Registration Dossier

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:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Test procedures cannot be subsumed under testing guideline, nevertheless are well documented and scientifically acceptable. Justification for Read Across is reported in the endpoint summary and in the Category Justification Report attached to the Section 13 of this dossier.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
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 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 tumors 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: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
From November 1973 to November 1975.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Test procedures cannot be subsumed under testing guideline, nevertheless are well documented and scientifically acceptable. Justification for Read Across is reported in the endpoint summary and in the Category Justification Report attached to the Section 13 of this dossier.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Principles of method if other than guideline:
50 male and 50 female rats received test substance administered for 2 years in the following concentrations with the feed: 0 (control), 100, 1000, 10000 ppm.
GLP compliance:
no
Remarks:
pre GLP
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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:

Method

50 male and 50 female rats 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 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
Study duration:
chronic
Species:
mouse

Additional information

In two combined chronic/carcinogenicity studies (Bomhart 1978), the test substances (CAS 4404-437 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).

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).

A further study was done in order to investigate whether the test substance (CAS 16470-24-9) 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 (details in the Category Justification Report attached to the Section 13 of the dossier).


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.

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).