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Description of key information

The repeated dose toxicity of the carboxylic acid component of the registered substance (tosyl salt) to rats was determined via the oral route of exposure in accordance with the OECD Guideline for Testing of Chemicals 422 (Short term repeated dose toxicity) and OECD TG 408 (Sub-chronic 90-day toxicity). As a result of the OECD 422, the NOEL and NOAEL for the test substance is defined as 400 mg/kg bw/day. The LOAEL for the test substance is defined as 1600 mg/kg bw/day. As a result of the OECD TG 408, the no-observed-adverse-effect-level (NOAEL) is considered 100 mg/kg bw/day for males and 1600 mg/kg bw/day for females.


No studies were conducted to determine the repeated dose toxicity via inhalation or dermal exposure as, owing to the physical properties and usage of the substance, it was considered unlikely that inhalation or dermal exposure would occur, therefore making it unjustifiable to perform further animal testing.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Remarks:
90 days
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 408. The study was conducted on 6-[(p-Tosyl)amino]hexanoic acid, which is the carboxylic acid component of the registered substance i.e. tosyl salt.
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The following read-across hypothesis is proposed for the registered substance:
The registered substance 6-[(p-tosyl)amino]hexanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) (target substance) is manufactured directly from 6-[(p-Tosyl)amino]hexanoic acid (source substance) by simple neutralisation with triethanolamine (TEA). Other than ionization of the carboxylic acid group, the 6-[(p-Tosyl)-amino]hexanoic acid remains chemically unchanged upon salt formation. In water, the acid and amine components of the target substance dissociate completely to the source substance and TEA and these two components behave essentially as independent substances. Moreover, it is hypothesised here that TEA is non-hazardous substance and the acid component of the salt that will have a more significant impact on the outcome of any (eco)toxicological or environmental tests. Hence, the toxicity data on the source substance will accurately represent the toxicity of the target substance.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Details are attached in Section 13 of the dataset

3. ANALOGUE APPROACH JUSTIFICATION
Details are attached in section 13 of the dataset

4. DATA MATRIX
Details are attached in section 13 of the dataset
Reason / purpose for cross-reference:
read-across source
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related clinical findings were revealed in any dose group.
In one female from the 100 mg/kg bw/day dose group (No.77), the body weight loss with a hunched posture and wheezing were recorded beginning day 16. The formulation aspiration is suggested as a likely cause of the observed clinical findings. The dose was failed to gavage for 5 days (at the period from day 16 to day 20, and the administration continued further without deviations with a positive body weight gain and the absence of wheezing. No associated gross observations were revealed in this female during scheduled necropsy.
In one female treated with 400 mg/kg bw/day dose (No.82), the unilateral chromodacryorrhea was recorded beginning from day 27 thought the all administration period. The ophthalmoscopy has revealed the corneal injury, which is not related to the test item.
In two males from the control group and low dose group, the non-treatment related focal alopecia was noted starting from the approximately eighth week of dosing.
Mortality:
no mortality observed
Description (incidence):
There was no morbidity and mortality of animals caused by the test item administration.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The absolute body weight of males was slightly dose-dependently decreased after approximately one-month dosing. To the end of the administration period, this statistically non-significant decrease was 3.5 % in the 100 mg/kg bw/day dose group (362 ± 26 g), 4.8 % in the 400 mg/kg bw/day dose group (357 ± 30 g), and 4.3 % in the 1600 mg/kg bw/day dose group (359 ± 26 g), respectively, compared to the control value (375 ± 33 g).

The body weight gain in gram was statistically reduced in the high dose group to the day 56 of dosing, and the total percentage gain was non-significantly decreased compared the value in the control group (155.6 ± 25.0 %) in all dose groups: 148.9 ± 26.6 %, 143.2 ± 15.4 %, and 143.4 ± 20.5 %. After three weeks of recovery in the 1600 mg/kg bw/day dose group, the decrease in body weight remained noticeable (371 ± 27 g versus 390 ± 43 g), and the total percentage body weight gain differed by 15.8% the value in the control group (177.1 ± 8.0 % versus 192.9 ± 20.8 %).

In females treated with the test item, body weight did not decrease, and even on the 1600 mg/kg bw/day dose, there was a slight increase in body weight: by 3.7 % (227 ± 24 g) compared to the control group (219 ± 18 g) by the end of the administration period. The increase in female body weight in the high dose group was non-significant, also noted after the recovery period, and is supposed to be non-adverse.
Changes in body weight did not correlate to the food consumption for both males and females but linked to the clinical chemistry findings
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean food consumption in the 100, 400, and 1600 mg/kg bw/day dose treated groups was approximately similar to that in the vehicle control group during all study days.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item related abnormalities in the eyes in all doses treated groups.
One female (No. 82) in the 400 mg/kg bw/day dose group had unilateral corneal injury that was considered incidental and not related to the test item administration.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related hematological changes were revealed in any dose groups. In males, hematological parameters were approximately similar in all groups. In females, the slight dose-dependent increase in hemoglobin level (HGB) can be noted. In the 1600 mg/kg bw/day dose group, this increase by 3.8 % (136 ± 6 g/L versus 131 ± 5 g/L) was associated to the same increase in hematocrit (HCT) value (0.409 ± 0.020 L/L versus 0.394 ± 0.014 L/L). These findings were slight, non-adverse, and presumably can be associated with increased diuresis in females and mild dehydration.
After the three-week high dose post-treatment, the hematological parameters were similar to the values in the control vehicle group in both males and females.

