Registration Dossier

Administrative data

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:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From November 1973 to November 1975.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted according to internationally accepted testing guidelines, well documented and scientifically acceptable. The analogous substance, part of the Stilbene Fluorescent Withening Agents, group 3, is the acid form of the disulphonated derivative dihydroxyethyl derivative. Justification for Read Across is detailed in the endpoint summary and in the Category Justification Report attached to the section 13.
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.
Limit test:
no
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 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 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.
Dose descriptor:
NOAEL
Effect level:
542 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: -
Dose descriptor:
NOAEL
Effect level:
779 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: -
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 eceived test substance administered for 2 years in the following concentrations with the feed: 0 (control), 100, 1000, 10000 ppm.

Results
Appearance, behavior, feed intake, body weights and mortality were not influenced in male and female animals of doses up to and including 10000 ppm. The animals in the dose groups to 10000 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 haematologicalinvestigations performed during and at the end of the test showed no dose of injuries. he clinical chemical analysis, sections and histopathological examinations revealed no evidence for treatment-related damage to the liver. Urinalysis, urea and creatinine concentrations in serum as well as macroscopic and histopathological organ findings did not indicateany influence.
NOAEL: 779 mg/kg bw/day (actual dose received) (female) NOAEL: 542 mg/kg bw/day (actual dose received) (male).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
542 mg/kg bw/day
Study duration:
chronic
Species:
rat

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

The substance under registration (CAS 16324-27-9) belongs to the category of Stilbene Fluorescent Whitening Agents, subgroup 3c.

This substance and all other members of this category do not show acute toxic effects after oral, inhalation and dermal administration. They are neither irritant to skin nor eyes, nor genotoxic in-vitro and in-vivo, nor sensitizing. Six over fourteen registered substances were tested for subchronic toxicity and four of them were tested for chronic toxicity up to two years with no relevant toxic effects.

Reliable data on repeated dose toxicity subacute and chronic after oral exposure of rats are available for CAS 16470-24-9 and CAS 4404-43, the tetrasulphonated sodium salt and disulphonic acid form, respectively.

In both the analogous the substitution on the triazino ring is different (dihydroxyethy) from that for the substance under registration CAS 16324-27-9 (monohydroxyethyl). The sulphonation degree of CAS 16324-27-9 and CAS 16470-24-9 is the same, while in the case of CAS 4404-43-7 it is a different.

In this respect the water solubilities of CAS 16324-27-9 and CAS 4404-43 are similar (140 vs 80 g/l, respectively), while in the case of CAS 16470-24-9 the solubility in water is higher (650 g/l). This difference in solubility does not impact on the adsorption since both substances are highly soluble and completely dissociated in water.

Based on the metabolic pathway proposed by the OECD Toolbox the same systemic effects can be expected also for 16324-27-9, being analogous the organic functional groups and metabolic pathway (see Category Justification Report attached to the section 13 of the dossier, for further details) The monohydroxyethyl derivative is in fact a metabolite of the dihydroxyethyl, according to the OECD Toolbox Liver metabolism simulation. Impurity profile has no influence on this read across, since substances contain about 10 % of organic by-products that are related to the similar production process: they are in fact di- substitution of the dihydroxyethylamino on the same triazine moiety or of the aniline part. In all cases the same metabolic pathway is expected for the impurities related to the two substances. It can be also stated that results on the diethylamino derivatives members can also be assumed as conservative, because impurities of the diethanolamino starting material can be present and have an undesirable effect on the long term studies, impact that at the toxicological lever is higher that the impact of monoethanolamine.

In the subacute toxicity study (according to OECD 407, RCC - Research and Consulting Company AG. 1988), the test substance CAS 16470-24-9 was administered to Wistar rats by gavage at the dose levels of 0, 50, 200, and 1000 mg/kg bw/day for 28 days. No compound related effects were recorded in mortality, clinical signs, food consumption, and urinalysis. Statistically decreased body weights were observed in female animals dosed at 1000 mg/kg bw/day during the whole treatment period. In addition the body weight gain of the same animals was decreased between days 15 and 18 when the results were compared to those of the animals of the control group and the groups dosed at 50 and 200 mg/kg bw/day. The assessment of haematological data indicated some effects at the end of the treatment when compared to the controls. These findings primarily reflect a slight haemolytic anaemia for rats dosed at 1000 mg/kg bw/day, whereas the changes noted in the lower dose groups were only marginal in nature and therefore not considered significant in toxicological terms. For biochemical data treatment-related effects were noted for rats dosed at 200 and/or 1000 mg/kg bw at the end of the treatment when compared to the controls. These findings primarily reflect changes of an adaptive nature due to an increased functional load on the liver; however, slight injury to liver tissue is to be considered for the high dose group as indicated by the moderate increase in enzyme activity (ASAT and ALP) for males dosed at 1000 mg/kg bw/day. Significant differences in absolute and/or relative liver, kidney and testes weights were observed in animals dosed at 200 and 1000 mg/kg bw/day, respectively. The observed differences in absolute and relative liver weights of the female rats of group dosed at 50 mg/kg bw/day were not considered to be unequivocal to test article treatment. The differences animals dosed at 200 mg/kg bw/day are not considered to be toxicologically relevant because no abnormalities in urinary and biochemical parameters, macroscopic and histopathological findings were observed in this group. After 28 days of treatment, minimal to slight hepatic fatty changes and minimal to slight renal tubular epithelial degeneration and necrosis, considered to be treatment-related, were diagnosed in most rats of group dosed at 1000 mg/kg bw/day. The other histopathological lesions observed in this study are commonly diagnosed in rats of this strain and age and are not considered to be related to treatment with the test substance. The effects found in the subacute toxicity study reflect changes of an adaptive nature up to 200 mg/kg bw/day. Adverse effects are only found in group dosed at 1000 mg/kg bw/day, thus the NOAEL for the subacute toxicity study can be set to 200 mg/kg bw/day.

In a chronic toxicity study (equivalent to OECD 453, Bomhard et al., 1978) the test substance CAS 16470-24-9 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. 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. Therefore, the NOAEL was determined for females at 709 mg/kg bw/day and for males at 521 mg/kg bw/day. This chronic study in the rat is acceptable and satisfies the guideline requirements for a chronic oral study OECD 453 in rats.

The same chronic study (Bomhard E. and Löser E., 1978) was also performed on the acid form of the disulphonated derivative (CAS 4404-43-7). 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).

Furthermore, a non-GLP, two years feeding study with 100, 1000 or 10,000 ppm of a commercial product containing CAS 4193-55-9 (purity unknown) was conducted in Sprague-Dawley rats (Procter and Gamble, 1974 - HPV program). Doses calculated from feed consumption are approximately 0, 5, 50 and 500 mg/kg bw/day). The original test report is not available, nevertheless the results reported in the HPV document confirm the outcomes for the two analogues. No differences in mortality rate, clinical signs, weight gain, blood chemistry, urinalysis or tumour incidence were noted between controls and treated animals. There was a dose-dependent increase of body/liver weight ratio in males (9.5 %, 17.9 % and 35 % at 100, 100 and 10000 ppm, respectively). The finding was without a histological correlate. Therefore, the finding is considered as adaptive and non-adverse. The resulting NOEL of the study is 1000 ppm. The NOAEL was 10000 ppm (app. 500 - 1000 mg/kg bw /day) for male and female rats. The original report is not available to the registrant.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Several studies have been reported and evaluated. A two year study on the acid form of the most similar substance has been considered as key and the related NOAEL selected for the Chemical Safety Assessment.

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).