
0	1/50	2.0	1/50	2.0
100	1/50	2.0	0/50	0.0
1000	1/50	2.0	0/50	0.0
10000	0/50	0.0	0/50	0.0
2 years
0	6/50	12.0	9/50	18.0
100	16/50	32.0*	8/50	16.0
1000	11/50	22.0	6/50	12.0
10000	6/50	16.2	8/50	16.0

* significance P < 0.05

Conclusions:

NOAEL: 779 mg/kg bw/day (actual dose received) (female)
NOAEL: 542 mg/kg bw/day (actual dose received) (male)

Executive summary:

The 50 male and 50 female rats eceived test substance administered for 2 years in the following concentrations with the feed: 0 (control), 100, 1000, 10000 ppm.

Results

Appearance, behavior, feed intake, body weights and mortality were not influenced in male and female animals of doses up to and including 10000 ppm.The animals in the dose groups to 10000 ppmdid notshow during the entire experimental period any treatment-related symptoms. The growth of the rats was not affected until the dose of 10000 ppm. The haematologicalinvestigations performed during and at the end of the test showed no dose of injuries.he clinical chemical analysis, sections and histopathological examinations revealed no evidence for treatment-related damage to the liver. Urinalysis, urea and creatinine concentrations in serum as well as macroscopic and histopathological organ findings did not indicateany influence.

NOAEL: 779 mg/kg bw/day (actual dose received) (female)NOAEL: 542 mg/kg bw/day (actual dose received) (male)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

542 mg/kg bw/day

Study duration:

chronic

Species:

rat

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:

other: read-across based on grouping od substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study carried out on an analogue substance belonging to the category of Stilbene Fluorescent Whitening Agents. The reliability of the source study is 1. Details for category approach are included into the IUCLID section 13.

GLP compliance:

no

Species:

mouse

Strain:

other: Skh: hairless-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Ciba-Geigy AG.
- Age at study initiation: 6 weeks.
- Housing: sindividualy in Macrolon cages, type I, with dust-free wood pellets.
- Diet: "ssniff'-Mausefutter", ad libitum and once per week sunflower seeds ad libitum.
- Water: tap water ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 2 °C; during the irradiation the temperature rose to a maximum of 29 °C.

Route of administration:

dermal

Vehicle:

other: water + 0.005 % Alkansulfonate (Emulgartor K30)

Details on exposure:

UV EXPOSURE
- Choice of the light source: the most important consideration for selecting a lamp is the similarity of the spectral distribution with the mean daylight, illuminant D 65 (DIN 503 3 Tail 7).
- Lamp: the "black lights" lamps by the type Philips TL 40 W/08, were selected.
- Lamp replacing: due to the wasting away of the lamps which may bring to a decrease of 10 %, the lamps were replaced after six months.
- Irradiation: ca 272 µW/cm2.

TEST SITE
- Area of exposure: 2 x 3 cm.
- Type of wrap if used: no wrap used.

TEST SOLUTION
- Solvent solution: an aqueous solution was prepared with 0.005 % of alkanesulfonate (emulsifier K 30), contained as the wetting agent. Then a solution of sodium bicarbonate and emulsifier K30 was preparade filling up to 20 liters with deionized water, so that per liter of solution 0.05 g of emulsifier K 30 and 0.084 g of sodium bicarbonate were included. Approximately every week, the solvent solution was freshly prepared.
- pH of solvent solution: always less than 8.1.
- Stock solution: were freshly prepared every 14 days and with the aqueous solvent before each application, dilute 1:10. All solutions were produced only in the absence of light with UV components and stored in the dark.
- Test concentration: 100 mg/l, administered at 0.03 ml three times per week .

Duration of treatment / exposure:

320 days.

Frequency of treatment:

UV-irradiation: daily 4 hours (seven times a week).
Test substance: three times per week (Monday, Wednesday and Friday).

Remarks:

Doses / Concentrations:
3 µg
Basis:
other: test material

No. of animals per sex per dose:

100 mice: 50 males and 50 females were treated. 100 untreated mice served as controls; as additional controls 50 (25M abd 25F) were used with acetone and 50 (25M and 25F) mice treated with wetting agents in aqueous solution.

