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Toxicological information

Carcinogenicity

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Description of key information

Oral NOAEL: 521 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:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test procedures cannot be subsumed under testing guideline, nevertheless are well documented and scientifically acceptable.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen, Germany.
- Age at study initiation: 28 - 32 days old.
- Weight at study initiation: 48 g, 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: 24 ± 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 in diet
No. of animals per sex per dose:
50 males and 50 females per dose.
100 animals in control group.
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 6 months; 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: determination of the hemoglobin content by cyanhaemiglobin, determination of the hematocrit with a micro-hematocrit centrifuge, calculation of the mean cell volume (MCV) and mean corpuscular hemoglobin content of the (MCH), count of reticulocytes after staining
with brilliant cresyl blue, thrombocytes (platelet) count in a Coulter Counter (model FN), assessment of complete blood count based on smears at 630 times magnification, at end of test determination of the prothrombin time.
The blood sugar determinations were made colorimetrically and enzymatically with glucose oxidase-peroxidase; the cholesterol determination was also performed.

CLINICAL CHEMISTRY
Clinical laboratory investigations were conducted in 5 male and 5 female rats in each dose at 1, 5, 6 and male 12 months; at the end of the experiment, the analysis were conducted to 10 male and female rats.
To test the liver function following enzymes in heparin plasma were determined: Alkaline phosphatase, glutamic-oxaloacetic transaminase and glutamic pyruvic transaminase.

URINALYSIS
The urine was collected in a 16-hour collection period; the semi-quantitatively determinations of glucose, protein, blood and pH were performed.
Microscopic examinations were performed sediment after centrifugation of urine. For examination of renal function, we determined urea and creatinine.
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, embedded in Paraplast and stained using hematoxylin-eosin (HE): aorta, eyes, small and large intestine, urinary bladder, heart, testis, pituitary gland, salivary gland, liver, lung, lymph nodes, stomach, spleen, epididymis, adrenal glands, kidneys, femur, esophagus, Ovarian, pancreatic, prostate, seminal vesicles, thyroid, skeletal muscle, sternum, trachea, brain, and uterus.
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).
In addition, tumor suspicious changes were evaluated in the same way the histopathological analysis.
Statistics:
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 %.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
comparable with the control group
Mortality:
mortality observed, treatment-related
Description (incidence):
comparable with the control group
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
no significant differences
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
nevertheless the differences from the control are limitated and random
Gross pathological findings:
no effects observed
Description (incidence and severity):
no evidence of treatment related changes in both rats dead before the end of the experiment and the rats killed at the end of the test
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
substance-related histopathological changes were not observed
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
tumours not treatment-related
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
Comparable in all groups: the bodyweight curve trend of both males and females is graphically superimposable to that traced by the trend of the control group.

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

HAEMATOLOGY
At the 1st month: differences from the control in the hemoglobin and hematocrit content (P < 0.05) were recorded only in the treated males rats groups of doses 100 and 1000 ppm (non dose-dependent); erythrocytes, the number of red blood cells, reticulocytes, white blood cells and platelets, MCH in value and mean cell volume (MCV) were not affected. All the females groups treated did no showed any difference.
At the 3rd month: the only differences from the control were recorded in the erytrocytes and thrombocytes (both P < 0.05) in the male rats group dosed at 1000 ppm.
At the 6th month: a decrease non dose-dependent in the leucocytes count was recorded in the male rats group dosed at 100 ppm (P < 0.05), in comparison with the control group. No difference was recorded in all the other treated groups.
At the 12th month: a decrease in the monocytes (P < 0.05) was observed in the males group dosed at 100 ppm. No other parameter, in no other group was affetcted.
At the 24th month: at the experiment conclusion only the number of reticulocytes in the male rats dosed at 1000 and 10000 ppm was significantly lower than in the control animals. The rest parameters are missing significant and dose-dependent differences. All the rest of parameters are missing significant and dose-dependent differences. In the analysis of complete blood count, no treatment-related changes in the leukocyte blood count at the dose levels up to 10000 ppm were observed.
Non pathological range blood sugar or cholesterol levels were determined at the dosed groups up to 10000 ppm.

CLINICAL CHEMESTRY
At the 1st month the activity of GOT and GPT transaminases was significantly (P < 0.05) increased in male rats of the dose group of 10000 ppm. In female rats, the GPT activity was significantly (P < 0.01) increased in groups at 1000 and 10000 ppm.
After 3 months of treatment the GOT activity in males is comparable with that in the control, while the GPT trensaminases showed difference in the dosed group at 100 ppm. In all the females groups no differences with the cobtrol were recorded.
All the liver function parameters investigated, in all treated groups, at all dosage (both males and females) were comparable to those of the control at the sixth month.
At the 12th month differences non dose-dependent in GOT activity were detected only in the females groups dosed at 100 and 1000 ppm (P < 0.05), while at the 24th months differences in GOT activity from the control were recorded in the male groups treated at 100 and 1000 (P < 0.05 and P < 0.01, respectively) ppm and differences in the total protein determined in serum were recorded in the female group dosed at 10000 ppm ( P < 0.01). Differences in the experiment were not significant.

URINALYSIS
The findings of the urinalysis after 1 -, 5 -, 6 -, 12 - and 24-month test period revealed no differences between control animals and treated rats up to the 1000 ppm dose. In none of the tested rats glucose, ketone-bodies or bilirubin were found in the urine. Urobilinogen content and pH-value of the treated animals did not differ significantly from the control animals. Positive blood and protein findings were found in approximately equal frequency in the treated as in the untreated rats. The examination of the sediment revealed no treatment-related effect.
The protein contents of the urine were increased in any group.

