reaction mass of Benzenesulfonic acid, 2,2'-(1,2-ethenediyl)bis[5-[[4-[bis(2- hydroxyethyl)amino]-6-(phenylamino)-1,3,5-triazin-2-yl]amino]-, disodium salt and Benzenesulfonic acid, 5-[[4-[bis(2-hydroxyethyl)amino]-6-(phenylamino)-1,3,5-triazin-2-yl]amino]-2-[2-[4-[[4-[(2-hydroxyethyl)amino]-6-(phenylamino)-1,3,5-triazin-2-yl]amino]-2-sulfophenyl]ethenyl]-, disodium salt

Carcinogenicity

Description of key information

No carcinogenic potential

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

Approximately from November, 1963 to January, 1966

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Test procedures cannot be subsumed under a testing guideline. The test procedures are well explained, nevertheless the purity of the substance is unknown. Justification for read across approach is given in the endpoint summary and in the read across justification report attached to the Section 13 of this dossier.

Principles of method if other than guideline:

Golden hamster were chosen to investigate the effects of prolonged oral administration of the product. One series of 15 males and one series of 15 females were treated for 24 months with 5 mg/kg/day of the test item in water. In order to study the reproductive function in treated animals, a treated male was mated with 5 treated females. The mating resulted in 30 newborns, of which 5 males and 5 females were selected for the successive experimental step. Furthermore, a male of the first generation treated with the test item was mated with 5 treated females of the first generation. 5 males and 5 females of the second generation were chosen for the study.

GLP compliance:

no

Remarks:

pre GLP

Species:

hamster

Strain:

other: Mesocricetus auratus

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Common name: Golden hamster. Unlike the rat, these animals do not spontaneously present pituitary tumor at the age of 2 years.
- Source: form breeding of Marseille C.N.R.S., where the breed was raised for generations.
- Housing: housed in metallic or plastic cages one to one, in well heated rooms.
- Diet: synthetic diet of Lacassagne MABI, ad libitum.

During the study four series of animals were used:
- one serie of 15 males of an initial weight of 30 grams. Experiment started on November 18th, 1963.
- one serie of 15 females of an initial weight of 30 grams. Experiment started on January 18th, 1964.
- one serie of 10 animals of first generation (5 males and 5 females); they were originated by males and females treated for 6 months. Experiment started on November 18th, 1964.
- one serie of 10 animals of second generation (5 males and 5 females); they were originated by males and females belonging to the first generation. Experiment started on June 18th, 1965.

Route of administration:

oral: unspecified

Vehicle:

water

Details on exposure:

Administration of test substance: oral, by means of a pipette

Duration of treatment / exposure:

24 months

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
5 mg/kg/day
Basis:
nominal in water

No. of animals per sex per dose:

one serie of 15 males and one serie of 15 females
one serie of 10 animals of first generation and one serie of 10 animals of second generation

Control animals:

yes

Details on study design:

In order to study the reproductive function in treated animals, a treated male was mated with 5 treated females.
5 males and 5 females of the newborns were selected for the successive experimental step.
Furthermore, a male of the fisrt generation treated with the test item was mated with 5 treated females of the first generation and 5 males and 5 females of the second generation were chosen for the study.

CONTROL
The results of examinations were compared with the effects observed in 80 animals used as control and placed under the same environmental and semi-synthetic alimentary conditions.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS
The evolution of the general condition and weight (systematic weighing all the animals every 15 days), of clinical behavior and the state of the hair system.

HAEMATOLOGY
The haematological examination was performed before sacrifice. A blood sample was taken for the electrophoretic examination.

Sacrifice and pathology:

GROSS PATHOLOGY
Animals spontaneously died or sacrificed were submitted to a complete autopsy, which includes the examination of the gastrointestinal digestive tract, cervical organs, thoracic organs, liver, biliary, pancreas, spleen, mesenteric lymph nodes, adrenals, adrenal glands and urinary channels, genital organs, mammary glands, skeleton, different segments of the brain, pituitary and skull base.

