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

Endpoint summary

Description of key information

NOAEL in male rats ≥ 250 mg/kg bw/day (based on the OECD 422 study on OB 3b-A)

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

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 OB 3b-A belongs to the category of Stilbene Fluorescent Whitening Agents.

As for the standard requirements of this registration, the repeated dose toxicity potential is assessed through Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening (OECD 422).

The test item was administered in the form of a solution in aqua pro iniectione to Wistar rats. Oral application by stomach tube was performed daily. The animals were without feed two hours before application and two hours after application of the test item. The study included 3 treated groups (doses 80, 250, 750 mg/kg/day) and one control group (vehicle only) and two satellite groups of animals - one control group (vehicle only) and one treated group (750 mg/kg/day).Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females.

Haematological examination of treated animals showed only sporadic significant differences in haematological parameters in treated animals compared to control animals.

In males statistically significantly decreased value of APTT at the dose level 80 mg/kg/day was recorded, but still in range of historical control. The differential percentage count of monocytes was slightly increased and the count of neutrophils was very slightly decreased in comparison with control group in males at the dose level 80 mg/kg/day (without statistical significance), but the value of monocytes count was over the historical control range. This exceed of the historical control range was due to a marked increase in the differential percentage count of monocytes and the concurrent decrease in the differential percentage count of neutrophils in one male at this dose level.

In the satellite treated males were recorded statistically significantly increased percentage count of lymphocytes statistically, significantly decreased count of monocytes and statistically significantly decreased value of reticulocytes in comparison with control satellite group. All values were in a range of historical satellite control.

No significant changes in haematological parameters were observed in treated groups of females. Only the percentage value of neutrophils was very slightly decreased at the dose level 250 mg/kg/day in comparison with control females (in range of historical control).

In the satellite treated females was noted the statistically significantly decreased value of reticulocytes, but still in range of the historical satellite control.

All findings in the haematological parameters observed in both sexes were considered to be of no toxicological significance. These isolated findings in haematological examination did not reveal toxic effect of the test item on administered animals.

During biochemical examination of animals, statistically significantly changed values were detected.

In males, significantly increased values of calcium ions (Ca) and decreased values of creatinine (Crea) were detected at all dose levels, without dose dependence. The decreased values of creatinine were out of the historical control range at the dose levels 80 and 750 mg/kg/day. The values of alanine aminotransferase (ALT) were dose-dependently increased in males at all dose levels, at the dose level 250 and 750 mg/kg/day with statistical significance, but reversible (not observed at the satellite treated males). The value of ALT was out of the historical control range at the highest dose level. This exceed of the historical control range was due to a marked increased value of ALT only in one male at the highest dose level. The value altered at all dose levels (statistically significantly) compared to control animals was cholinesterase (CHE) – decreased at the dose level 80 mg/kg/day and increased at the dose levels 250 and 750 mg/kg/day (in range of the historical control range). Also the values of bile acids (BA) were increased at all treated groups, but without statistical significance (in range of historical control).

Significantly decreased values of chlorides ions were reported in males at the dose levels 250 and 750 mg/kg/day, but still in range of historical control range. In males at the dose level 250 mg/kg/day was detected statistically significantly increased value of phosphorus (in the historical control range). All these changes in biochemical parameters in males were reversible, not observed in satellite treated males. A statistically significant decreased values of total protein, albumin and triglycerides were recorded in satellite treated males in comparison with the satellite control males, but in range of the historical satellite control. This could be associated with microscopical changes in liver - hepatitis. The values of other biochemical parameters of satellite treated males were similar to the satellite control group.

During the biochemical examination of females, significantly changed biochemical values were recorded only sporadically in treated females. Significantly increased value of total cholesterol was reported in females at the dose level 250 mg/kg/day (in historical control range). Significantly increased values of calcium ions were recorded in females at the dose levels 250 and 750 mg/kg/day, but also in range of the historical control. Values of ALT and AST were insignificantly increased and out of the historical control range at the dose level 750 mg/kg/day. These exceeds of the historical control ranges were due to a marked increased values of ALT and AST in one female at this dose level (female No. 170). The value of triglycerides was out of the historical control range for the same reason at the dose level 250 mg/kg/day, the value of triglycerides was marked increased only in one female at this dose level. Other biochemical parameters of treated females were comparable to the control group.

Statistically significantly decreased value of cholinesterase and increased value of total cholesterol were noted in satellite treated females in comparison with the satellite control females (in range of the historical satellite control). Values of other biochemical parameters of treated satellite females were comparable to the control group.

The biochemical examination showed a toxicologically significant effect of the test item on the treated males. The increased values of ALT were caused by liver damage in males. Decreased creatinine levels may be associated with decreased synthetic liver function. Also increased values of cholinesterase, bile acids may be associated with the findings reported during the histological examination of liver.

Biochemical examination of treated females did not show a toxicologically significant effect of the test item.

Urinalysis did not reveal any statistically significant changes of pH. The presence of proteins, blood and leucocytes were recorded in treated males as well as in control males and were not associated with the application of the test item.

During biometry of organs in males and females, significant changes of absolute and relative weights of organs related to the administration of the test item were not recorded.

During the macroscopic examination, no findings related to the test item treatment were found out.  