COAGULATION:
In females treated with the test item, the shortening of activated partial thromboplastin time (APTT) was observed. This effect was dose-dependent with statistical significance in the 400 mg/kg bw/day dose group (14.7 ± 1.7 s, p < 0.05) and 1600 mg/kg bw/day dose group (14.6 ± 1.3 s, p < 0.01) compared the control value (16.2 ± 1.0 s). Prothrombin time (PT) did not significantly change in females; however, the mean value in the 1600 mg/kg bw/day dose group was slightly decreased (17.4 ± 1.7 s versus 18.4 ± 1.8 s in the control group). In the recovery female high dose subgroup, the coagulation parameters were similar to the control group.
In males, there were no significant changes in the APTT, PT, and fibrinogen

Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males from the 400 and 1600 mg/kg bw/day dose groups had elevated urea level, respectively, by 16.9 % (p < 0.01) and 12.4 % (p < 0.05 using paired t-test) (10.4 ± 0.9 and 10.0 ± 1.4 versus 8.9 ± 0.6 mmol/L in the control group). In addition, in the 1600 mg/kg bw/day treated males, the statistically significant decrease in creatinine (by 9.5 %, p < 0.05) and increase in urea:creatinine ratio (174 ± 21 versus 142 ± 13, p < 0.01) were observed. In contrast to the urea level, the increase in the urea:creatinine ratio was dose-dependent, and the change in these parameters is considered to be related to the test item. Urea and serum creatinine are routinely used to measure kidney damage; however, creatinine is a poor indicator of renal function in the situation of its reduction. Elevated urea:creatinine ratio may be recorded in the context of the catabolic state due to starvation or corticosteroids. The relative kidney weight was increased in the high dose male group; however, the histological alterations associated with the altered renal function were observed only in single males. Moreover, males in the 1600 mg/kg bw/day dose had a slightly non-significant increase in total creatine kinase (by 21.3 % versus control group) and decrease in glucose level (4.5 ± 0.3 mmol/L versus 5.2 ± 0.7 mmol/L in the control group, p < 0.05) associated with a slight decrease in body weight and unchanged food intake. The glucose was reduced dose-dependently with statistical significance observed in the 400 and 1600 mg/kg bw/day male groups that considered test item-related change. No significant change in protein levels was observed in the test item treated males.
In addition, in males of the 1600 mg/kg bw/day dose group, the decrease in cholesterol (by 14.2 %, p < 0.05), triglycerides (by 31.0 %, p < 0.01) was observed with visible tendency to the decrease in Low-Density Lipoproteins (LDL) (by 20.0 %, not significant due large variability). These changes were dose-dependent and considered as test item-related. Notably, after a withdrawal period, lipid levels in the high dose group recovered and were even slightly higher than control levels (not significant).
Interestingly, in females of all test item treated groups, there were no significant changes in serum chemistry parameters. However, it is noticeable that the mean cholesterol and High-Density Lipoproteins (HDL) level was slightly increased (not significantly) in the high dose group (2.58 ± 0.49 mmol/L and 0.86 ± 0.13 mmol/L versus 2.32 ± 0.41 mmol/L and 0.79 ± 0.10 mmol/L in the control group) following the tendency towards a decrease in cholesterol and HDL in the 400 mg/kg bw/day dose group (respectively, 2.08 ± 0.63 mmol/L and 0.71 ± 0.20 mmol/L) as well as a slight decrease in glucose (4.7 ± 0.6 mmol/L versus 5.0 ± 0.4 mmol/L). Such an inverse dynamic of lipid levels in females was non-significant; however, it correlated with the dynamics of body weight and microscopic findings in the kidneys and liver in the test item treated females. Females in the 1600 mg/kg bw/day dose group had a slightly increased body weight (by 3.7 %) to the end of the administration period, hepatocellular hypertrophy, and kidneys without pigmentation in tubules. Whereas 400 mg/kg bw/day dose female group showed the most incidence and severity of renal tubular pigmentation (presumable lipofuscinosis), there was no noticeable hypertrophy of hepatocytes.
So, the main clinical chemistry findings in male and female rats treated with ASCplus are related to serum lipids change. These observations are supposed to be due to the test item (or its primary metabolites) influence on cholesterol exchange via peroxisome proliferator-activated receptors, PPARs (discussed below); they were reversible and non-adverse. The revealed sex difference in lipid change is considered to be associated with a faster and more pronounced activation of the liver's metabolic enzyme system in females. No clinical chemistry findings were found to be biomarkers of any adverse changes in the liver and kidney.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In the 1600 mg/kg bw/day dose group, the statistically significant increase in the urobilinogen level was observed in both males and females (p < 0.01, p < 0.05), that considered as test item related. Moreover, in males of this group, the pH was decreased (p < 0.01) compared to the control value. The excretion of urobilinogen is increased during liver damage or hemolysis. In animals treated with the test item, there were no hemoglobinemia and serum bilirubin changes. On the other hand, the excretion of urobilinogen is a pH-dependent process [Levy M. et al,1968] and, at least in males, an increase in urinary urobilinogen in the high dose group correlated with a decrease in urine pH.
In the high dose recovery subgroup, the median value of urobilinogen still remained increased, but not significantly. Moreover, the increased specific gravity was noted in post-treated males with unclear relation to the test item.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional Observation Battery testing were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the vehicle control group on study day 88 (end of the treatment period). After a three-week recovery period, the FOB parameters in 1600 mg/kg bw/day dose group were also comparable with the control group.

