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Key value for chemical safety assessment

Additional information

No repeated dose toxicity studies are available for sodium tert-butanolate. Sodium tert-butanolate is the salt of tert-butyl alcohol ion and the sodium metal cation. In the presence of water it reacts under formation of tert-butyl alcohol and sodium hydroxide (NaOH). For that reason, a weight of evidence approach with read-across to tert-butyl alcohol was performed. Here, 2 oral subchronic studies as well as 2 subacute and 2 subchronic inhalation studies are available.

Oral studies:

The 13-week study was performed in rats and mice according to GLP (NTP, 1995). Here, tert-butyl alcohol was administered to groups of 10 male and 10 female F344 rats and B6C3F1 mice through the drinking water in following doses: 2.5, 5, 10, 20, and 40 mg/mL for 13 weeks. The average received doses for rats were ca. 230, 490, 840,1520, or 3610 mg/kg body weight (male) and ca.

290, 590, 850, 1560, or 3620 mg/kg body weight (female). Mice received 350, 640, 1590, 3940, or 8210 mg/kg bw (males) and ca. 500, 820, 1660, 6430, or 11620 mg/kg (females).

In the 40 mg/mL group 10/10 male and 6/10 female rats died, as well as 6/10 male mice and 4/10 female mice. In addition, one female mouse of the 2.5 mg/mL group and one female mouse of the 20 mg/mL group died. The body weights of the male rats were significantly reduced in the 5 mg/mL (6%), 10 mg/mL (17 %), and 20 mg/mL (25 %) and in female animals only after treatment with 40 mg/mL (44 %). Male mice showed a significantly reduced body weight gain in the 20 mg/mL (30 %) and 40 mg/mL (54 %) groups and females in the highest dose (27 %).

The observed clinical symptoms in rats and mice were ataxia, hypoactivity, hyperactivity, and emaciation. Changes in organ weights accompanied the changes in body weights and were therefore not regarded as specific effects.

Gross-pathological examination resulted hyperplasia of the urinary tract epithelium of all male and three female mice at the dose of 40 mg/mL and in six male animals receiving 20 mg/mL. A chronic inflammation of the urinary bladder could be detected in all males and six females receiving 40 mg/mL and six male animals at 20 mg/mL. For rats, abnormalities in the kidneys, urethra and bladder-mainly hyperplasia and inflammation in the bladder-were regarded as substance-related. This inflammation resulted in increased numbers of macrophages, lymphocytes, and plasma cells in the lamina propria. Especially in male, tert-butyl alcohol induced an increase in the spontaneaus nephropathy and mineralization processes in the kidneys in all dose groups. But evidence was found of a2u-globulin nephropathy indicated by an increase in the number of hyaline droplets and hyaline crystals. Since a2u nephropathy is not relevant for human beings, findings from male rats were not regarded as adverse. There was an increase in the incidence of nephropathy in female rats at 10, 20, and 40 mg/mL indicating a second mechanism for the occurring nephropathy. The low effect concentrations in male rats might be the result of an additive effect of both mechanisms. Therefore, the effect level of the female animals was used for the determination of the NOAEL. The NOAEL for the urinary tract in females is 5 mg/mL (ca. 590 mg/kg body weight).

Inhalation studies:

Two 18-day studies were performed in rats and mice under GLP (NTP, 1997).

5 Animals/dose/gender were exposed 12 days to vapour of tert-butyl alcohol for 6 h/day for 5 days/week. Applied doses were: 1.36, 2.7, 5.3, 10.6, 21.2 mg/L.

All mice and rats of the high dose died on day 2 and one male mouse exposed to 10.6 mg/L died on day 3. Mice and rats of all other groups survived. In 10.6 mg/L groups, mice were prostrate following the first exposure through day 3 of the study. Clinical signs were observed post-exposure and included hypoactivity, hyperactivity, ataxia, urogenital wetness, rapid respiration.

In 10.6 mg/L rats, thymus weights were decreased but other significant organ weight differences were considered related to lower final mean body weights. In gross-pathology no treatment-related findings could be observed. Relative liver weights at 10.6 mg/L were increased and absolute and relative thymus weights of 10.6 mg/L females were significantly lower compared to controls. Other significant differences in organ weights were considered not related to treatment.