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

yes, sham-exposed

Positive control:

Historical data with 8-Methoxypsoralen.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS
The mice were checked daily.

BODY WEIGHT
Body weight determinations were made every 14 days.

DERMAL CHANGES
Once a month all skin changes were accurately determined.

SKIN NEOPLASMS
All skin neoplasms were recorded as soon as they were clearly visible to the bare eye.
The criteria for assessing the carcinogenic effect were: time of occurrence of first tumors, total number of tumors, tumor number per animal, number of animals with tumors, tumor growth, histology of tumors.

Sacrifice and pathology:

At the end of the exposure perio the mice were killed, dissected and all skin growths examined histologically.
All the treated areas of skin, including the occurred neoplasms, were inserted for histological examination.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

observed neoplasis were not test-item induced

Details on results:

CLINICAL SIGNS AND MORTALITY
The treatment was well tolerated by all mice (except for the induction of skin tumors by the end of the experiments merely by UV irradiation). Neither the UV radiation or the additional treatment with the test compound resulted in premature death of animals, with the only exception that after a 10-month test period, the mice began to die of their UV-induced skin tumors, but only occasionally.

BODY WEIGHT AND WEIGHT GAIN
Weight gain were recorded without difference in the experimental group and in the control groups.

SKIN CHANGES
In the experimental group and the control groups occasionally individual animals showed in the back erythema, and in few cases slight necrosis. The erythema and necrosis was equal weak in the experimental group and the control groups.

HISTOPATHOLOGY: NEOPLASTIC
Almost all mice used (ca 80 %) developed neoplasms of the skin, and especially at the level of the middle of the back; the smaller part of these neoplasms were skin cancers in the rest it was virtually only papillomas.
With regard to the occurrence of the first skin tumors per animal and the neoplasms as a whole, the number of animals with malignant and the number of animals with benign skin tumors as well as benign and malignant overall skin neoplasm, no evidence of a tumor-promoting effect of the test item were revealed.

Relevance of carcinogenic effects / potential:

Non carcinogen.

Mortality

	N. of animals	Sex	mice died (in % of animals used)
Test item	50	M	8
	50	F	2
untreated	50	M	8
	50	F	2
wetting agent	25	M	12
	25	F	0
acetone	25	M	12
	25	F	0

Tumours

	N. of animals	Sex	Skin tumors (% of animals used)
			malignant	benign	total
Test item	50	M	52	142	194
	50	F	52	182	234
untreated	50	M	60	148	208
	50	F	56	210	266
wetting agent	25	M	56	172	228
	25	F	56	160	216
acetone	25	M	48	124	172
	25	F	76	188	264

	N. of animals	Sex	Animals with skin tumors (in% of animals used)
			malignant	benign
Test item	50	M	44	68
	50	F	40	84
untreated	50	M	52	68
	50	F	46	68
wetting agent	25	M	36	80
	25	F	44	76
acetone	25	M	48	80
	25	F	44	96

Conclusions:

The test substance does not contribute to carcinogenicity induced by UV-irradiation.

Executive summary:

Method

The UV irradiation was administered in rats four hours daily (seven times a week); the treatment with test susbtance was conduced three times per week with 0.03 ml at 0.01 % on an area of ​​2 x 3 cm. Each dose group included 100 mice (50 male, 50 female). 100 untreated mice served as controls; as additional controls 50 were used with acetone and 50 mice treated with wetting agents in aqueous solution.

The treatment was stopped after 320 trial days, killed the mice, dissected and all skin growths examined histologically.