Locomotor activity patterns (horizontal movements and vertical activity) registered for 6 minutes were unaffected by test item administration at all doses when evaluated on day 88 (end of the treatment period) in males and females. After a three-week post-treatment period, the parameters of locomotor activity in the 1600 mg/kg bw/day dose group were comparable with the control group in both males and females.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute kidneys weight was non-significantly but notably increased in males and females in the 1600 mg/kg bw/day group (males, 2.2722 ± 0.2454 g versus 2.1581 ± 0.2184 g; females, 1.3453 ± 0.1141 g versus 1.2620 ± 0.1119 g) while the change in relative to body weight value was significant in males (by 9.3 %, 0.6645 ± 0.0348 versus 0.6078 ± 0.0370 g/100 g, p < 0.01), and females (by 9.5 %, 0.6114 ± 0.0191 versus 0.5887 ± 0.0242 g/100 g, p < 0.05). In the recovery high dose subgroup, the mean relative weight of kidneys in males remained slightly increased, but without statistical difference from the control group. The change in weight of kidneys in the high dose group was correlated to the clinical pathology and pathomorphology observations and considered to be test item-related.
The weight of the liver was increased by 9.5 % in the 1600 mg/kg bw/day treated males (relative to body weight, 3.0916 ± 0.1513 versus 2.8232 ± 0.1766 g/100 g, p < 0.001). In females, the change in liver weight was more pronounced. The increase in absolute weight was dose-dependent with a significant change relative control group by 25.8 % in the high dose group (7.2479 ± 0.6628 g versus 5.7599 ± 0.4665 g, p < 0.0001). Females in the 400 and 1600 mg/kg bw/day dose group had the significantly increased relative liver weight by 8.0 % and 22.2 %, respectively, (2.9091 ± 0.2219 g/100 g, p < 0.05, and 3.2927 ± 0.1317 g/100 g, p < 0.001) compared to the control group (2.6940 ± 0.2228 g/100 g). The relative to brain weight value of the liver weight was also significantly increased in the 1600 mg/kg bw/day female group. Change in the liver weight correlated to the clinical pathology and histological findings; however, it is supposed to be caused by adaptive activation of metabolic enzymes and considered as non-adverse. After the recovery period, the mean relative liver weight remained slightly increased, but without statistically difference from the control group.
In the 400 mg/kg bw/day dose female group, the relative weight of thymus was statistically increased by 23.6 % compared to the control group (p < 0.05). The relative weight of thymus in the high dose group was approximately similar to the control value. Non-dose-dependent change in the thymus weight was not correlated to the hematological observations and considered of unclear relationship to the test item. There was no test item related histological findings in the thymus of the high dose treated animals.
In 1600 mg/kg bw/day dose recovery males, the slight decrease in the brain weight (p < 0.05 for absolute value) and heart weight (p < 0.05 for relative to body weight value) was registered. No correlations in the histology of these organs in the high dose main subgroup were revealed. These changes can be associated with the decrease in body weight of high dose treated males and considered non-adverse. The slight increase in the relative to brain testes weight (g/100 g of brain weight, right testis: 101.07 ± 2.48 versus 92.00 ± 5.56 in the control group) and adrenals weight (g/100 g of brain weight: 2.748 ± 0.292 versus 2.370 ± 0.194) in the high dose recovery males can be caused partially by a decrease in the brain weight value and regarded to be of uncler relationship to the test item.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The gross finding related to the test item was revealed in one female (No.85) from the 400 mg/kg bw/day dose group. This female had dark kidneys associated with a tubular brown pigmentation in the cortex of moderate grade (presumably lipofuscin accumulation). Accumulation of pigment in cortex tubular was observed in a dose-dependent manner in males and was most pronounced in 400 mg/kg bw/day treated females.
Other gross findings revealed during necropsy are considered not related to the test item.
Macroscopic findings in the thymus, lymph nodes, spleen, and thyroids were noted in the test item-treated groups and the control group; they did not correlate to organ weight changes, clinical pathology, or any microscopic observations.
One female (No. 82) in the 400 mg/kg bw/day dose group had unilateral corneal injury discussed above, focal alteration of texture of urinary bladder, and unilaterally enlarged ovary. These findings were considered incidental and not related to the test item administration.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related histological alterations were observed in liver, kidneys, thyroid glands, testes, and male mammary gland.

The findings of unclear relation to the test item were observed in adrenals, pituitary gland, female salivary glands, lymph nodes, spleen, and glandular stomach.

