Hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-[(2-cyanoethyl)[2-(2-hydroxyethoxy)ethyl]amino]-1,3,5-triazine-4,2-diyl]imino]]bis[benzene-1,4-disulphonate]

Endpoint summary

Description of key information

Oral NOAEL (28d) (male and female): 200 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

From April the 04th to May the 16th, 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

Justification for read across is detailed in section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted on 12 May, 1981

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH Wiga, Sulzfeld, West Germany.
- Age at study initiation: ca. 6 weeks.
- Weight at study initiation: males 169 - 220 g; females 132 - 167 g
- Total no. of animals: 20 males and 20 females.
- No. animals per group: 5 males and 5 females.
- Accomodation: housed 5 to a cage (same sex) in stainless steel suspended cages with wire mesh floors.
- Diet: free access to standard pelleted laboratory animal diet; certificate of analysis performed.
- Water: tap water ad libitum; certificate of analysis performed.
- Acclimatisation: fourteen days (7 days pre and 7 days post randomisation). Veterinary examination performed.
- Identification: earmark and tattoo.
- Randomisation: computer-generated random algorithm according to body weight.

ENVIRONMENTAL CONDITIONS
- Temperature: 18 - 22 °C
- Relative humidity: 35 - 80 %
- Air changes: 7.5 air change per hour.
- Photoperiod: 12 hours artificial light / 12 hours dark.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

TEST ARTICLE PREPARATION
- Method: weighed into a glass flask on an analytical balance and the vehicle (w/w) added.
- Frequency of formulation: daily immediately prior dosing.
- Storage temperature of food: at ambient temperature.

DIET PREPARATION
- Concentration in vehicle: 5 ml/kg bw

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Analysis of formulations: samples of formulations prepared during weeks 1 and 5.
Concentration of the test substance in vehicle was determined on day 2 and 29. Actual concentrations of preparations were in agreement with the treatment levels.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, approximately at the same time each day; 7 days per week.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (control)

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

MORTALITY
Twice daily.

CLINICAL OBSERVATIONS
At least once daily.

BODY WEIGHT
Weekly and on the day preceding termination, prior to overnight fasting.

FOOD CONSUMPTION
Weekly.

HAEMATOLOGY
Blood samples were collected under light ether anesthesia immediately prior to post mortem examination, between 8.30 and 10.30 a.m.. The animals were fasted overnight before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and
collected into tubes prepared with EDTA for haematoiogicai parameters (0.6 ml) and untreated tubes for clinical biochemistry parameters (>2 0 ml).
Parameters: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, red cell distribution width, platelet count, total leucocyte count, differential leucocyte count.

CLINICAL CHEMISTRY
Blood samples were collected under light ether anesthesia immediately prior to post mortem examination, between 8.30 and 10.30 a.m.. The animals were fasted overnight before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and
collected into tubes prepared with EDTA for haematoiogicai parameters (0.6 ml) and untreated tubes for clinical biochemistry parameters (>2 0 ml).
Parameters: glucose, urea, creatinine, bilirubin total, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, sodium, potassium, chloride, calcium, phosphorus, protein total, protein albumin.

Sacrifice and pathology:

NECROPSY
All animals were necropsied and descriptions of all macroscopic abnormalities recorded. All animals survived to the end of the observation period (day 29) and were anaesthetised with pentobarbitone and killed by exsanguination.
Samples of the following tissues (and any noted gross abnormalities) and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution: adrenal glands, heart, kidneys, liver, spleen, stomach and testes.

ORGAN WEIGHTS
The following organ weights (and terminal body weight) were recorded at termination on the scheduled dates of necropsy listed: adrenal glands, heart, kidneys, liver, spleen and testes

HISTOPATHOLOGY
All organ and tissue samples, as defined below, were processed, embedded and were cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.
Slides of adrenals, heart, kidneys, liver and spleen, collected at termination from all animals of the control and high dose group, any macroscopically noted abnormalities and slides of kidneys from the intermediate groups were examined by a pathologist.

