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Administrative data

Description of key information

NOAEL (oral, rat female, 51 days) = 100 mg/kg bw/day

NOAEL (oral, rat male, 30 days) = 300 mg/kg bw/day

NOAEL (oral, rat, 28 days) = 200 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 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 repeated dose toxicity of the substance was evaluated in a subacute 28-day oral toxicity study according to the OECD Guideline 407 (1981) and method B.7 of the Directive 84/449 EEC. The substance was administered daily by oral gavage to SPF-bred Wistar rats of both sexes for a period of 28 days. Test animals were randomly assigned to dose levels of 50, 200 and 1000 mg/kg bw/day and a control group which received only the vehicle (carboxymethyl cellulose (4 %) in distilled water) (5 males and 5 females per group).
Observations for mortality and clinical signs were recorded daily; food consumption and body weights were measured weekly. At four weeks, ophthalmic examinations, blood samples for haematology and clinical biochemistry, and urinalyses were performed. All test animals were sacrificed at four weeks; at necropsy, organ weights were recorded and macroscopic and microscopic histopathologic examinations were performed.
Females administered the highest dose recorded a slight decrease in erythrocyte count, haemoglobin concentration and haematocrit value; males administered the highest dose recorded a slight increase in phosphorus and potassium. Both males and females administered the highest dose recorded a moderate increase in urobilinogen which was attributed to direct interaction with urine discolouration; however this finding was considered adaptive and not of toxicological significance. 
A significant increase in absolute kidney weights was observed among males administered with 1000 mg/kg bw/day and females administered 200 mg/kg bw/day; and increased kidney to body weight ratios were observed among females administered 50 and 200 mg/kg bw/day, however these findings were considered to be of a spontaneous nature and not related to treatment.
Macroscopic pathology findings included discolouration. Faeces and gastrointestinal contents of most test animals in groups administered 200 and 1000 mg/kg bw/day were discoloured black between day 7 and the end of treatment period. Black-brown foci in the lungs were observed in 2, 4 and 1 test animals from dosage levels 50, 200 and 1000 mg/kg bw/day, respectively, however a dose-response relationship cannot be established as incidence did not increase with dose. Mesenteric lymph nodes displayed black-brown discolouration in one test animal which received 200 mg/kg bw/day, and 7 which received 1000 mg/kg bw/day, however these findings were considered to be of a spontaneous nature and common among rats of this strain and age.
No treatment-related histopathological findings were recorded, however a spurious amount of brownish pigment was found within macrophages in the lung and mesenteric lymph nodes of some rates which was attributed to accidental inhalation of the substance.
Based on the results observed, the NOAEL for the substance in male and female rats is 200 mg/kg bw/day. Therefore, the lowest adverse effect level (LOAEL) can be considered to be 1000 mg/kg bw/day.

Further data for general toxixcity is available from the screening test performed according to the OECD Guideline 421 (2016). Four groups containing 10 male and 10 female rats each were administered the test item in water softened by reverse osmosis (10 mL/kg of body weight) for 30 days (males) and at least 51 days (females). The test item was given to Sprague Dawley rats by oral administration (gavage) at dosages of 100, 300 and 1000 mg/kg/day. The control group received only the vehicle.

Non pregnant females and females with total litter loss were dosed up to the day before necropsy. The following investigations for general toxiccity on parental animals were performed in all groups: mortality check, clinical signs, body weight, body weight gain, food consumption and thyroid hormone determination only for males, macroscopic observations, organ weights. The histopathological evaluation was performed on the following organs/tissues: adrenal glands, epididymides, lungs, cervical and mesenteric lymph nodes, kidneys, liver,mammary gland, ovaries with oviducts, seminal vesicles with coagulating glands, testes, thyroid, uterus, cervix and vagina performed on all males and females from the control and high dose groups, as well as on all abnormalities detected during post mortem observation at the end of the treatment period. On the basis of treatment-related findings detected in the mesenteric lymph nodes and lungs of high dose treated animals of both sexes, the histopathological evaluation of these organs was extended to the low and middose animals of both sexes.