Liver
Microvesicular vacuolation in hepatocyte cytoplasm was increased in 1600 mg/kg bw/day treated males as well in 400 and 1600 mg/kg bw/day treated females with a significantly higher incidence in 1600 mg/kg bw/day female group compared to the control females (p < 0.05). The specific morphological features of the cytoplasmic vacuolation are sufficiently consistent with lipid accumulation to warrant a presumptive diagnosis of fatty change. Fatty change may be due to perturbations in lipid metabolism and disposition, correlated to the clinical serum chemistry, and considered test item related but non-adverse.
In the 1600 mg/kg bw/day female group, the hepatocyte hypertrophy of slight to moderate grade was observed with significantly increased incidence (p < 0.05). It is noteworthy that hepatocyte hypertrophy was revealed only in high-dosed females, which correlated to the significant increase in absolute and relative liver weight in this group. The increase in relative liver weight in males of the 1600 mg/kg bw/day group and females of the 400 mg/kg bw/day group was less pronounced and correlated to the increased incidence of fatty change. There were no associated changes in the serum enzymes considered to be biomarkers of hepatotoxicity (ALP, ALT, AST, GGT, GLDG) in both female and male treated rats.
Interestingly, 1600 mg/kg bw/day treated males had decreased body weight correlated to lipids metabolism perturbations in this group. Females from the high dose group developing hepatocyte hypertrophy had the reversible serum lipids level and increased body weight. The hepatocyte hypertrophy was not observed after a 3-weeks post-treatment period, whereas the liver weight remained slightly increased. Hypertrophy of hepatocytes on xenobiotics develops due to metabolic enzyme induction, increase in mitochondria and peroxisomes [Hall A. et al, 2012; Thoolen B. et al, 2010]. Hepatocellular hypertrophy in females can cause sex-dependent changes in serum lipids and not dose-dependent microscopic findings in kidneys, is considered adaptive and non-adverse.
In one female (No.91) from the 1600 mg/kg bw/day dose group, the focal hyperplasia of oval cells was observed. Oval cell proliferation is considered to arise from terminal ductule epithelial cells (canal of Hering cells). It can be a rare spontaneous lesion in rats but can be observed following xenobiotic-induced hepatic injury. However, the uniqueness of this finding among test item treated group does not allow concluding its test item relation. A similar finding was observed in the control male of the recovery subgroup.

Kidney
The tubular brown pigmentation in the cortex (presumably lipofuscin accumulation) was found in 4 males from the 1600 mg/kg bw/day dose group and in single males from 100 and 400 mg/kg bw/day dose groups. In females, pigment accumulation of minimal grade was found in two control animals; however, four females had the lipofuscinosis in more severe grade in the 400 mg/kg bw/day dose group. In one female (no.85), the moderate grade of pigment correlated to the dark kidney noted during necropsy.
Lipofuscin is found in the kidneys of most laboratory rodents, especially rats, is most often localized to the proximal tubules, and considered of limited or no functional significance [Frazier K. et al, 2012]. However, there is a clear dependence of lipofuscinosis on the test item in our study. This finding shows the sex-dependence presumably related to differences in metabolic enzyme activation (see Discussions).
The mineralization in the renal pelvis was noted only in 400 mg/kg bw/day treated females, which correlated to more pronounced lipofuscinosis in this group. Mineralization in the kidney occurs spontaneously in laboratory animals with a dietary imbalance of calcium/phosphorus ratio, particularly in female rats and severity increase with age. There can be a much higher prevalence of spontaneous mineralization in the outer stripe of the outer medulla (as we can see in this study, see Table S19-3). The mineralization in 400 mg/kg bw/day treated females was more severe with calculus in the renal pelvis, which was considered test item-related.
Interestingly, that no test item related findings were revealed in the kidney of the high dose treated females with hepatocyte hypertrophy. There is an assumption that the early metabolic deactivation of the substance by the hypertrophied liver neutralizes the main test-related effects in 1600 mg/kg bw/day female group.
The signs of chronic progressive nephropathy (CPN) of minimal to slight grade (tubule basophilia, tubule dilation with hyaline casts in the cortex, OSOM, and ISOM) were observed in approximately similar frequency, including the control group. CPN is one of the most common spontaneous lesions in rats, and male rats are more severely affected than female rats. In females, the incidence and severity of CPN have not been exacerbated by the test item administration. In one male from the 1600 mg/kg bw/day dose group, CPN findings were more severe with glomerulosclerosis and tubular hypertrophy. Renal tubule hypertrophy is often observed in more severe CNP cases and is believed to be a compensatory mechanism related to a decline in renal function. One another male from the high dose group had focal tubular necrosis of slight grade.
Microscopic findings in kidneys of 1600 mg/kg bw/day administered males correlated to the clinical pathology changes and increase in kidneys weight in males of this group. Unlike lipofuscinosis, exacerbation of CNP in treated males was not reversible; in the 1600 mg/kg bw/day recovery subgroup, such findings were more severe, which can be potentially adverse.

Thyroids
In males and females of the 1600 mg/kg bw/day dose group, the slight decrease in the T3 was observed after 90 days of test item administration and a more pronounced decrease in T4 level in high dose animals. The increase in TSH in the high dose group due to the hypothalamo-pituitary-thyroid feedback was slight, and no indications of TSH-mediated thyroid gland activation were found in its histology. There were no findings of thyroid hypertrophy, or follicular hyperplasia, and changes in thyroids weight.
In the 400 and 1600 mg/kg bw/day dose groups, the increase in incidence and severity of multifocal C-cell hyperplasia was observed. Diffuse C-cell hyperplasia represents a physiological or pathophysiological response to the stimulation of calcitonin-producing cells, such as chronic hypercalcemia.
It can be assumed that C-cell hyperplasia reflects a disturbance in calcium balance associated with renal dysfunction and indirect parathyroid hormone stimulation on the high dose of the test item acting on kidney PPAR. However, there are no enough data to make this conclusion. The calcium level in serum of test item treated males and females was not changed. Despite the absence of explainable reasons for C-cell hyperplasia, its relationship with the test item cannot be excluded. But this change was slight and is not considered adverse.
In one female from the 1600 mg/kg bw/day group, follicular atrophy was revealed. This finding can reflect the decrease in thyroid hormones; however, it was of minimal grade and considered non-adverse without toxicological importance.