Statistics:

The following statistical methods were used to analyse the body weight, food consumption, organ weights and clinical laboratory data: univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups.
The Steel-test (many—one rank test) was applied when the data could not be assumed to follow a normal distribution.
All tests were two-sided and in all cases p<0.05 was accepted as the lowest level of significance.
Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs noted that were considered related to treatment with the substance.
Incidental findings that were noted included one rat with alopecia and encrustations and one incidence of respiratory distress.

Mortality:

no mortality observed

Description (incidence):

There was no mortality during the course of treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights and body weight gain by rats receiving 1000 mg/kg/day were decreased when compared with control values over the 4 weeks of treatment (although this difference did not achieve a level of statistical significance in males at any time).
Body weights and body weight gain by rats receiving 50 or 200 mg/kg/day remained essentially similar to those of the controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Females receiving 1000 mg/kg/day had apparently reduced food consumption in comparison with controls. However, this difference was not clear after adjustment for body weight. Differences in food consumption, therefore, probably reflect the differences in body weight noted.
There were no differences in the food consumption by treated males and females receiving 50 or 200 mg/kg/day before and after correction for body weight, compared to controls.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no differences of toxicological significance between treated rats and controls.
The incidental haematological parameters that did achieve a level of statistical significance i.e. increased neutrophils and decreased lymphocytes in females receiving 1000 mg/kg/day, were considered not to be of biological significance and within the range normally seen in rats of this age and strain.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment related changes in serum biochemistry were considered to be centred around electrolyte levels. However, there were no clearly consistent alterations between males and females. Treated females and males receiving 1000 mg/kg/day had statistically significantly decreased potassium levels, whereas in males receiving 200 mg/kg/day, increased potassium values were noted. Treated males had calcium levels that were significantly decreased, but this effect was not noted in females.
Chloride levels in females receiving 50 or 1000 mg/kg/day were significantly decreased compared to controls and although there was evidence of decreased chloride levels in treated males, this difference did not achieve a level of statistical significance. Males receiving 200 mg/kg/day had statistically significantly decreased sodium values, but this effect was not seen in treated females and did not achieve a level of statistical significance in the other treated males.
Any other clinical biochemistry values that achieved a level of statistical significance when compared with control values such as changes in serum enzymes and protein values, were considered within the range normally seen in rats of this age and strain and not to of toxicological significance.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Kidney weights of rats receiving 1000 mg/kg were statistically significantly greater when compared with controls after adjustment for body weight.
The increase in testis weights of males receiving 200 mg/kg/day (the increase in males receiving 1000 mg/kg/day can be discounted as it attains a level of significance only after adjustment for body weight and testis weight is not directly related to body weight) and the increased spleen weight (after adjustment for body weight) in females receiving 1000 mg/kg/day were considered to have arisen fortuitously and in the absence of supportive clinical pathological or histopathological evidence to be of no toxicological significance.
Organ weights of rats receiving 50 or Females receiving 200 mg/kg were similar to those of controls before and after allowance for body weight.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

A macroscopically noted abnormality that was considered to be related to treatment was pale discolouration of the kidneys. This was noted in 2/5 males and 4/5 females receiving 1000 mg/kg/day, but not noted among rats of the other treated groups.
Incidental findings that were also noted included pelvic dilation of the kidneys in one rat, haemorrhage in the mandibular lymph nodes of one rat and haemorrhage in the stomach of one rat.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males and females receiving 1000 mg/kg/day showed slight renal tubular necrosis. Renal tubular inclusion bodies were noted in males and females receiving 1000 mg/kg/day.
These inclusion bodies were absent in control females, but present, in small numbers, in control males and in males receiving 50 or 200 mg/kg/day. The incidence and severity of these inclusion bodies in male controls and those receiving 50 or 200 mg/kg/day was considered within the normal background range

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

gross pathology

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

NOAEL (28d) (male and female): 200 mg/kg bw/day

Executive summary:

A subacute 28-day toxicity study was conducted with test item; the substance was administered daily by gavage to SPF-bred Sprague-Dawley rats. 4 groups, each comprising 5 males and 5 females were selected and treated at the following dose levels: 0 (control), 50, 200 and 1000 mg/kg bw/day.

At 50 mg/kg/day changes in serum electrolyte levels (decreased potassium and chloride in Females only and decreased calcium in males only) were recorded.