HISTOPATHOLOGY
All the autopsied animals have were subjected to a complte histological examination, which regarded: at least three fragments of the stomach, three fragments of the small intestine and of the large intestine, two/three liver fragments, one/two fragments of pleen, one fragment of pancreas, two fragments of kidneys, two fragments of adrenals (two slides of adrenal gathered by the solar plexus), two fragments of testicles or ovaries, fragments of uterine bladder-prostate, two/three fragments of lung, one cardiac fragment, three slides of the brain (for the examination of the fronto-parietal cortex, the striers coros, thalamus, of hypothalamus, cerebellum and midbrain sub-thalamic formations), one slide passing through the sphenoid bone and forward pituitary and the trigeminal ganglion, one slide of sternum and of the femoral bone for the bone marrow examination, a slide og the lymphatic mesenteric ganglion.

All the fragments were fixed using Bouin solution (including brain to use the Gomori staining) and paraffin. The obtained preparations were stained with the Masson's trichrome stain or the Toluidine blue stain, or with the Gomori method, or with the Mac Manus method, or with several of these methods.

Details on results:

CLINICAL SIGNS AND MORTALITY
MALES: two animals died before the end of the experiment (H.15162 died one month and six days before the end of the experiment; H. 15163 died one month and four days before the end of the experiment). None of the animals treated showed hair removal.
FEMALES: two animals died before the end of the experiment (H.15177 and H. 15178).

BODY WEIGHT AND WEIGHT GAIN
MALES: the weight curve of treated animals resulted to be identical to that for the normal animals. At the sacrifice, the treated males (which had an initial weight of 40 grams) weighed 98 - 120 grams; females weighed 95 - 120 grams. The same individual variability was recorded also in the control animals.

HISTOPATHOLOGY: NEOPLASTIC
MALES: no tumours were recorded.

ANIMALS DIED BEFORE THE END OF THE EXPERIMENT - MALES
H.15162: it died on 1965.12.12 and weighed 100 grams. The macroscopic examination revealed a bilateral testicular atrophy; all the remaining bowels had a normal aspect. Absence of auditory, pituitary, testis and kidney tumours. The histological examination revealed:
- lungs: the lymphoid tissue was well developed, without any signs of bronchitis.
- liver: absence of any pathologic sign.
- spleen: the lymphoid tissue was well developed. Into the red pulp, the reticulo-histiocytic system was well developed. Presence of many eosinophil cells.
- pancreas: normal aspect.
- kidneys: absence of any pathologic sign.
- adrenals: normal aspect.
- stomach and intestine: normal aspect.
- testicles: precence of many aplastic seminiferous tubes. The interstitial gland was hyperplastic.
- pituitary: absence of any abnormalities.
- thyroid: normal aspect.
- thymus: normal aspect.
- lymphatic ganglia: absence of any sign of reactions.
Absence of any other pathological, inflammatory, degenerative visceral process.

H.15163: it died on 1965.12.14 and weighed 105 grams. The macroscopic examination revealed a pulmonary emphysema and a bilateral testicular atrophy. Absence of auditory, pituitary, testis and kidney tumours. The histological examination revealed:
- lungs: presence of foci at large bubbles due to the emphysema. No signs of bronchitis.
- liver: absence of any abnormalities.
- spleen: the lymphoid tissue was well developed. Into the red pulp blood pigment was scarce. Absence of plasmacytosis.
- pancreas: normal aspect of the endocrine and exocrine tissue.
- kidneys: absence of any pathologic sign.
- adrenals: normal aspect.
- stomach and intestine: normal aspect.
- testicles: precence of many aplastic seminiferous tubes. The interstitial gland was not hyperplastic.
- pituitary: normal aspect.
- thyroid: normal aspect.
- thymus: normal aspect.
- lymphatic ganglia: absence of any sign of reactions.
Absence of any other pathological, inflammatory, degenerative visceral process.

ANIMALS DIED BEFORE THE END OF THE EXPERIMENT - FEMALES
H.15177: it died on 1965.12.25 and weighed 100 grams. The macroscopic examination revealed pulmonary emphysema and distinct colouration of kidneys. All the remaining bowels had a normal aspect. Absence of auditory, pituitary, genital and adrenal tumour. The histological examination revealed:
- lungs: the lymphoid tissue was well developed, without bronchial distention.
- liver: lymphocytic infiltrates in Kiernan's spaces.
- spleen: absence of any sign of reactions.
- pancreas: normal aspect of the endocrine and exocrine tissue.
- kidneys: nephrosis microcystic appearance with the presence of many distended urinary tubes, containing hyaline product.
- adrenals: histo-physiological normal aspect.
- stomach and intestine: normal aspect.
- uterus and ovaries: absence of any pathologic sign.
- pituitary: normal aspect.
- thyroid: normal aspect.
- thymus: normal aspect.
- lymphatic ganglia: absence of any sign of reactions.
Absence of any other pathological, inflammatory, degenerative visceral process.