Testes
Hyperplasia of Leydig cells was found in two males from 100 mg/kg bw/day dose group, and four males from each of 400 and 1600 mg/kg bw/day dose group. These findings were multifocal, minimal to slight grade, and considered to be a physiologic response to hormonal imbalance. It is known that interstitial cell hyperplasia can be induced in rats by any chemical that increases circulating levels of luteinizing hormone (LH) via disruption of the hypothalamic-pituitary axis followed by stimulation of steroidogenic Leydig cell function. It is assumed that the test item and/or its metabolites (4-(tosylamino)butyric acid and 2-[(4-methylphenyl)sulfonylamino]acetic acid) acts on peroxisome proliferator-activated receptor α (PPARα), which is highly expressed in liver and renal proximal tubules, as well as in Leydig cells [Gazouli M. et al, 2002; Guan Y., 2004]. The “classic” PPARα-agonists aryloxyisobutyric acid derivates (clofibrate and its analogs) are clinically proven lipid-lowering drugs. Some peroxisome proliferators, including clofibric acid, are known to lower serum testosterone levels causing testicular atrophy and impaired spermatogenesis [Gazouli et al., 2002; Harada Y. et al, 2016]. It was shown a critical role for PPARα in testicular dysfunction due to disrupted cholesterol/testosterone homeostasis in Leydig cells [Harada Y. et al, 2016]. To maintain normal reproductive function, a feedback mechanism is present, where decreased circulating testosterone increases circulating LH that stimulates testosterone synthesis and hyperplasia of Leydig cells. The physiological stimulation of steroidogenic Leydig cell function can be adverse, considering Leydig cell tumor outcome [Kotula-Balak M. et al, 2020].
The tubular degeneration/atrophy was revealed in one male (No.44) from the 1600 mg/kg bw/day dose group and one male (No. 36) in the 400 mg/kg bw/day dose group. The male from the high dose group had a slight change with partial germ cell depletion in some tubules of one testis. Male from 400 mg/kg bw/day dose group had a more severe bilateral observation with atrophy of some tubules correlated to the decrease in relative testes individual weight. Tubular degeneration is a common manifestation of toxicologic injury to the testis. However, it also can be seen as a low incidence background finding in rats and mice. This finding was revealed in the medium and high dose group that correlated to the increased incidence of Leydig cells hyperplasia on these doses and, presumably, can be caused by impaired testosterone as noted above. Therefore, the relationship of tubular degeneration/atrophy in testes of males in the 400 and 1600 mg/kg bw/day dose group with the test item is not excluded.
The mean testes weight (absolute and related) was not changed after 90 days administration, but in 1600 mg/kg bw/day dose recovery males, the testes weight was slightly increased relative to brain weight, which can be partially explained by the slightly decreased value of brain weight in this group. Simultaneously, the increase in the relative weight of the testes after the post-treatment period may reflect the rebound of testosterone level.
There was no test item related changes in the epididymides, seminal vesicles with coagulating glands, and prostate. In the prostate of males from the 1600 mg/kg bw/day dose group, two mononuclear infiltration incidents of the ventral lobe were observed. Focal extravasation of inflammatory cells into the interstitial tissue or the acinar lumen is common in the prostate of rodents of unknown etiology. This finding was unfrequent, slight, and considered as not treatment-related.

Mammary glands (male)
In two males (No. 38 and No. 39) from the 1600 mg/kg bw/day group and two males (No. 18 and No.22) from the 100 mg/kg bw/day group, the lobule atrophy of moderate grade was observed with a noticeable smaller of alveolar profiles and slight tubulo-alveolar appearance. The change of mammary gland with signs of feminization was associated with the hyperplasia of Leydig cells in high dose treated males, and it is supposed to be test item related due to the effects of decreased androgens via disruption of the hypothalamic-pituitary axis. The absence of such a finding on the 400 mg/kg bw dose is not entirely clear, but no change in the mammary gland was found in the control males.

Salivary glands (females)
The incidence of alteration in granular secretory ducts was slightly increased in all doses treated females and especially in the 400 mg/kg bw/day dose group. The submandibular salivary gland is sexually dimorphic and shows increased granularity of the convoluted (granular) ducts in males compared to females. When females have increased androgen levels, their granular ducts of the submandibular glands acquire male morphology. Both male and female salivary glands have androgen receptors AR, estrogen receptors ER‐β, and progesterone receptors PR [Konttinen et al., 2010], are sensitive to hormonal disbalances during stress; apparent cyclic fluctuations are observed in tissue morphology depending on the phases of the estrous cycle [Rybakova M.G., 1979]. As speculation, increased androgenic background in the test item treated females can be due to androgen/estrogen hormonal disturbance. However, the change from the control group was weak, which does not allow conclusions about its test item relation.

