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

Endpoint summary

Description of key information

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

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 1973 to November 1975.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test procedures are well documented and scientifically acceptable.

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.

Limit test:

no

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 oral 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.

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

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

no significant or dose-dependent differences from the control

Urinalysis findings:

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

Details on results:

CLINICAL SIGNS AND MORTALITY
During the experimental period no differences in appearance and behaviour 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 groups 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 the 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.

Dose descriptor:

NOAEL

Effect level:

542 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Dose descriptor:

NOAEL

Effect level:

779 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Critical effects observed:

not specified

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

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

Results

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 until the dose of 10000 ppm. 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 indicate any 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

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In the chronic toxicity study (equivalent to OECD 453, Bomhard, 1978) the test substance CAS 4404-43-7 was administered to 50 Wistar rats/sex/dose in diet at dose levels of 0, 100, 1000, 10000 ppm (10000 ppm = 709 mg/kg bw/day for females and 521 mg/kg bw/day for males) for 24 months. 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 of 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 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. NOAEL: 779 mg/kg bw/day (actual dose received) (female) NOAEL: 542 mg/kg bw/day (actual dose received) (male).

In the 14 days study a NOAEL was not identified because no effects were observed up to 250 mg/kg bw/day, which probably was the highest dose tested (Gloxhuber et al., 1962). In the same literature report was mentioned also a subcutaneous test in rats performed over one year: no adverse effects were observed at the dose tested of 1.5 mg/kg.

Reference

^[1]Gloxhuber, Hecht and Kimmerle. Toxikologische Untersuchungen mit Aufhellern (Blankophor Von Typen). Archiv für Toxikologie 19, 302--312 (1962)

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Test procedures are well documented and scientifically acceptable.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), 3.9 Specific target organ toxicity - repeated exposure section, substances are classified as specific target organ toxicants following repeated exposure by the use of expert judgement, on the basis of the weight of all evidence available, including the use of recommended guidance values which take into account the duration of exposure and the dose/concentration which produced the effect(s), and are placed in one of two categories, depending upon the nature and severity of the effect(s) observed.

Classification in Category 2 is applicable, when significant toxic effects observed in a 90-day repeated-dose study conducted in experimental animals are seen to occur within the guidance value ranges as:

- oral (rat): 10 < C ≤ 100 mg/kg bw/day

The guidance values refer to effects seen in a standard 90-day toxicity study conducted in rats.

The No Observed Adverse Effect Level was established at 542 mg/kg bw/day, on the basis of the results from the chronic study of 24 months, on rats.

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