H.15178: it died on 1965.12.29 and weighed 98 grams. The macroscopic examination revealed distinct colouration of kidneys. All the remaining bowels had a normal aspect. Absence of auditory, pituitary, genital and adrenal tumour. The histological examination revealed:
- lungs: histopathological normal aspect.
- liver: absence of any pathology.
- spleen: absence of any abnormalities.
- pancreas: normal aspect.
- kidneys: many distended urinary tubes containing eosinophilic product.
- adrenals: normal aspect.
- stomach and intestine: normal aspect.
- uterus and ovaries: absence of any pathologic sign.
- pituitary: normal aspect.
- thyroid: absence of any abnormalities.
- thymus: normal aspect.
- lymphatic ganglia: absence of any sign of reactions.
Absence of any other pathological, inflammatory, degenerative visceral process.

Relevance of carcinogenic effects / potential:

No treatment related increase in tumour incidence, as compared to controls.

SUMMARY OF MALES EXAMINATIONS

Absence of pituitary tumours.

Absence of hearing tumours.

Absence of subcutaneous sarcoma.

Absence of adrenal tumours.

No testicular tumour; testicular atrophy was recorded in three animals.

Hepatic inflammatory infiltrates were observed in two animals.

Spleen reactions were recorded in one animal.

Pulmonary emphysema, without inflammatory reactions was recorded in two animals.

Nephrosis were found in four animals.

SUMMARY OF FEMALES EXAMINATIONS

Absence of subcutaneous sarcoma.

Absence of pituitary tumours.

Absence of hearing tumours.

Absence of adrenal tumours.

No ovary and uterine tumours. Uterine hypertrophy was observed in one animal.

Pulmonary hyperplasia tumouriforme was recorded in one animal.

Pulmonary emphysema, without inflammatory reactions was recorded in four animals.

A hepatitis zonal processes was observed in one animal.

Hepatic inflammatory phenomena were recorded in two animals.

Spleen reactions were recorded in one animal.

Nephrosis were found in four animals.

REPRODUCTIVE FUNCTIONS

The founders' mating process resulted in 30 newborns of normal constitution, while the first generation's mating process resulted in 26 newborns of normal constitution.

None of the 10 animals of the first generation died spontaneously and none presented any clinic disturbance. They did not result sterile.

The 10 animals belonging to the second generation grew up normally and not presented any clinic disturbance.

No signs of leukemia were recorded in the animals.

All animals of this experimental set present normal leukocyte and electrophoretic formulas.

Conclusions:

No malignant tumour was recorded in 30 animals treated, thus the test item resulted to be non carcinogenic.
Furthermore, the investigated substance did not promote the malignant tumour development, which were observed in the control animals.
No signs of the reproductive impairments were observed and no teratogenic reactions were recorded. The test item did not shown the capability to induce visceral damage.

Executive summary:

Golden hamster were chosen to investigate the effects of prolonged oral administration of the product. One serie of 15 males and one serie of 15 females were treated for 24 months with 5 mg/kg/day of the test item in water.

The evolution of the general condition and weight (systematic weighing all the animals every 15 days), of clinical behavior and the state of the hair system were recorded. The haematological examination was performed before sacrifice. A blood sample was taken for the electrophoretic examination. Animals spontaneously died or sacrificed were submitted to a complete autopsy and to histological examination.

The results of examinations were compared with the effects observed in 80 animals used as control and placed under the same environmental and semi-synthetic alimentary conditions.

In order to study the reproductive function in treated animals, a treated male was mated with 5 treated females. The mating resulted in 30 newborns of normal constitution; 5 males and 5 females of these 30 newborns were selected for the successive experimental step (none died spontaneously during the experiment). They did not present any abnormalities. Furthermore, a male of the fisrt generation treated with the test item was mated with 5 treated females of the first generation. This mating resulted in 26 newborns of normal constitution. 5 males and 5 females of the second generation were chosen for the study. They grew up normally and they did not present any abnormalities.

No signs of leukemia were recorded in the animals and all of them present normal leukocyte and electrophoretic formulas.