Adrenals and Pituitary
In the 1600 mg/kg bw/day male group, the slight increase in the incidence of vacuolation in zona fasciculata was observed; one male (No. 40) had the maximal of noted severity of this finding. Cortical vacuolation is increased during fatty change due to degeneration or lipid retention of the cortical cells. A variety of xenobiotics can increase lipid deposition and vacuolation in the adrenal cortex, usually by inhibiting steroid synthesis; adaptive hypertrophy can also be observed [Harvey P. et al, 2007]. The cortical hypertrophy was noted in single males from each group included control.
The complex effects of the test item in this study may be attributed to a sex-dependent alteration in cholesterol and steroid synthesis that discussed below. In male No.40, the moderate grade of cortical vacuolation was correlated to the incidence of microfocal hyperplasia of endocrine cells of the pars intermedia of the pituitary gland, which can be a consequence of hypothalamic-pituitary feedback ACTH activation. The relative weight of adrenals (relative to brain) was statistically increased in the high dose recovery males, but it can due to a decrease in body weight and brain weight and not test item related. An increase in cortical vacuolation was not dose-dependent, not correlated to the cortical hypertrophy, and was observed in the approximately same frequency in recovery control males.
Thus, the slight increase in adrenal cortical vacuolation in the high dose treated males, and one incidence of hyperplasia in the pars intermedia of the pituitary gland are slight, non-adverse and without clear relation to the test item.
In pituitary pars distalis, no changes of gonadotropin-producing and thyrotropin-producing cells were observed in the test item treated animals; there were no significant changes in the pituitary weight.

Lymph Nodes, Spleen, and Glandular Stomach
A slightly higher incidence of lymphoid hyperplasia was observed in mandibular lymph nodes in the 1600 mg/kg bw/day dose-treated males and females. In one male from this group, hyperplasia in spleen white pulp of moderate grade was observed. These findings reflect a change in lymphocyte kinetics due to antigen stimulation [Willard-Mack C. et al, 2019] .
In one male in 1600 mg/kg bw/day dose group, the macrophage accumulation of slight grade was noted in the mesenteric lymph node. Macrophages accumulate and form aggregates when they cannot completely degrade ingested macromolecules.
In the stomach, the increased incidence of infiltration of deep mucosa with inflammation cells was observed in the 1600 mg/kg bw/day dose group (male and female). A minimal/mild infiltration of eosinophilic leucocytes is common in the submucosa of the glandular stomach. The slightly high infiltration observed mainly in a deep mucosa can be potentially due to the local pro-inflammation/immunogen effect of the test item.
Findings in lymph nodes, spleen, and glandular stomach were of slight grade or observed as an individual case, did not correlate to the clinical pathology or gross observations and changes in organ weight (spleen), was not associated to the inflammation of the tissues, considered to be non-adverse and of unclear relation to the treatment.
The remaining histological changes not discussed above were considered to be incidental findings or related to some aspect of physiological condition and variability, experimental manipulation other than the administration of the test item. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue changes.

A summary of the FINDINGS THAT ARE and ARE NOT CONSIDERED TO BE ASSOCIATED WITH ADMINISTRATION OF THE TEST ITEM is provided in the attached TABLE 1 (Background information).
Dose descriptor:
NOAEL
Effect level:
1 600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
urinalysis
Critical effects observed:
no
Conclusions:
The source test item as a suspension in the vehicle (distilled water), was administered by gavage once daily to three groups of Sprague-Dawley rats starting from the age of 6-7 weeks at the doses 100, 400 and 1600 mg/kg body weight (kg/bw/day). A concurrent vehicle control group received the vehicle on a comparable regiment and in the same volume of 10 mL/kg bw. Each group consisted of 10 males and 10 females used for 90-day dosing and euthanized on the day 91. An additional satellite group of six animals per sex was included in the control and the top dose group for observation after the treatment period for the potential reversibility or persistence of any toxic effects and was euthanized at day 112.

There was no morbidity and mortality of animals caused by the test item administration. No test item-related clinical external observations, ophthalmologic findings, and functional findings of neurotoxicity evaluated during FOB testing were revealed in any dose group.
Male rats appeared to be more sensitive to the test substance in this study, therefore, under the conditions of this study, the no-observed-adverse-effect-level (NOAEL) is considered 100 mg/kg bw/day for males and 1600 mg/kg bw/day for females.
Executive summary:

The possible health hazards likely to arise from repeated exposure of the test animals to the source test item 6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid (ASCplus®, CAS 78521-39-8) over a prolonged period of time covering post-weaning maturation and growth into adulthood, was evaluated over 90-days by an OECD TG 414 study.


The test item as a suspension in the vehicle (distilled water), was administered by gavage once daily to three groups of Sprague-Dawley rats starting from the age of 6-7 weeks at the doses 100, 400 and 1600 mg/kg body weight (kg/bw/day). A concurrent vehicle control group received the vehicle on a comparable regiment and in the same volume of 10 mL/kg bw. Each group consisted of 10 males and 10 females used for 90-day dosing and euthanized on the day 91. An additional satellite group of six animals per sex was included in the control and the top dose group for observation after the treatment period for the potential reversibility or persistence of any toxic effects and was euthanized at day 112. All animals were observed twice daily for mortality and morbidity. Detailed clinical observations, body weights, and food consumption were recorded weekly. Ophthalmic examinations were done once in the pre-treatment period and at the end of the in-life period. Functional Observational Battery (FOB) and locomotor activity data were recorded for half males and females from each group at the end of the dosing period and in recovery subgroup. Clinical pathology evaluations (hematology, coagulation, serum chemistry, and thyroid hormones T3, T4, and TSH) were performed in all males and females at the end of an in-life phase. Urinalysis determination was performed in all animals before scheduled euthanasia. Complete necropsies were conducted on all animals, and selected organs were weighed. Tissues were examined microscopically from all males and females in the vehicle control and high-dose groups from the main subgroup; lower doses level groups and satellite subgroup were evaluated for presumptive target organs.


