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Description of key information

NOAEL in male and female rats = 80 mg/kg bw/day (based on the OECD 422 study on OB 3a-DSA)

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
80 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 1-DSA belongs to the category of Stilbene Fluorescent Whitening Agents. The repeated dose toxicity of this category of substances was extensively explored and data on the toxic effects, after oral repeated exposure, are available on the substance in itself as well as for similar substances belonging to the same category. Details on the Read Across approach for category are reported in a document attached in IUCLID section 13.

The potential to cause toxic effects after repeated oral administration in rats of the substance under registration was investigating ina 90-day feeding study((in 1969 by TNO). No sub-acute repeated dose studies were performed on this substance.

No details on purity are provided. The substance was mixed with the diet and administered to Wistar rats at 0, 0.2 %, 1 % and 5 %. Dose calculations were not provided in the report but food consumption data and body weights are reported. Thus, using this information it was possible to estimate for week 12 of the study for the male that 0.2 % group corresponded to 115 mg/kg bw/day and for the female 120 mg/kg bw/day. For the 1 % group the dose was for males 723 mg/kg bw/day and for females 754 mg/kg bw/day. The 5 % group animals did not survive past week 9 of the study. The body weight in the 1 % groups was significantly reduced and at 0.2 % slightly lower. Relative brain, liver and kidney weights were increased at 1 % in both sexes. Testicle weight was decreased in the 1 % group. The serum enzyme levels for glutamic-oxaloacetic transaminase (SGOT) and alkaline phosphatase (SAP) were increased at 1 %. For the differential blood cell counts the percentage of neutrophils was significantly increased and the percentage of lymphocytes was significantly decreased in males at 1 %. Gross and microscopic examinations revealed severe toxic tubular nephrosis in both sexes and testicular atrophy in all males at 1 %. A number of seminiferous tubules only contained sertoli cells and occasional spermatogonia, which contained irregular vacuolated cytoplasm. Partial inhibition of spermatogenesis occurred in other tubules. No pathological changes occurred at 0.2 %.

The NOAEL was identified at 0.2 % (about 120 mg/kg bw/day),but the study may seemcontroversial:the outcomes should be confirmed. Now a Klimish score R2 is assigned: the purity of the substance is not reported and the findings found, mainly in terms adverse effects on fertility parameters, were not confirmed by other available long-term studies either on OB of the same subgroup (1-MSA) and on other category’s members (even with the same degree of sulfonation), irrespective whether the oral administration happened by feed or by gavage.

On the contrary,the adverse effects on kidneyswere also reported in the new performedcombined repeated dose with reproduction and developmental toxicity studieson OB 2-DSA and OB 3a-DSA, highlighting an influence of the highest degree of sulfonation on the excretion apparatus. No effectsin terms of target organshave been seen in any other member of the category at any dose.  

Currently, as supporting information, also available studies carried out according to OECD 422 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening) on analogues 2-DSA and 3a-DSA are presented.

Repeated oral administration of OB 2-DSA at doses of 150, 300 and 600 mg/kg bw/day to rats by gavage did not cause the death of animals. Test item treatment did not produce clinical changes in health status of animals, did not affect the normal growth of males and females.

The haematological examination did not reveal toxic effect on administered animals. The isolated findings were found in all dose levels. These findings were not associated with any pathological and/or histopathological findings of haematogenous organs. Haematological examination in males did not reveal changes of the red blood components in dosed groups in comparison with the control group. Only one statistically significant difference in haemocoagulation parameter - increased value of fibrinogen at the dose level 600 mg/kg/day was recorded. Sporadic changes in the values of white blood components were recorded in treated groups of males. Statistically significantly increased value of lymphocytes and decreased values of neutrophils were recorded in dose groups 150 and 300 mg/kg/day. Decreased value of eosinophils was recorded in males at the dose level 300 mg/kg. All changes were reversible. No statistically significantly differences were recorded in satellite treated group of males compared to satellite control group.

Haematological examination in females showed the changes in values of red blood components in treated groups of females. Statistically significantly increased value of RBC associated with increased value of Hct and decreased MCV were recorded in females at the dose level 600 mg/kg/day in comparison with the control group of females. The values of haemocoagulation parameters were not significantly affected by the test item treatment. The white blood components were not affected by the test item treatment. On the contrary, a decreased value of RBC and associated decreased value of Hct and Hgb were recorded in satellite treated females in comparison with the satellite control females.

However, all haematological values of treated animals (males and females) were in a historical control range.

The biochemical examination of treated animals showed a toxicologically significant effects on creatinine concentration in serum (irreversibly increased values in males and females) and increased concentration of chloride ions in satellite animals. These finding can be associated with the findings reported during the histological examination of kidneys.