No mortality occurred during the study. The only clinical sign recorded during the treatment period was black staining on the tail (slight to marked) in all treated males and in females (slight to moderate) receiving the dose level ≥300 mg/kg/day. No adverse effects were observed in body weight or body weight gain of treated animals compared to control group. Food consumption of parental animals of both sexes was unaffected by treatment. Statistically significant increases of triiodothyronine and thyroxine were recorded in males dosed at 1000 mg/kg/day (46 % and 12 %, respectively). Similar increases were also recorded in some animals receiving 100 and 300 mg/kg/day, with no statistical significance nor clear dose-relation. Since changes were of low severity, with no dose-relation nor concurrent alteration of thyroid stimulating hormone, the above increases were considered to be of no toxicological relevance. No relevant changes were observed on terminal body weight, absolute and relative organ weights of treated animals, when compared to the controls.

An increased incidence of dark colour and/or areas of cervical and mesenteric lymph nodes, lungs and intestinal tract (caecum, colon, ileum and rectum) were noted in treated animals of both sexes. An increased incidence of reduced size of thymus was observed in high dose treated females, when compared to the controls.

Treatment-related lesions were noted in mesenteric lymph nodes of males and females receiving test item at 1000 mg/kg/day, in the lungs of males dosed at 1000 mg/kg/day and in females dosed at ≥300 mg/kg/day.

The findings consisted in the presence of black pigment laden macrophages in the mesenteric lymph nodes in all high dose males and females, and intracytoplasmic black granular pigment in the alveolar macrophages, associated or not with chronic inflammatory reactions in the lungs reported in males dosed at 1000 mg/kg/day and females dosed at ≥300 mg/kg/day.

Therefore, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 300 mg/kg/day for males and 100 mg/kg/day for females, based on the results of this study.

The NOAEL determined for general toxicity in parental females during the OECD 421 (51 days, 100 mg/kg bw day) is selected as the most representative starting dose based on exposure route, study duration, presence of adverse effects, parameters observed. In addition, it is the lowest available value obtained in the available experimental studies. Therefore, it is used as key value for Chemical Safety Assessment .

Justification for classification or non-classification

According to the CLP Regulation (EC) no. 1272/2008, substances are classified as specific target organ toxicants following repeated exposure, and are placed in one of two categories, depending upon the nature and severity of the effect(s) observed. 

Substances that have produced significant toxicity in humans or that, on the basis of evidence from studies in experimental animals, can be presumed to have the potential to produce significant toxicity in humans following repeated exposure, are classified in Category 1 for target organ toxicity (repeated exposure). Classification in Category 1 is applicable when significant toxic effects are observed in a 90-day repeated-dose animal study at low concentrations (< 10 mg/kg bw/day in oral studies, and < 20 mg/kg bw/day in dermal studies) (CLP Regulation (EC) no. 1272/2008: Annex 1, Part 3, Table 3.9.2).

Substances which can be presumed to have the potential to be harmful to human health following repeated exposure, based on evidence from animal studies, are classified in Category 2. Classification in Category 2 is applicable when significant toxic effects are observed in a 90-day repeated-dose animal study at generally moderate exposure concentrations (10 to 100 mg/kg bw/day in oral studies, and 20 to 200 mg/kg bw/day in dermal studies) (CLP Regulation (EC) no. 1272/2008: Annex 1, Part 3, Table 3.9.3).

The 90-day guidance values 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. Based on this relation, the equivalent guidance value calculated for a 50-day study results to be approximately 180 mg/kg bw/day. Therefore, starting from the lowest available NOAEL of 100 mg/kg for general toxicity to females obtained during the OECD 421 study (exposure of approx. 50 days), a LOAEL of 300 mg/kg bw/day can be considered. This value results to be above the calculated equivalent guidance value (50 day-exposure). Therefore, no classification of the substance is warranted for specific target organ toxicity: repeated exposure.