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Description of key information

NOAEL in male rats 250 mg/kg bw/day (based on the sub-acute 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 2-A 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.

For the target substance a sub-acute study, a summary of asub-chronicstudy and achronicstudy are available.

A subacute 28-day toxicity study was performed according to the OECD guideline 407, under GLP conditions, administering the test substance daily by gavage to SPF-bred Wistar rats (report n. 288505, 1991). The study was comprised of four groups dosed at 0, 20, 200 and 1000 mg/kg.

No premature death occurred. There were no toxicologically relevant significant effects on absolute or relative food consumption, on the body weight in the test article treated animals when the results were compared to those of the controls.

No clinical signs were observed during the entire study period. No clinical abnormalities were found on ophthalmoscopy.

The assessment of hematology, clinical biochemical and urinalysis data indicated no changes of toxicological significance at termination of the treatment nor at the end of the treatment-free recovery period.

No changes in absolute and relative organ weights were observed after 4 weeks of treatment nor at termination of the treatment-free recovery period when the results from the animals of the test article treated groups were compared to those of the control animals.

No treatment related findings were recorded at termination after 4 weeks of treatment nor at termination of the treatment-free recovery period.

Based on the results obtained the "no-adverse-effect-level" of the test substance is 1000 mg/kg body weight for male and female rats when administered orally by gavage for a period of 28 days followed by a treatment-free 14-day recovery period.

The various statistically significant differences observed (increase/decrease) in food consumption (absolute/relative) and organ weights (relative) were considered to be of no toxicological relevance and did therefore not provide any evidence of toxicity at any of the dose levels tested.

Thesub-chronictoxicity of the test item was investigated in a feeding study with rats. 25 male and female animals received for 90 days 50, 500 and 5000 ppm of the test substance, corresponding to 250 to 500 mg/kg. Body weight and food consumption were monitored continuously; blood urea from the transaminases SGPT, red and white blood cells, haemoglobin and differential count were measured at regular intervals. After 3 months the animals were killed and examined macroscopically. The weight of spleen, adrenal glands, kidneys, testes/ovaries and liver were measured in 10 animals and recorded as percent of body weight. The removed organs were used for histological examination.

The analyses did not reveal significant abnormalities respect to the control. The No Adverse Effect Level can be stated at 5000 ppm for both male and female rats.

In the chronic study, male and female rats received test for 2 years with the feed (report n. 7234, 1978),

in concentrations of 0, 100, 1000 and 10000 ppm. The treated animals showed no treatment-related symptoms during the entire experimental period in any dose group. Growth, feed intake and mortality were unaffected. The hematological investigation activities carried out during and at the end of the experiment showed no injuries.

Despite 1 month after the blood test revealed dose-dependently and significantly elevated (P <0.05) platelet levels in females, this was not considered compound-related toxic effect, since the investigation at all remaining time points did not confirm these findings and all values ​​are within the norm.

At all the doses tested the clinical chemistry analysis, sections and histopathological studies revealed no evidence of treatment-related damage to the liver.

One month after the start of the experiment in male rats of the dose group 10000 ppm significantly increased GPT activity was measured; it was not considered as sign of damage, since all values ​​are in the range of physiological variation and the investigation into the later trial dates did not report corresponding findings.

Urinalysis, urea and creatinine concentrations in serum as well as macroscopic, gravimetric and histopathological findings did not indicate kidney injuries. No reference to a specific damage that might be caused by the drug was revealed by the gross pathology analysis. Significant differences found in organ weights are considered to be due to chance or due to differences in animal weights, because the haematological, clinical chemical and histopathol analysis revealed no effects correlated.

Histopathological examination of the brain, pituitary, eyes, lymph nodes, salivary glands, thyroid, aorta, heart, lung, spleen, pancreas, esophagus, stomach, intestine, adrenal glands, bladder, testes, epididymides, prostate, seminal vesicle, uterus, ovaries, skeletal muscle and bone showed the usual age-related, spontaneous alterations. Changes which might be associated with the drug administration, were not revealed.

From the nature, site and frequency of observed benign and malignant tumors, a carcinogenic effect of substance was excluded. Doses up to 10000 ppm (included) were thus tolerated without any damage. The NOAEL value found, converted in mg/kg bw/day, was established to be ≥ 523.9 mg/kg bw/day for males and ≥ 790.6 mg/kg bw/day for females.

A subacute toxicity study is available on OB 3a-MSA. However, the findings are questionable anda Klimish score R3 was assigned to this test.Indeed, a deeper and longer study, according to the OECD guideline 408, on this substance denied any toxicological effects on liver, kidney, blood and testes as target tissues.

In particular,the oral administration of3a-MSAto 3 treated groups (doses 50, 150, 450 mg/kg of body weight/day) of rats by gavage for a period of 90 consecutive days did not cause mortality related with the test item treatment. No effect of the test item on the body weight, food consumption and water consumption were recorded during the study. Slight changes of these parameters were without toxicological importance.