Histological examination recorded changes related to the test item treatment only in the males. The focal polymorphonuclear inflammation (hepatitis) was detected in liver of five males at the dose levels 750 mg/kg/day. This finding was recorded only in one female at the highest dose level. This finding was not fully reversible, because of occurrence of the focal polymorphonuclear necrotic inflammation in two satellite males. The histopathological examination of the genital organs revealed the tubular degeneration of testicles in one control male and in three males at the dose level 750 mg/kg/day. Because the spermiogenesis of the males was not affected with the test item treatment, this change was considered as non-specific, unrelated to the effect of the test item treatment. The incidence of other microscopical findings were in most cases spontaneous, agonal changes, or sporadic, not associated with the test item treatment.

According to the study results, the NOAEL value for repeated dose toxicity in males was established as 250 mg/kg body weight/day and in females was established as 750 mg/kg body weight/day.

Recently, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening (OECD 422), following GLP’s principles, were also performed on other category’s members.

OB 4-MSA was tested at doses of 80, 250 and 750 mg/kg/bw and no toxicologically adverse effects of the test item on growth of animals, food consumption and health condition of animals were detected in both sexes.

Repeated oral administration of the test item, to rats by gavage did not cause any mortality (except one male from the dose level 250 mg/kg/day). This death was accidental and not treatment-related.

The body weight of parental males and females was not affected by the test item administration and no treatment-related changes of haematological parameters were found in both sexes.

Biochemical examination in treated animals of both sexes showed following statistically significant differences: increased value of protein total in the middle (250 mg/kg/day) and highest dose levels (750 mg/kg/day), decreased concentration of sodium ions in the highest dose level, delayed increased concentration of sodium ions (opposite to main highest dose) and delayed decreased activities of ALT, AST and ALP in satellite males. In females the activity of ALT was statistically significantly increased in the lowest and middle dose levels, activity of AST was increased in the lowest dose level and value of sodium ions was increased in satellite treated females. All biochemical parameters were in a range of historical control limits.  

Biometry of organs showed statistically significantly decreased absolute weight of spleen in males in the lowest dose level and reversibly significantly increased relative weight of epididymis in males in the highest dose level. In females; reversibly significantly decreased absolute and relative weight of ovaries in all treated groups, delayed statistically significantly increased absolute and relative weight of pituitary gland (satellite treated females) and statistically significantly increased relative weight of kidneys in the lowest and highest dose level were recorded. All changed weights of organs in treated and satellite treated animals have no biological or toxicological significance.

No findings related with the test item treatment were detected during the histopathological examination of organs in animals at the highest dose level and satellite treated animals.

A further Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening is available and was carried out on OB 3a-A(Na), with the same conditions and doses as the one performed for OB 4-MSA.

Repeated oral administration of the test item, to rats by gavage did not cause any mortality (except one male from the dose level 250 mg/kg/day). This death was accidental and not treatment-related.

The body weight of parental males and females was not affected by the test item administration.

Evaluation of the absolute and relative weight of reproductive organs of male and female, as well as the weight of pituitary and thyroid gland, did not reveal any statistically significant differences in organ weight.

Test item treatment did not affect male ability to produce sperm that can fertilise eggs and ability of females to become pregnant.

No adverse effect of the test item treatment was observed during examination of thyroxine and rat thyroid stimulating hormone blood concentration in males.  

Histological examination did not reveal negative effect of the test item on collected organs of reproductive organs (pituitary and thyroid gland). Spermatogenesis in the testes of the high dose administered males was without any pathological findings. Epididymides of both control and high dose males were without any pathological findings. Sporadic findings were of spontaneous origin. The administration of test item did not cause pathological changes in the organs. Examination of sperm motility and morphology in treated parental males did not show any differences in comparison with the control males. The administration of test item did not cause damage of sperms.

The number of implantations was comparable between treated and control groups. The test item did not affect the number of pups and their development.

Under test condition, the NOAEL (No Observed Adverse Effect Level) value was established as 750 mg/kg body weight/day.

In conclusion, the NOAEL values found in all the studies above mentioned confirmed an analogous behavior of this category of substances after prolonged repeated oral exposure. For all category members, a conservative value was however selected from the study carried out on the substance under registration, according to guideline OECD 422, where the NOAEL for male rats was established to be 250 mg/kg bw/day.

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 No Observed Adverse Effect Level was established at 250 mg/kg bw/day, on the basis of the results from an OECD 422 study on rats. Therefore, the LOAEL can be extrapolated as 750 mg/kg bw/day. The duration of the treatment was 49 days for males and 54 for females.

As reported in the CLP guidance, Annex I: 3.9.2.9.5. "The guidance values refer to effects seen in a standard 90-day toxicity study conducted in rats. They can be used as a basis to extrapolate equivalent guidance values for toxicity studies of greater or lesser duration, using dose/exposure time extrapolation similar to Haber’s rule for inhalation, which states essentially that the effective dose is directly proportional to the exposure concentration and the duration of exposure. The assessment shall be done on a case-by-case basis; for a 28-day study the guidance values below is increased by a factor of three."

For this reason, taken into account the duration of treatment of males, an assessment of 1.8 was applied and the extrapolated limit values for classification were adapted as follows:oral (rat): 18 < C ≤ 180 mg/kg bw/day.

In conclusion, as the LOAEL value falls above the upper limit of classification, the substance is not classified for repeated dose toxicity according to the CLP Regulation (EC 1272/2008).