Reaction mass of 2,6-bis(1-phenylethyl)phenol and 2,4,6-tris(1-phenylethyl)phenol

Endpoint summary

Description of key information

The key study for this endpoint is the 90-day portion of a 90-day/36-week study in rats by administration in the diet, with NOAEL 1000 ppm, i.e. approximately 40 mg/kg (males) or 50 mg/kg (females), based on statistically significant effects on body weight gain (both sexes) and liver weight (females).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Dose descriptor:

NOAEL

40 mg/kg bw/day

Additional information

Wingstay S (Styrenated phenol) was tested for its oral repeat-dose toxicity in rats of unspecified strain and age, by dietary administration, in two successive phases of the same experimental study, over 90 days (Food and Drug Research Laboratories study No.81351, 1961) or 36 weeks (Food and Drug Research Laboratories study No.81351, 1962). The 90-day phase was of validity 2 according to Klimisch criteria, whereas the 36-week phase was of validity 3 due to limited investigations (neither clinical pathology nor histopathology were included).

 

Following 90 days of dietary administration at concentrations of 100, 316, 1000, 3160, 10000 ppm, body weight gain was significantly affected at 3160 ppm and 10000 ppm, together with a decreased food efficiency at the highest dose level. Relative liver weight was significantly increased from 3160 ppm for females and at 10000 ppm for males. No associated liver abnormalities were detected at microscopic examination. An increased incidence of focal thyroid hyperplasia was observed at10000ppm. The NOAEL was therefore considered to be 1000 ppm, corresponding to approximately 40 or 50 mg/kg, for males or females, respectively.

 

Following 36 weeks of dietary administration at the same dose levels, body weight was also adversely affected at10000 ppm, but not at 3160 ppm. Significantly increased relative liver weight was also observed at10000 ppm, but not at 3160 ppm. Relative kidney weight was also increased at the highest dose level. No histopathological examination was performed after 36 weeks of dosing. The NOAEL was therefore 3160 ppm, corresponding to approximately 126.4 or 158 mg/kg, for males or females, respectively.

The discrepancy between NOAELs may be attributed to a lower number of animals in the 36-week phase (25 rats per dose and gender, 50 controls per gender) than in the 12-week phase (30 rats per dose and gender, 60 controls per gender), lowering the statistical power of endpoint comparisons (body and organ weights), and therefore resulting in a lower NOAEL despite a higher dosing duration. Because overall, for the endpoints taken into consideration for setting the NOAELs (body weight, liver and kidney weights), the changes versus controls were of similar magnitude between weeks 12 and 36 (except for liver weight in females given 3160 ppm which were increased with a higher magnitude at week 12 than at week 36).

Given the discrepancy between NOAELs and the difference of reliability between studies, a NOAEL of 1000 ppm (corresponding to approximately 40 or 50 mg/kg, for males or females, respectively) for oral subchronic administration in rats should be considered for safety concerns. At higher dose levels, relevant effects consisted of decreased body weight gain, increased liver and kidney weights, and focal thyroid hyperplasia.

In order to further understand the effects observed in the repeated dose toxicity studies, TSP, 2,6-DSP and 2,4-DSP were tested in an in vitro assessment on the transactivation of the rat thyroid hormones receptors (rTRα, rTRβ). TSP, 2,6-DSP and 2,4-DSP were also tested on mouse and human xenosensors Pregnane X Receptors (mPXR, hPXR) and Mouse and human Constitutive Androstan Receptors (mCAR, hCAR) that are involved in thyroid hormones metabolism and elimination. Robust Study Summaries of these studies can be found in the section on “Specific investigations”.

The results show that 2,4,6-TSP, 2,4-DSP and 2,6-DSP did not show agonist or antagonist effects on rTRα and rTRβ, suggesting that they do not have deleterious effect on thyroid hormone signaling. The tested compounds did not show a significant effect on hCAR transactivation.

However, the tested compounds showed a significant transactivation of mPXR, hPXR and mCAR xenosensor with EC50 values in the range of 1-5 μM. Compared to known xenobiotics (enzymatic inducers) the tested compounds are to be considered potent.

In rodents, mPXR and mCAR activation will result in an increased T3/T4 elimination. While in humans only hPXR will be activated, and thus the impact will be less. The increased T3/T4 turnover will lead to decreased T3/T4 plasma levels. Via the negative feedback system to the hypothalamic and pituitary gland the body will try to compensate. As a consequence, T3/T4 production will be stimulated to correct the plasma levels. The increased production will result in a higher follicular activity and TSH pituitary cells activity, leading to follicular hyperplasia associated with an increased thyroid weight and an increased pituitary size and weight.

This will be the case in rodents. In humans, however, this will not occur because of the Thyroid Binding Protein (TBP) (Leghait et al., 2010; Roques et al., 2013). This protein serves as a buffer as it binds T3/T4 and in this way stores the hormones. While the stimulation is already less in humans, as only hPXR is activated, the activation can be compensated by TBP. While in rodents both mPXR and mCAR are activated and they do not express TBP, they will be more sensitive to changes in the thyroid hormones metabolism.

In conclusion, the in vitro assessment provides a possible explanation for the thyroid hyperplasia observed in the repeated dose toxicity tests. The results furthermore indicate that the stimulation of the thyroid will be less in humans as compared to rodents.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; glandular: thyroids; urogenital: kidneys

Justification for classification or non-classification

The guidance values for classification of a substance as harmful on repeated exposure by the oral route according to the criteria of Annex VI Directive 67/748/EEC (R48/22) relate to severe effects reported at 50 mg/kg bw/day or below in a 90-day study. The equivalent guidance values for classification according to EU/GHS criteria (STOT Category 2) relate to effects reported at 10-100 mg/kg bw/day in a 90-day study.

No classification is warranted as to repeated dose toxicity, because:

• the oral route is of limited relevance to the human situation,
• adverse effects in rats were noted only at high dose levels (from approximately 130 mg/kg)
• and no major organ function impairment was evidenced.

TSP/DSP is not classified for repeat-dose toxicity according to the criteria of Annex VI Directive 67/748/EEC or UN/EU GHS.