At 200 mg/kg/day changes in serum electrolyte levels (decreased potassium in females only, decreased calcium and sodium in males only and increased potassium in males only) were recorded.

At 1000 mg/kg/day the effects observed were decreased body weight and body weight gain; changes in serum electrolyte levels

(decreased potassium in males and females decreased calcium in males only and decreased chloride in females only); macroscopically observed pale discolouration of the kidneys; increased kidney weights; and microscopically observed renal tubular necrosis and renal inclusion bodies.

Treatment with test item produced changes in serum electrolyte parameters that could be supported by the changes noted microscopically in the kidneys, although no microscopic lesions were noted in the intermediate groups.

A no effect level (NOEL) could not be definitively determined, but it must be considered that the serum electrolyte changes in the intermediate dose groups have no corroborative microscopical evidence and given the inconsistent nature of these changes, the toxicological significance may be considered doubtful.

Conclusion

NOAEL (28d) (male and female): 200 mg/kg bw/day

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

From December the 05th, 1989 to January the 16th, 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

Justification for read across is detailed in section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH Wiga, Sulzfeld, West Germany.
- Age at study initiation: ca. 6 weeks.
- Weight at study initiation: males 124 - 230 g; females 126 - 175 g
- Total no. of animals: 40 males and 40 females.
- No. animals per group: 10 males and 10 females.
- Diet: free access to standard pelleted laboratory animal diet; certificate of analysis performed.
- Water: tap water ad libitum; certificate of analysis performed.
- Acclimatisation: fourteen days (7 days pre and 7 days post randomisation). Veterinary examination performed.
- Accomodation: housed 5 to a cage (same sex) in stainless steel suspended cages with wire mesh floors.
- Identification: earmark and tattoo.
- Randomisation: computer-generated random algorithm according to body weight.

ENVIRONMENTAL CONDITIONS
- Temperature: 18 - 21 °C
- Humidity: 47 - 71 %
- Air changes: 15 ACH
- Photoperiod: 12 hours dark.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

TEST ARTICLE PREPARATION
- Method: weighed into a glass flask on an analytical balance and the vehicle (w/w) added.
- Frequency of formulation: daily immediately prior dosing.
- Homogeneity of test: following shaking the test substance formed a solution in water.
- Storage temperature of food: at ambient temperature.
- Dose selection rationale: based on data from previous studies in rats.

DIET PREPARATION
- Concentration in vehicle: 5 ml/kg b.w.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days.

Frequency of treatment:

Once daily, approximately at the same time each day; 7 days per week.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (control)

Dose / conc.:

2 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

MORTALITY
Twice daily.

CLINICAL OBSERVATIONS
At least once daily.

BODY WEIGHT
Checked weekly and on the day preceding termination, prior to overnight fasting.

FOOD CONSUMPTION
Checked weekly.

CLINICAL BIOCHEMISTRY
Blood samples were collected under light ether anesthesia immediately prior to post mortem examination, between 8.00 and 10.00 a.m. The animals were fasted overnight before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0.6 ml).
- Parameters: sodium, potassium, chloride, calcium, phosphorus

Sacrifice and pathology:

SACRIFICE
At the end of the study (day 29) all animals were sacrificed by carbon dioxide asphyxiation following blood sampling.

Statistics:

Used to analyse the body weight and clinical laboratory data: univariate one-way analysis of variance, steel-test, dunnett-test.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity or behavioral changes over the 29-day observation period that were considered to be related to treatment.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred over the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the 4 weeks study period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no differences in food consumption before and after allowance for body weight between treated and control animals.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no differences in serum electrolyte levels noted between control and treated rats.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity or behavioral changes.

Dose descriptor:

NOEL

Effect level:

>= 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

NOEL (28d) (male and female) ≥ 50 mg/kg bw/day

Executive summary:

A subacute 28-day toxicity study was conducted with test item ; the substance was administered daily by gavage to SPF-bred Sprague-Dawley rats. The study was intended to examine a possible effect of the test substance on serum electrolyte levels at dose levels of 50 mg/kg/day or lower. A total of 4 groups, each comprising 10 males and 10 females were selected and treated at the following dose levels: 0 (control), 2, 10 and 50 mg/kg bw/day.