Results

The UV radiation generated in the experimental group and in the control groups caused neoplasms of the skin in the about 80 % of the mice

. Treatment with the test compound had no effect on tumor formation by UV irradiation; that was evident at the time to occurrence of tumors in the number of animal with tumors in the total number of occurring tumors and the growth behavior of the tumors. The UV irradiation cutaneous treatment with the test item also had no detectable influence on the behavior, appearance, weight gain, and the survival times of mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

chronic

Species:

mouse

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), 3.6 Carcinogenicity section, carcinogen means a substance, which induce cancer or increase its incidence. Substances, which have induced benign and malignant tumours in well performed experimental studies on animals are considered also to be presumed or suspected human carcinogens unless there is strong evidence that the mechanism of tumour formation is not relevant for humans. For the purpose of the classification for carcinogenicity, substances are allocated to one of two categories (known or presumed human carcinogens and Suspected human carcinogens) based on strength of evidence and additional considerations (weight of evidence). In certain instances, route-specific classification may be warranted, if it can be conclusively proved that no other route of exposure exhibits the hazard.

The combined chronic/carcinogenicity studies available did not provide any evidence of carcinogenicity.

In conclusion, the available experimental data are adequate for classification and labelling and the substance is not classified for carcinogenicity according to the CLP Regulation (EC 1272/2008).

Additional information

In two combined chronic/carcinogenicity studies (Bomhart 1978), the test substances (CAS 4404-43-7 and CAS 16470-24-9) were administered to Wistar rats/sex/dose in diet at dose levels of 0, 100, 1000, 10000 ppm for 24 months. There were no compound related effects in mortality, clinical signs, body weight, food consumption, haematology, clinical chemistry, urinalysis, organ weights, or gross and histological pathology.

In the test with CAS 4404-43-7, the acid form of the disulphonated derivative dihydroxyethyl derivative, the haematological investigations performed during and at the end of the test showed no dose of injuries. The clinical chemical analysis, sections and histopathological examinations revealed no evidence for treatment-related damage to the liver. Urinalysis, urea and creatinine concentrations in serum as well as macroscopic and histopathological organ findings did not indicateany influence. The No Observed Effect Level was set at 779 mg/kg bw/day (actual dose received) for females and at 542 mg/kg bw/day for males (Bomhard E. and Löser E., 1978). The tested substance contains the same organic functional groups then the substance under registration (CAS 4193-55-9), but due to the sulphonation/salification degree it is less soluble; this property makes the Read Across substance a conservative representative because of the potential higher bioavailability (details in the Category Justification Report attached to the Section 13).

In the test conducted on the analogous dihydroxyethyl derivative tetrasulphonated sodium salt (CAS 16470-24-9), appearance, behaviour, feed intake, body weights and mortality were not influenced in male and female animals of doses up to and including 10000 ppm. The animals in the dose groups to 10000 ppm did not show during the entire experimental period any treatment-related symptoms. The growth of the rats was not affected and the haematological investigations performed showed no dose of injuries; also the clinical chemical analysis, sections and histopathological examinations revealed no evidence for treatment-related damage to the liver. Furthermore, the from nature, localization, abundance and time of occurrence of the identified benign and malignant tumours was no evidence of a carcinogenic effect of the test item. The NOAEL was set at 709 mg/kg bw/day (actual dose received) for females and at 521 mg/kg bw/day (actual dose received) for males (Bomhard et al., 1978). The substance tested has the same organic functionalities than the CAS 4193-55-9, with a higher sulphonation degree.

A further study was done in order to investigate whether the test substance has a carcinogenic effect onto skin under light exposure (Strinhoff D. 1979). Photocarcinogenesis testing involved pretreating hairless mouse skin with the test compounds, 8 -methoxypsoralen (8 -MOP; known phototoxic agent), or solvent only before each daily exposure to simulated solar ultraviolet light. In terms of tumour yield and tumour development time, photocarcinogenesis was enhanced by 8 -MOP, but not by test substances.

Based on the similarities in toxicological behaviour for all members of the category, the results of the described studies can be considered as a reference also for the substance under registration.

Justification for selection of carcinogenicity via oral route endpoint:

Test procedures cannot be subsumed under testing guideline, nevertheless are well documented and scientifically acceptable.

Justification for selection of carcinogenicity via dermal route endpoint:

Test procedures cannot be subsumed under a testing guideline, nevertheless are well documented and scientifically acceptable.