No clinical findings or changes of urine parameters revealed harmfulinfluence of the test item. Haematological examination showed sporadic findings in males: decreased value of fibrinogen (middle and highest doses) and increased value of eosinophiles (lowest and middle doses). Sporadic changes were detected in females: increased total erythrocyte count and value of platelets count (lowest dose), decreased value oftotalleucocyte count (middle dose) and decreased value of fibrinogen and value of APTT (highest dose). All changes were reversible, not dose-dependent and all values were in a historical control range.

Biochemical examination showed reversibly increased value of albumin in males at all dose levels. In both sexes the value of glucose was decreased (reversibly) at all dose levels in males and at the middle and highest dose levels in females. Value of ALP was reversibly decreased in all dosed groups of males and females Also the value of triglycerides in females was reversibly decreased in all dosed groups. All biochemical parameters were in a historical control range. No significantly changed value was detected in satellite treated males and females.

Examination of thyroid hormones of treated group of animals in comparison with the control animals did not reveal adverse effect of the test item administration.

Biometry of organs showed significantly decreased absolute weight of testes and increased relative weight of liver in satellite males only.

Notoxicologically significantfindings were detected during the histopathological examination of organs in animals at the highest dose level and satellite treated animals. Sporadic findings recorded in adrenal glands, heart, liver and thymus were not related with test item treatment.  

No adverse treatment-related response that results in change in morphology, physiology or growth of an animal organism was recorded. The NOAEL in male and female rats for this test item was established as 450 mg/kg body weight/day.

An additional 70d study is available for OB 3a-MSA (Kimmerle, 1967). Groups of 6 male and 6 female rats were given during 10 weeks (5 doses/week) 30, 60, 120, 250 or 500 mg of the test substance/kg as a solution in oil administered by gavage. A further group of 12 male and 12 female rats received daily 5.0 ml of oil/kg (control). During the experimental period, the condition of the animals was observed daily, body weights (all animals) were weekly recorded; blood and Harn status (3 males and 3 females per dose) were analyzed at 14 -days, and at the end of the test, the prothrombin time (3 males and 3 female per group) and in all animals the SGPT and SGOT Transaminases and SDH were determined 24 hours after the last administration, the animals were sacrificed with ether and the internal organs (liver, kidneys, adrenals, thyroid, spleen, ovary, testis, lung) macroscopically examined and weighed.

No adverse effects were recorded: the animals of the experimental groups showed no apparent symptoms of poisoning and behaved similarly to the control animals.

The body weight is not significantly altered in the experimental groups compared with the control groups. The complete blood count, the checks of liver function tests and the urine tests did reveal no pathological findings.

After killing the rats, the internal organs in the experimental groups compared with the control groups did not appear changed and a significant dose-dependent change in the absolute and relative organ weights could not be determined (P > 0.1) in the experimental groups compared with the control group.

The NOAEL value established in this study was therefore ≥ 500 mg/kg bw/day, in line with the above-mentioned study.

The analogue substance OB 1-MSA was tested for its potential to cause toxic effects after repeated oral administration in rats, according to the OECD guideline 408, under GLP conditions (report n. 20-159, 2020).

In a dose-range finding experiment, animals were administered by oral gavage at 100, 300 and 1000 mg/kg bw/day. No animal died during the application period.

Clinical observations did not detect any impact of the test item on the health conditions of animals at any dose level.

The administration of the test item did not induce treatment-related toxicologically significant changes.

No macroscopical changes were observed during necropsy of treated animals.

Based on the results obtained in the DRFE and all available data on category’ members (following described), the main study was carried out following an oral administration by gavage at 120, 350 and 750 mg/kg bw/day.

The administration to rats for a period of 90 consecutive days at the lowest dose level did not cause mortality related with the test item treatment.

No effect of the test item on the body weight, food consumption and water consumption were recorded during the study. Slight changes of these parameters were without toxicological importance.

No clinical findings or changes of urine parameters revealing influence of the test item were recorded.

Haematological examination showed no findings in males. Sporadic changes were measured in females: prolonged decreased total erythrocyte count, reversible increased value of platelets count (lowest and highest doses), increased mean corpuscular volume (lowest and middle doses) and reversible decreased prothrombin time (highest dose). These differences were without dose response relationship and without correlation with other manifestations of toxicity.

Biochemical examination showed irreversible increased concentration of inorganic phosphorus in males (middle and highest doses). In females, inorganic phosphorus concentration was decreased only at the lowest dose level. Concentration of chloride ions was increased only in females (middle and highest dose levels). Value of sodium ions was decreased in both sexes of satellite animals. Other changes of measured biochemical parameters observed were without dose dependence, sex correlation and without association with related toxicologically significant endpoints.

Biometry of organs showed significantly decreased absolute and relative weight of spleen in females. These changes persisted in satellite females but without statistical significance. This effect is isolated. Changes in other related parameters usually associated with the effect of concern were not observed: no significant changes of red and white blood parameters and no histopathological findings.

No toxicologically significant findings were detected during the histopathological examination of organs in animals at the highest dose level and satellite treated animals. Sporadic findings in epidydimis, testis and thymus were not related with test item treatment.