Four animals died spontaneously before the end of the experiment. No animals present subcutaneous or liver sarcoma; none of the treated animals presented pituitary tumour. No animals presented testicular tumour. Testicular atrophy was noted in 3 animals; the same processes were observed also in the control animals. No females presented ovarian or uterine tumours. Ovarian hypertrophy was observed in only one animal; uterine hypertrophy was not recorded. Pulmonary hyperplasia tumouriforme was recorded in one female and the same process was observed in control animals. Pulmonary emphysema, without inflammatory reactions, was recorded in two males and in four femlaes.

A hepatitis zonal processes was observed in one female; hepatic inflammatory phenomena were recorded in two males and in two females.

Spleen reactions were recorded in one male and in one female. Nephrosis were found in eight animals (four males and four females) and such phenomena were also observed in the control animals. No signs of leukemia were recorded in the animals.

Conclusion

No malignant tumour was recorded in 30 animals treated, thus the test item resulted to be non carcinogenic.

Furthermore, the investigated substance did not promote the malignant tumour development, which were observed in the control animals.

No signs of the reproductive impairments were observed and no teratogenic reactions were recorded. The test item did not shown the capability to induce visceral damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

5 mg/kg bw/day

Study duration:

chronic

Species:

other: hamster and rat

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

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

In conclusion, the substance does not meet the criteria to be classified for carcinogenicity, according to the CLP Regulation (EC 1272/2008).

Additional information

Golden hamster were chosen to investigate the effects of prolonged oral administration of the analogue substance 07. One serie of 15 males and one serie of 15 females were treated for 24 months with 5 mg/kg/day of the test item in water. The evolution of the general condition and weight (systematic weighing all the animals every 15 days), of clinical behaviour and the state of the hair system were recorded. The haematological examination was performed before sacrifice. A blood sample was taken for the electrophoretic examination. Animals spontaneously died or sacrificed were submitted to a complete autopsy and to histological examination. The results of examinations were compared with the effects observed in 80 animals used as control and placed under the same environmental and semi-synthetic alimentary conditions.

In order to study the reproductive function in treated animals, a treated male was mated with 5 treated females. The mating resulted in 30 newborns of normal constitution; 5 males and 5 females of these 30 newborns were selected for the successive experimental step (none died spontaneously during the experiment). They did not present any abnormalities. Furthermore, a male of the fisrt generation treated with the test item was mated with 5 treated females of the first generation. This mating resulted in 26 newborns of normal constitution. 5 males and 5 females of the second generation were chosen for the study. They grew up normally and they did not present any abnormalities.

No signs of leukemia were recorded in the animals and all of them present normal leukocyte and electrophoretic formulas. Four animals died spontaneously before the end of the experiment. No animals present subcutaneous or liver sarcoma; none of the treated animals presented pituitary tumour. No animals presented testicular tumour. Testicular atrophy was noted in 3 animals; the same processes were observed also in the control animals. No females presented ovarian or uterine tumours. Ovarian hypertrophy was observed in only one animal; uterine hypertrophy was not recorded. Pulmonary hyperplasia tumouriforme was recorded in one female and the same process was observed in control animals. Pulmonary emphysema, without inflammatory reactions, was recorded in two males and in four females.

A hepatitis zonal processes was observed in one female; hepatic inflammatory phenomena were recorded in two males and in two females.

Spleen reactions were recorded in one male and in one female. Nephrosis were found in eight animals (four males and four females) and such phenomena were also observed in the control animals. No signs of leukemia were recorded in the animals.

No malignant tumour was recorded in 30 animals treated, thus the test item resulted to be non carcinogenic. Furthermore, the investigated substance did not promote the malignant tumour development, which were observed in the control animals. No signs of the reproductive impairments were observed and no teratogenic reactions were recorded. The test item did not shown the capability to induce visceral damage (Mosinger, 1966).

A further investigation was performed administering the same substance for 24 months to 30 Winstar rats at 5 mg/kg/day. The purpose of the investigation was examine the evolution of the general condition and of the clinical behaviour. Animals were submitted to a complete autopsy and a complete histological examination was performed. The reproductive function was assessed studying the first and the second generation. Sarcomatous malignant tumour was identified in one animal as a reticulo-sarcoma. A breast adenoma, pituitary adenoma and benign testicular tumours in two animals were also recorded. The test item did not show any carcinogenic potential; it did not cause more malignant tumours than those spontaneously observed in control animals (Mosinger).