ORGAN WEIGHTS
In comparison to the control group significantly different organ weights were recorded. In male and female rats, the kidney weights were not increased significantly (P < 0.01) up to 10000 ppm. The remaining organ weight differences have to be regarded as limitated and random.

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
Substance-related histopathological changes were not observed.
In the heart region of the animal 464 (F, 10000 ppm) was found a marked suppurative myocarditis. The animal 359 (F, 1000 ppm) showed pyelonephritis with low-gradiger hyperplasia (Hyper) of the renal pelvic epithelium.
Tumors that would be attributed to the substance were not detected.
_ Cardiovascular and respiratory apparatus: symptoms of chronic inflammation of different degrees were seen in the lungs and tracheae in animals of all groups; nevertheless they are common in old, conventionally held rats.
_Liver: in some animals of different groups of extramedullary hematopoiesis characters (EMH) were seen in the liver in an average small extent. Pathological fatty infiltration were observed in the liver. In many animals bile ducts were altered. Other sporadic effects on singolar animals were seen.
_Genito urinary system: the majority of rats showed renal senile changes of different degrees .In the testes of individuals of different groups atrophy and Spermiogranulome was observed. In the ovaries of many female cysts were not present.
_Adrenal, thyroid and pituitary: in the thyroid and pituitary glands of individual animals of different groups Epithelmeta-neoplasms were observed.
Eyes, intestine, bone, bone marrow, lymph nodes, spleen, pancreas, parotid gland and skeletal muscle showed only treatment-independent individual findings.

In some animals of different groups of extramedullary hematopoiesis characters (EMH) were seen in the liver in an average small extent. Pathological fatty infiltration were observed in the liver.
All tumors detected were considered to be spontaneous, not treatment-related.
Relevance of carcinogenic effects / potential:
Non carcinogen
Dose descriptor:
NOAEL
Effect level:
709 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: overall effects
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
Dose descriptor:
NOAEL
Effect level:
521 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: overall effects
Remarks on result:
other: Effect type: carcinogenicity (migrated information)

Number of tumors

0 ppm  10000 ppm  1000 ppm  100 ppm 
male  female  male  female  male  female  male  female 
N. of total tumors  77 76 43  39  42  43  31  40 
N. of malign tumors  30  23  15  16  15  11 

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 16.48 22.43 - -
100 16.49 22.44 3.84 5.23
1000 16.44 22.36 38.4 52.24
10000 16.61 22.6 382.77 520.78

female

0 12.5 17.01 - -
100 12.54 17.06 5.16 7.02
1000 12.79 17.4 50.96 69.33
10000 12.86 17.52 521.3 709.25

Mortality

Dose

ppm
N. dead / N rat

Male
% N. dead / N rat

Female
%

1 year

0 2/100 2 0/100 0
100 1/50 2 1/50 2
1000 3/50 6 0/50 0
10000 0/50 0 0/50 0

2 years

0 37/100 37 25/100 25
100 23/50 46 17/50 34
1000 25/50 50 14/50 28
10000 16/50 32 17/50 34
Conclusions:
From nature, localization, abundance and time of occurrence of the identified benign and malignant tumors was no evidence of a carcinogenic effect of the test item. Thus, the doses of 1000 ppm was well tollerated.
Executive summary:

Method

The 50 male and 50 female rats (100 animals in the control group) received 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 ppm did not show during the entire experimental period any treatment-related symptoms. The growth of the rats was not affected until the dose of 10000 ppm. The haematological investigations performed during and at the end of the test showed no dose of injuries. The significantly lower reticulocyte numbers in the blood of the male rats dosed at 1000 and 10000 ppm after 24 months was not considered as compound-related toxic effect. The studies of male animals to the remaining time points and all investigations in the female animals showed no evidence for a corresponding findings. 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 indicate any influence on the kidneys because the incidence is low (< 10 %) and the significantly increased kidney weights in male and female rats in the 10000 ppm dose group was not regarded as an expression of injury. Blood sugar and cholesterol levels were not substance-related alterated.
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. Thus, the doses of 1000 ppm was well tollerated.

NOAEL: 709 mg/kg bw/day (actual dose received) (female)

NOAEL: 521 mg/kg bw/day (actual dose received) (male)

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
521 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:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test procedures cannot be subsumed under a testing guideline, nevertheless are well documented and scientifically acceptable.
GLP compliance:
no
Species:
mouse
Strain:
other: Skh: hairless-1
Sex:
male/female
Details on test animals 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 % Alkansulphonate (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 tumours.
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 tumours (% 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 tumours (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 contribut to cancerogenicity 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 tumour formation by UV irradiation; that was evident at the time to occurrence of tumours in the number of animal with tumours in the total number of occurring tumors and the growth behaviour of the tumours. The UV irradiation cutaneous treatment with the test item also had no detectable influence on the behaviour, appearance, weight gain, and the survival times of mice.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
chronic
Species:
mouse

Additional information

In a combined chronic / carcinogenicity studies (Bomhart 1978), the test substances was administered to 50 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. The NOAEL is for females and males 10000 ppm/day. The various statistically significant differences observed with CAS 16470-24-9 (increase/decrease) in organ weights (absolute in males), number of reticulocytes/trombocytes, and ALAT (GPT) activities and serum protein were considered to be of no toxicological relevance and did therefore not provide any evidence of toxicity of the test substance at any of the dose levels tested. At the doses tested, there were in tested substances not a treatment related increase in tumour incidence when compared to controls.

Further studies were done in order to investigate whether the test substance has a carcinogenic effect onto skin under light exposure.

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


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 they are well documented and scientifically acceptable.

Justification for classification or non-classification

According to CLP regulation (EC1272/2008), 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 CLP Regulation (EC1272/2008).