The NOAEL (No Observed Adverse Effect Level) value for male and female rats was established as 750 mg/kg body weight/day.

The value of NOAEL was established on the basis of no adverse effects found during haematological, biochemical and histopathological examination. Biometric changes of the spleen do not correlate with other toxicologically important endpoints.

 An additional test carried out on an analogue substance, OB 5-A, according to the OECD guideline 408 and under GLP (report n. 20-184, 2020), was used in weight of evidence within a read-across approach.

The oral administration of the test item did not cause mortality.

No effects of the test item on the body weight, food consumption and water consumption were recorded during the study. Slight changes of these parameters were without toxicological importance.

No influence of the test item on clinical status of treated animals and satellite treated animals was recorded. No findings were detected during the histopathological examination of liver, spleen and kidneys of animals at the highest dose level and satellite treated animals.

All changes detected during the haematological and biochemical examinations were reversible (except decreased activity of ALP in males and delayed decreased activity of ALT and AST in males). All these changes observed in animals had probably an adaptive character - the organism's response to the test item treatment. A longer recovery time would probably be required to completely eliminate the altered hepatic enzyme activity in males.

All changed values of biochemical parameters in satellite treated animals have no biological or toxicological significance.

The value of NOAEL was established as 750 mg/kg body weight/day for both males and females.

Different Combined Repeated dose with Reproduction and Developmental toxicity studies are furthermore available.

As for OB 3a-A(Na) and OB 4-MSA, both studies did not show toxic effects and the NOAEL values found were ≥ 750 mg/kg bw/day in both cases

On the contrary, toxicologically significant changes, mainly in treated males, were recorded in the study carried out on OB 3b-A. Damage of liver, confirmed by the histological examination and evaluation of biochemical parameters, was observed in males at the highest dose level, i.e. 750 mg/kg/day. The statistically significantly increased value of alanine aminotransferase and statistically significantly decreased value of creatinine (out of the historical control ranges) was recorded in males at the same dose. Histopathological finding – the focal polymorphonuclear inflammation in the liver (hepatitis) – was reported in males at the dose level 750 mg/kg/day and only in one female at this dose level. This finding was not fully reversible. Two satellite high dose males showed also this lesion (focal polymorphonuclear necrotic inflammation) at the end of recovery period.

 

Accordingly, 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.

 

Moreover, experiments investigating the effects of long-term feeding administration of OB3a-A (free acid), OB 3a-A(Na) and OB 3a-MSA are available.

The study onOB3a-A(free acid)was conducted following a procedure similar to the OECD TG 453 and the substance was administered via the diet to rats (report n. 7396, 1978). The kidney weights of female animals at mid- and high-dose groups increased with respect to the control group. No evidence of a carcinogenic effect was identified and no other relevant toxicological effects were reported, a NOAEL equivalent to 779 mg/kg bw/day for females and 542 mg/kg bw/day for males, which were the highest doses tested, was set.

As for OB3a-A(Na), three groups of 60 male and 60 female rats received the compound thoroughly mixed in the feed along with a fourth group of as control, for two years (1974). After death or sacrifice, all animals were examined grossly and almost all microscopically. Histopatological examination of all twenty rats sacrificed at six months was negative.

Of the twenty animals sacrificed at mid-term (12 months), there was one male rat at 10000 PPM rat with chronic mild nephritis (chronic cystitis) and two female control rats with adenocarcinoma of the mammary glands.

As the experiment progressed more and more pathology became evident; the most striking clinical finding was the observation of adenocarcinomas of the mammary glands.

The incidence was high among the four groups, excluding a correlation to the compound administration. Other effects observed were nephritis, pituitary adenomas and some pathologies/lesions, known to occur spontaneously in the albino rat and frequently in old rats, thus, no treatment related. The NOAEL value found under this test conditions was established to be ≥ 542 mg/kg bw/day for males and ≥ 779 mg/kg bw/day for females.

As per OB 3a-MSA, the 50 male and 50 female rats (100 animals in the control group) received test substance administered for 2 years (report n. 7214, 1978).

The animals in the highest dose groups did not show during the entire experimental period any treatment-related symptoms. The growth of the rats was not affected. The haematological investigations performed during and at the end of the test showed no dose of injuries. The studies of male animals to the remaining time points and all investigations in the female animals showed no evidence for a corresponding finding. 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 on the kidneys because the incidence is low (<10%) and the significantly increased kidney weights in male and female rats in the highest dose group was not regarded as an expression of injury. Blood sugar and cholesterol levels were unchanged by test item administration.

The sections of dead and killed rats did not show reference to a specific ending damage that could be caused by the substance. Histopathological examination showed the usual age-related spontaneous alterations. The NOAEL value found under this test conditions was established to be ≥ 521 mg/kg bw/day for males and ≥ 709 mg/kg bw/day for females.

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 OB 3b-A, 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 NOAEL and the LOAEL values of the study were established at 250 mg/kg bw/day and 750 mg/kg bw/day respectively, on the basis of the results from an OECD 422 study on rats. 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 NOAEL 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).