Moreover, during the biochemical examination of males, significantly irreversibly changed values (decreased) of Alanine aminotranferase (ALT) and reversibly decreased Urea (BUN) in all dosed groups of males were recorded. Decreased value of Glucose (GLU) in males at the dose level 300 and 600 mg/kg/day and decreased concentration of Calcium ions (Ca) in males at the dose levels 150 and 300 mg/kg/day were recorded in comparison with the control group of males. The value of Creatinine (Crea) was irreversibly increased in males at the dose level 600 mg/kg/day. In satellite males a statistically significant increased value of Creatinine (Crea) and concentration of Chloride (Cl) ions were recorded in satellite treated males compared to control males. Significantly decreased value of Alanine aminotranferase (ALT) and Cholinesterase (CHE) were also recorded in satellite treated males.

As for females, statistically significantly changed biochemical values were recorded only sporadically. Increased value of Total Cholesterol (T-Chol) was reported in females at the dose level 150 and 600 mg/kg/day and increased value of Triglycerides (TG) was recorded in females at the dose level 150 mg/kg/day only. Decreased value of Glucose (GLU) and concentration of sodium ions (Na) were recorded in females at the dose level 150 mg/kg/day only. In satellite treated females, increased values of Creatinine (Crea) and Chloride (Cl) ions and decreased value of Alanine aminotranferase (ALT) were noted in satellite treated females in comparison with the satellite control females.

Although the value of Creatinine (Crea) was not increased statistically significantly in females, dose dependence and elevated values in treated groups compared to control were recorded. This trend was confirmed by a statistically significantly increased value of creatinine reported in a satellite treated females.

All values except the value of Triglycerides were in a historical control range. Increased value of Triglycerides (TG) were out of historical control range in all dosed groups of females.

The examination of urine parameters showed the presence of blood and leucocytes in urine. An increased pH of urine (statistically significantly) was detected in males at the dose level 150 mg/kg/day. Specific gravity was increased in males at the dose levels 300 and 600 mg/kg/day in comparison with the control group of males. Also presence of blood was recorded in males at the dose level 300 and 600 mg/kg/day. The presence of leucocytes was recorded in treated males as well as in control males, but in males at the dose level 300 and 600 was presence of leucocytes in the urine more frequent. In satellite males the volume and pH of urine was statistically significantly decreased in treated males. The blood in urine was recorded in one satellite treated male only.

The test item had toxicology significant effect on weight of organs of treated animals. The weight of kidneys (absolute and relative) was significantly increased in all dose groups of females and this increase was irreversible. This was confirmed by pathological and histopathological examination, where the findings would indicate a toxic effect of the test item on kidneys of dosed animals. In males, statistically significantly decreased absolute and relative weight of spleen were recorded at the dose levels 150 and 600 mg/kg/day. Absolute weight of liver was decreased insignificantly, but relative weight of liver was decreased significantly in males at all dose levels. Increased absolute weight of liver was recorded in dosed females. Statistically significant increasing was recorded at the dose levels 150 and 300 mg/kg/day and insignificantly increased value at the dose level 600 mg/kg/day. Relative weight of liver was significantly increased in females at all dose groups. In satellite treated females, a statistically significant changes in absolute and relative weight of kidneys were recorded in treated animals in comparison with the control satellite animals. 

During the macroscopic examination, findings related to the test item treatment were found out. Changed colour of kidneys.

Histological examination revealed minimal to marked signs of tubular necrosis in kidneys of rats administered by highest dose of test item (600 mg/kg).This lesion was subsequently revealed in some rats from the middle and low dose groups (300, respective 150 mg/kg). This lesion was irreversible during recovery period and was histologically characterized by a variety of findings: dilatation of cortical tubules lined by flattened epithelium, sometimes was epithelium of basophilic color as a sign of reparation. Further, amorphous eosinophilic material or cellular debris were present in the lumen of some tubules, and in some cases chronic interstitial inflammation was found. This basic pattern was accompanied also by tubular vacuolation and presence of hyaline casts.

Histological examination of kidneys was expanded in the case of macroscopic findings in kidneys (very light colour) of test animals. Tubular necrosis in kidneys was also reported in 2 male of the middle group and in 7 females of the low and middle group.

Accordingly, the NOAEL value for repeated dose toxicity in males was established as 150 mg/kg/day and in females was established to be less than 150 mg/kg/day.

The secondtest carried out according to the OECD guideline 422 and under GLP on the analogue substance,OB 3a-DSA,supports these outcomes in terms of target organs and was used in weight of evidence within a read-across approach. This derivative was tested at doses of 80, 250 and 750 mg/kg/day.

Decreases of body weight were reported and were marked in the highest dose level in both sexes and can be considered as biologically significant. No effect of the test item on the food consumption was recorded during the study. Slight changes of these parameters were without toxicological importance.