Male rats appeared to be more sensitive to the test substance in this study.


Majority of morphological and histopathological effects in kidney and liver appeared in the highest dose group (1600 mg/Kg bw); hence, those effects are not relevant for repeated dose target organ classification (STOT RE) in the GHS/CLP criteria. Majority of effects that appeared at low and intermediate doses were mild clinical chemistry effects such as glucose and urea levels and are most likely indications of minor metabolic changes without any noticeable organ dysfunction. Thyroid hormone related changes only appeared at the highest dose and were not accompanied by TSH change and any other alteration in thyroid gland; hence, do not represent any significant treatment related specific target organ effects up to at least dose level of 400 mg/kg.


Hyperplasia of Leydig cells that was observed in some animals at low and intermediate doses, was most likely a result of hormonal imbalance due to disrupted cholesterol homeostasis and it should be considered an adverse effect. However, hyperplastic findings were minimal to slight grade and were not accompanied by any other major adverse effects in testes up to the highest tested concentrations of 1600 mg/kg bw.


In conclusion, although the NOAEL derived for the substance in male rats is 100 mg/kg bw/day, the adverse effects observed in this study are not severe enough to justify classification in Specific target organ toxicity - repeated (STOT-RE) category under GHS and CLP.


The test substance (source) was the carboxylic acid component of the registered substance (tosyl salt). Read-across between the tosyl salt carboxylic acid (6-[(p-Tosyl)amino]hexanoic acid) and the registered substance is considered justified as the registered substance is manufactured directly from 6-[(p-Tosyl)amino]hexanoic acid by simple neutralisation with triethanolamine (TEA). Other than ionization of the carboxylic acid group, the 6-[(p-Tosyl)amino]hexanoic acid remains chemically unchanged upon salt formation. In water, the acid and amine components of 6-[(p-Tosyl)amino]hexanoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) dissociate completely and behave essentially as independent substances. Since TEA can be considered non-hazardous, it is the acid component of the salt that will have a more significant impact on the outcome of any (eco)toxicological or environmental tests. The pKa of the carboxylic acid group in 6-[(p-Tosyl)amino]hexanoic acid (pKa = 4.90) is the same in the free acid as it is in the TEA salt. As a result, 6-[(p-Tosyl)amino]hexanoic acid will respond to changes of pH in the same way whether it is in the salt form or as the parent carboxylic acid and hence it’s bioavailability will be the same (Further justifications for the support of read-across hypothesis are attached in Section 13).


 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
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 via the oral route of the carboxylic acid component of the registered substance (tosyl salt) to rats was determined in accordance with the OECD Guideline for Testing of Chemicals 422 and the OECD TG 408.


In the OECD TG 422 study, doses of 0, 100, 400, and 1600 mg/kg bw/day of the test substance were administered by stomach tube to groups of 12 male rats for 54 days. The test-article was formulated in drinking water and administered in 10 ml/kg bw. At dosages of 100 and 400 mg/kg bw/day the animals showed no differences to the control animals (NOEL). All examined organs and tissues showed a normal histological structure. 1600 mg/kg bw/day may have induced an influence on the body weight gain of the males (and the survival of pups until day 4 after birth). The NOEL and NOAEL for the test substance is defined as 400 mg/kg bw/day. The LOEL for the test substance is defined as 1600 mg/kg bw/day.


In the OECD TG 408 study, doses of 0, 100, 400, and 1600 mg/kg bw/day of the test substance were administered by stomach tube to 3 groups of 10 male and 10 female rats for 90-day dosing and euthanized on the day 91. The test-article was formulated in drinking water and administered in 10 ml/kg bw. Male rats appeared to be more sensitive to the test substance in this study. Majority of morphological and histopathological effects in kidney and liver appeared in the highest dose group (1600 mg/Kg bw). Majority of effects that appeared at low and intermediate doses were mild clinical chemistry effects such as glucose and urea levels and are most likely indications of minor metabolic changes without any noticeable organ dysfunction. Thyroid hormone related changes only appeared at the highest dose and were not accompanied by TSH change and any other alteration in thyroid gland; hence, do not represent any significant treatment related specific target organ effects up to at least dose level of 400 mg/kg.


Hyperplasia of Leydig cells that was observed in some animals at low and intermediate doses, was most likely a result of hormonal imbalance due to disrupted cholesterol homeostasis and it should be considered an adverse effect. However, hyperplastic findings were minimal to slight grade and were not accompanied by any other major adverse effects in testes up to the highest tested concentrations of 1600 mg/kg bw. The NOEL and NOAEL for the test substance is defined as 100 mg/kg bw/day in males and 1600 mg/kg bw/day in females.