A "No Observed Effect Level" could not be established in the preliminary assay, as result of slight and doubtful electrolyte disturbances at the lowest treatment level (50 mg/kg/day); therefore, an additional oral 28 day study was initiated in order to examine serum electrolyte levels following treatment at 0, 2, 10 or 50 mg/kg/day.

No mortality occurred over the study period and there were no clinical signs of toxicity or behavioral changes over the 28-day observation period that were considered to be related to treatment. Body weights and body weight gain of treated animals remained in the same range as controls over the 4 weeks study period. There were no differences in food consumption before and after allowance for body weight between treated and control animals. There were no clinical signs of toxicity or behavioral changes.

Conclusion

NOEL (28d) (male and female) ≥ 50 mg/kg bw/day

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

There is no information about the repeated dose toxicity potential of Optical Brightener 380, thus the available data on the structural analogous Similar Substance 01 have been taken into consideration for the assessment. The read across approach can be considered reliable and appropriate to investigate the property (details for the approach are included into the IUCLID section 13).

A subacute 28-day toxicity study was conducted with Similar Substance 01; the substance was administered daily by gavage to SPF-bred Sprague-Dawley rats. 4 groups, each comprising 5 males and 5 females were selected and treated at the following dose levels: 0 (control), 50, 200 and 1000 mg/kg bw/day.

At 50 mg/kg/day changes in serum electrolyte levels (decreased potassium and chloride in Females only and decreased calcium in males only) were recorded.

At 200 mg/kg/day changes in serum electrolyte levels (decreased potassium in females only, decreased calcium and sodium in males only and increased potassium in males only) were recorded.

At 1000 mg/kg/day the effects observed were decreased body weight and body weight gain; changes in serum electrolyte levels (decreased potassium in males and females decreased calcium in males only and decreased chloride in females only); macroscopically observed pale discolouration of the kidneys; increased kidney weights; and microscopically observed renal tubular necrosis and renal inclusion bodies.

Treatment with test item produced changes in serum electrolyte parameters that could be supported by the changes noted microscopically in the kidneys, although no microscopic lesions were noted in the intermediate groups.

A no effect level (NOEL) could not be definitively determined, but it must be considered that the serum electrolyte changes in the intermediate dose groups have no corroborative microscopical evidence and given the inconsistent nature of these changes, the toxicological significance may be considered doubtful.

In order to clarify the study outcomes, a second subacute 28-day toxicity study was conducted with Similar Substance 01; the substance was administered daily by gavage to SPF-bred Sprague-Dawley rats. The study was intended to examine a possible effect of the test substance on serum electrolyte levels at dose levels of 50 mg/kg/day or lower. A total of 4 groups, each comprising 10 males and 10 females were selected and treated at the following dose levels: 0 (control), 2, 10 and 50 mg/kg bw/day.

No mortality occurred over the study period and there were no clinical signs of toxicity or behavioural changes over the 28-day observation period that were considered to be related to treatment. Body weights and body weight gain of treated animals remained in the same range as controls over the 4 weeks study period. There were no differences in food consumption before and after allowance for body weight between treated and control animals. There were no clinical signs of toxicity or behavioural changes.

The No Observed Effect Level was established to be equal/higher than 50 mg/kg bw/day.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 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.

In order to help reach a decision about whether a substance shall be classified or not, and to what degree it shall be classified (Category 1 or Category 2), dose/concentration ‘guidance values’ are provided for consideration of the dose/concentration which has been shown to produce significant health effects. The guidance values refer to effects seen in a standard 90-day toxicity study conducted in rats. Nevertheless, they can be used as a basis to extrapolate equivalent guidance values for toxicity studies of greater or lesser duration (the assessment shall be done on a case-by- case basis). For example, for 28-day study the guidance values are increased by a factor of three, thus the limit for sub-acute studies should be 300 mg/kg bw/day.

In the specific case, the No Observed Adverse Effect Level was established at 200 mg/kg bw/day; on the basis of the results from the subacute study on rats, it can be reasonably expected that relevant effects would not be seen at concentrations lower than 300 mg/kg bw/day.

In conclusion, the substance does not meet the criteria to be classified for repeated dose toxicity, according to the CLP Regulation (EC) No 1272/2008.