No clinical findings revealed influence of the test item on clinical status of treated animals and satellite treated animals were recorded.

Statistically significant differences were registered during the haematological examination in red blood components – irreversible increased value of platelet count and delayed decreased value of mean corpuscular volume in males, delayed decreased value of total erythrocyte count, accompanied with decreased values of haemoglobin and haematocrite in satellite treated females. Statistically significantly increased value of fibrinogen in males at the middle and highest dose levels was also recorded. 

In white blood components, statistically significantly increased total leucocyte count in males in the highest dose level and in females in all treated groups was recorded. Decreased percentage portion of monocytes in males in all treated group (without dose dependence) and percentage portion of eosinophils in males in the highest dose level and in satellite treated males were observed. 

The values of total leucocyte count (WBC) and fibrinogen in males were out of the historical control limits.

Increased values of WBC may be related to the organism´s reaction to the test item administration. This effect was reversible, no significant change of value of WBC was observed in satellite treated animals.

Biochemical examination showed following statistically significant differences in males: increased value of creatinine (dose-dependent, irreversible) in the middle and highest dose level; in males in the highest dose level this increasing was out of historical control limits, irreversible increased activity of AST, decreased activity of ALP in all treated dose levels, decreased value of bilirubin total in the middle and highest dose levels, decreased value of protein total, albumin and bile acid in the highest dose level and delayed increased value of cholinesterase.  

In females the values of cholesterol total, triglycerides and inorganic phosphorus (dose dependently) were statistically significantly increased in all treated groups. Increased concentration of inorganic phosphorus was recorded also in satellite treated females; this value was out of historical control limits. Statistically significantly increased value of glucose in middle and highest dose levels was detected (dose dependently). Activity of AST was statistically significantly increased in females in the highest dose level. Concentration of potassium ions in females in the lowest dose level (80 mg/kg/day) was statistically significantly decreased. Delayed increasing of values of calcium ions and bile acid were detected in satellite treated females.  

Irreversible increased concentration of creatinine in males and irreversible increased concentration of inorganic phosphorus in females can be considered as biologically significant and can be related to the histopathological findings of kidneys (tubular necrosis), that were found out during the histopathological examination.

The examination of urine parameters in males showed statistically significantly decreased urine volume in lowest and highest dose levels and in satellite treated males. Moreover, presence of protein in highest dose level and leucocyte in all treated groups were recorded. The presence of blood was recorded in males of middle and highest dose levels.

Changed urine parameters can be related to histopathological findings of kidneys that were found out during the histopathological examination.

Biometry of organs showed statistically significantly delayed increased absolute weight of spleen in both sexes, statistically significantly increased absolute weight of kidneys in females at the middle and highest dose levels and in satellite treated animals of both sexes.

Relative weight of kidneys was increased in males in the highest dose level and in satellite treated males and also in females in the middle, highest dose levels and in satellite treated females. This increasing of kidneys was irreversible. In spleen, relative weight was irreversibly statistically significantly increased in satellite treated males and dose-dependently, irreversibly in females at all treated groups. Relative weight of liver was increased in females in the middle and highest dose levels (irreversibly).

Irreversible increased relative weight of kidneys in males and females can be related to histopathological findings of kidneys that were found out during the histopathological examination.

 

Histopathological examination showed minimal to marked signs of tubular necrosis in kidneys in two males in the middle dose level, six males in the highest dose level and in six males in the satellite treated males. The same finding was found out in three females in the middle dose level, six females in the highest dose level and two satellite treated females. 

This lesion was histologically characterized by variety of findings: dilatation of cortical tubules lined by flattened epithelium, sometimes was epithelium of basophilic colour as a sign of reparation. Further, amorphous eosinophilic material or cellular debris were present in the lumen of tubules, and in some cases chronic interstitial inflammation was found. This basic pattern was accompanied also by tubular vacuolation and presence of hyaline casts.

Histological examination of kidneys was expanded in the case of macroscopic findings in kidneys (very light colour) of test animals. Totally the tubular necrosis on kidneys was recorded in four males in the middle, twelve males in the highest dose level, in seven females in the middle dose levels and eight females in the highest dose level.

Based on these findings, the NOAEL for repeated dose toxicity in males and females was established as 80 mg/kg body weight/day.

In conclusion, the NOAEL values found in all the studies above mentioned are in line with the one obtained for the target substance, confirming an analogous behavior for the disulfonated subcategory after prolonged repeated oral exposure.In this respect, a conservative value was selected from the study carried out on OB 3a-DSA, according to OECD TG 422, were the NOAEL was established to be 80 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 80 mg/kg bw/day, on the basis of the results from an OECD 422 study on rats. The LOAEL is therefore extrapolated as 250 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).