In addition, two 13-week inhalation studies were performed under GLP (NTP 1997).

Here, 10 F344 rats and 10 B6C3F1 mice per dose/gender were exposed for 6 hours daily, 5 days/week for 13 weeks to tert-butyl alcohol with the following doses: 135, 270, 540, 1080 or 2100 ppm.

In rats, no mortality occurred and treatment had no effects on body weight gains. Clinical signs were emaciation and hypoactivity and were observed only in the 2100 ppm females. In mice, 1/10 male animals died at 2100 ppm and body weight gain at 1080 and 2100 ppm in females was reduced.

In male rats, a significant decrease in haematocrit, haemoglobin level and erythrocyte counts occurred indicating a slight anaemia, which was characterized as normocytic, normochromic and non-regenerative. Alkaline phosphatase activity was decreased in the male rats receiving 1080 and 2100 ppm. The pH value of the urine was decreased at 1080 (female) and 2100 ppm (male and female). In male mice, neutrophil counts at a dose of 2100 ppm were increased in the hematology.

In the gross pathological examinations of mice and rats, no treatment-related findings could be observed. In rats, the absolute and relative kidney weights were significantly increased in the male animals receiving 1080 ppm and 2100 ppm. In females receiving 1080 and 2100 ppm, relative liver weights were increased; in the 2100 ppm group, additionally the relative kidney weights were increased. In female mice, relative liver weights at 1080 and 2100 ppm were increased. In mice, no microscopic findings were observed in the histopathology. In histological examination of rats no findings were observed for the females. In male animals, the severity of spontaneaus chronic nephropathy increased dose-dependently from 1 (minimal) in the control group to 1.4 in 135 ppm group to 2 (mild) in the 2100 ppm group. No accumulation of hyaline droplets was detected.

The NOAEC for male rats was below 135 ppm due to increased severity of spontaneous nephropathy. Although in this study no hyaline droplets could be found, study results published by Borghoff et al., 2001 showed concentration-dependent accumulation of protein droplets in male rats after inhalation exposure to tert-butyl alcohol. This was accompanied by a slightly increased a2u-globulin staining and cell proliferation missing in female rats and indicating an a2u-nephropathy. A minimal anemia, evidenced by a slight decrease in hematocrit values, hemoglobin concentrations, and erythrocyte counts. The mean cell volume, mean cell hemoglobin concentration, and reticulocyte and nucleated erythrocyte counts were not altered, indicating the anemia was normocytic, normochromic, and nonresponsive. For that reason, the NOAEC for female rats was used, which was 540 ppm based on liver and kidney weights.

The NOAEC for mice was also 540 ppm due to increased liver weights and neutrophil counts and decreased body weight gain.

The rat spontaneous chronic nephropathy and its human relevance in the safety evaluation is a current issue in discussion and in evaluation by the MAK commission. The publication of Hard et al., 2009, describes very well the theme and shows a direct comparison of rat and human nephropathy with its existing significant differences. Rat nephropathy can be influenced by natural and physiological parameters including protein-rich diet or male hormones and it strongly differs in the biology and pathology compared to human nephropathy. Human nephropathy is based on immunological and inflammatory processes with shrunken kidneys in the end-stage in contrast to rat nephropathy which additionally, especially in males, is characterised by hyaline droplet accumulation. In humans, it also could not be shown that nephropathy can be exacerbated by chemicals as it was shown in rats in the NTP-studies, indicating that it is unlikely that chemical induced exacerbations in rat can be considered as a reliable indication of a health hazard for humans. In consideration of the actual discussion, the data received in the NTP studies regarding the kidneys cannot be considered as human-relevant and the current MAK-value (including the NOAELs) might undergo a re-evaluation.

Justification for classification or non-classification

The determined NOAELs for oral (590 mg/kg) and for inhalation route (1.6 mg/L) received from tert-butyl alcohol exceed the obligatory classification limits. Therefore, no classification is proposed according to EU directive 67/548/EEC and EU regulation (EC) NO. 1272/2008 (CLP).