Read-across between the tosyl salt carboxylic acid (6-[(p-Tosyl)amino]hexanoic acid) and the registered substance is considered justified as the registered substance is manufactured directly from 6-[(p-Tosyl)amino]hexanoic acid by simple neutralisation with triethanolamine (TEA). Other than ionization of the carboxylic acid group, the 6-[(p-Tosyl)amino]hexanoic acid remains chemically unchanged upon salt formation. In water, the acid and amine components of 6-[(p-Tosyl)amino]hexanoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) dissociate completely and behave essentially as independent substances. Since TEA can be considered non-hazardous, it is the acid component of the salt that will have a more significant impact on the outcome of any (eco)toxicological or environmental tests. The pKa of the carboxylic acid group in 6-[(p-Tosyl)amino]hexanoic acid (pKa = 4.90) is the same in the free acid as it is in the TEA salt. As a result, 6-[(p-Tosyl)amino]hexanoic acid will respond to changes of pH in the same way whether it is in the salt form or as the parent carboxylic acid and hence it’s bioavailability will be the same.


In a chronic two year study on rats using the triethanolamine component of the tosyl salt, the test substance was found to be toxic to the kidneys at a concentration of 1000 mg/kg bw/day. Kidney weights were greatly increased, and major histopathological changes were observed in the kidneys, suggesting a dose-related acceleration of chronic nephropathy, which is common in ageing Fischer 344 rats. A variety of tumors developed in all groups, including the control group, and all tumors observed were histologically similar to spontaneous tumors in this strain of rats. No statistically significant increase of the incidence of any tumor was observed in the treated groups of both sexes by the chi-square test. In this study, however, there was an increase in nephrotoxicity, which appeared to have an adverse effect on the life expectancy of the treated animals, especially of females. These tumors, however, have been observed spontaneously in this strain of rats, and their incidences in the control group of the present study were lower than those of our historical controls. These results may indicate that a positive trend in the occurrence of these tumors is not attributable to triethanolamine administration. The toxicity of the test substance to the rats would appear to be related to an acceleration of normal ageing and tumour formation in this species of rat.


In conclusion, the registered substance (tosyl salt) is not considered toxic via repeated oral exposure and does not need to be classified as such according to EU CLP criteria. Although in the OECD 414 the NOAEL derived for the substance in male rats is 100 mg/kg bw/day, the adverse effects observed in this study are not severe enough to justify classification in Specific target organ toxicity - repeated (STOT-RE) category under GHS and CLP


 


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:


Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 422 and OECD TG 408. The study was conducted on 6-[(p-Tosyl)amino]hexanoic acid, which is the carboxylic acid component of the registered substance i.e. tosyl salt. The NOEL and NOAEL for the test substance is defined as 400 mg/kg bw/day. The LOEL for the test substance is defined as 1600 mg/kg bw/day in the OECD 422 and 100 mg/kg bw/day in males and 1600 mg/kg bw/day in females in the OECD 408.


Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:


This study is not deemed necessary as the substance does not solidify readily and when it does it is a waxy solid and does not form inhalable particles. The substance has also been estimated to have a very low vapour pressure via modelling using the EPIsuite modelling program. The substance is provided to customers only in formulated products and is not isolated. Therefore, inhalation is not considered to be a significant route of exposure to the registered substance, in accordance with REACH Annex XI, Column 2. The results of a repeated dose oral toxicity study will be provided (section 7.5.1). It is therefore considered unjustified to perform further animal testing.


 


Justification for selection of repeated dose toxicity inhalation - local effects endpoint:


This study is not deemed necessary as the substance does not solidify readily and when it does it is a waxy solid and does not form inhalable particles. The substance has also been estimated to have a very low vapour pressure via modelling using the EPIsuite modelling program. The substance is provided to customers only in formulated products and is not isolated. Therefore, inhalation is not considered to be a significant route of exposure to the registered substance, in accordance with REACH Annex XI, Column 2. The results of a repeated dose oral toxicity study will be provided (section 7.5.1). It is therefore considered unjustified to perform further animal testing.


 


Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:


This study is not considered necessary as repeated dose toxicity via the oral route will be provided (section 7.5.1), in accordance with REACH Annex IX, Column 2. Dermal exposure to the substance is not considered likely as the substance is supplied in an aqueous formulation without isolation of the substance. In addition, the substance is directly added to the formulated product without isolation so skin contact is not considered a major exposure route to this substance. Therefore, further animal testing is considered to be unjustified.


 


Justification for selection of repeated dose toxicity dermal - local effects endpoint:


This study is not considered necessary as repeated dose toxicity via the oral route will be provided (section 7.5.1), in accordance with REACH Annex IX, Column 2. Dermal exposure to the substance is not considered likely as the substance is supplied in an aqueous formulation without isolation of the substance. In addition, the substance is directly added to the formulated product without isolation so skin contact is not considered a major exposure route to this substance. Therefore, further animal testing is considered to be unjustified.

Justification for classification or non-classification

The carboxylic acid component of the registered substance was not found to cause any toxic effects after repeated oral exposure in rats. Kidney toxicity was seen at a dose of 1000 mg/kg bw/day in a study using the triethanolamine component of the registered substance. However, this toxicity was considered an acceleration of the normal ageing process of the tested species of rat. The registered substance was not considered to cause toxicity after repeated oral exposure.

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