Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, 2-ethylhexyl ester

Endpoint summary

Description of key information

The structural analogue substance induces liver metabolism which results in activation of the liver-thyroid axis in rats and effects on liver at fairly low doses (NOAEL = 5 mg/kg bw for subacute oral toxicity) (RCC, 1991). Mice are less sensitive and showed adaptive effects on liver with a NOAEL of 150 mg/kg bw for exposure for 12 - 18 weeks (Huntington, 2001).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

13.REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to Read-Across Justification Document in chapter 13.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to Read-Across Justification Document in chapter 13.

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to Read-Across Justification Document in chapter 13.

4. DATA MATRIX
Please refer to Read-Across Justification Document in chapter 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Remarks:

Rat

Effect level:

5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see remark

Remarks on result:

other: CAS 125643-61-0

Key result

Dose descriptor:

NOAEL

Remarks:

Mouse

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: CAS 125643-61-0

Key result

Dose descriptor:

NOAEL

Remarks:

Rat RF 5 d

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no edverse effects observed

Remarks on result:

other: CAS 125643-61-0

Key result

Dose descriptor:

NOAEL

Remarks:

Rat RF 14 d

Effect level:

5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: CAS 125643-61-0

Dose descriptor:

NOAEL

Remarks:

chronic study

Effect level:

64 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Remarks on result:

other: CAS 2082-79-3

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (actual dose received)

System:

other: liver-thryroid axis in rat

Organ:

liver

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant study

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

Assessment of structural analogue substance (EC 406-040-9)

In the available subacute oral toxicity study, the substance was administered by gavage to Albino, SPF-bred, Wistar rats at doses of 0, 5, 50 and 250 mg/kg bw/day daily during 29 consecutive days for males and 30 days for females (RCC, 1991). The study design included one main group and one recovery group, for a total of 10 males and 10 females at each dose level. From the 10 rats per sex in each dosage group, 5 were designated for sacrifice at the end of the treatment and 5 were designated for sacrifice after a 2-week treatment-free (recovery) period. No mortality and no treatment related clinical signs were observed. The liver and the thyroid gland were identified as target organs by microscopical findings. The NOAEL is below 5 mg/kg bw/day for the males and is 5 mg/kg bw/day for the females.

The species-specific mechanisms causing the thyroidal stimulation following uptake of 2,6-substituted phenols (and similar compounds) in rats have been described in standard textbooks and are generally acknowledged by the scientific community (Davies, D.T. (1996). Thyroid Endocrinoloy. Animal Clinical Chemistry: A primer for Toxicologists. Ed: A.O. Evans,and Francis,). From basic research performed over several years, it became clear, that the stimulation of glycuronidyl-transferase synthesis is used to facilitate excretion of 2,6-substituted phenolic antioxidants. However, glycuronidyl-transferase is also able to promote the excretion of thyroid hormones. This has remarkable consequences for the physiology of the rat upon exposure to phenolic antioxidants:

- In the rat, the thyroid hormones T3and T4 are not bound to a specific carrier protein in the plasma. Therefore, increased levels of glycuronidyl-transferase (induced e.g. by 2,6-substituted phenolic antioxidants) speed up the elimination of T3and T4. This causes a drop of the plasma concentrations of T3and T4. By feedback mechanism, reduced levels of T3and T4hormone subsequently lead to a stimulation of the thyroid to produce new hormone. The chronic stimulation of the thyroid can cause hyperplasia and can lead in extreme cases to thyroid tumours.

- In primates, about 90% of the thyroid hormones T3and T4are bound to a specific protein (thyroxine binding protein) in the plasma. Due to this protein, primates are protected from significant changes of thyroid hormone levels in the plasma, even in the presence of high levels of glycuronidyl-transferase. Consequently, in primates thyroid hormone concen­trations are not affected, no feedback mechanisms are induced to produce new T3and T4and no thyroid hyperplasia occurs.

 

Human experience with phenolic antioxidants similar to EC no 406-040-9

The active, bioavailable substructure of EC no. 406-040-9 is closely related to 2’,6’-di-tertiary butylmethylphenole (=butylated hydroxytoluene, BHT). BHT is a direct food additive used world-wide since decades and causes - similar to EC 406-040-9 - a stimulation of the thyroid in rodents. BHT is known to be safe for humans due to the long-lasting experience and due to the well investigated mechanisms of toxicity (see above).

Data from a preliminary study and a one-generation study in mice (Huntington 2001 a and b)

The effects of EC no. 406-040-9 have been investigated in the course of a range-finder study and the subsequent one-generation reproductive toxicity study in the mouse. The mouse model was chosen in agreement with the German Competent Authority to avoid the masking of possible human-relevant effects by massive non-relevant rat-specific effects. Although both rat and mouse don’t own thyroxine binding proteins, the effects of phenolic antioxidants on the excretion rate of T3and T4in the mouse are known to be slighter than in the rat. It was therefore expected that the mouse model allows the evaluation of human-relevant effects. To gain additional information on the effects on the metabolic and hormonal status, blood samples were taken in the preliminary study (2001b) to assess the thyroid function (by measuring T3and T4). Additionally, at termination liver samples were assessed for peroxisomal palmitoyl CoA-oxidase and microsomal lauric acid 12-hydroxylase activity.

- In the preliminary reproductive toxicity study (2001b) the substance was administered by gavage at dosages of 50, 150 or 500 mg/kg bw/day. To mice for 15 days prior to pairing and until termination, after waning, of the filial F1 generation. Dosages up to the top dose had no obvious adverse effects on general condition, thyroid function, mating performance, fertility or the progress of gestation or lactation in the treated mice. Furthermore, littering and survival and development of the F1 progeny were similarly unaffected. Parental liver weights at 500 mg/kg bw/day were high and hepatic lauric acid 12-hydroxylase activity was increased at this dosage for the males and females and at 150 mg/kg bw/day for the females only. The substance seems to act as a weak inducer of the cytochrome P450 CYP4A subfamily. There was no evidence that the substance was acting as a peroxisome proliferator in the mouse after seven weeks of treatment. Based on these observations the NOAEL was established at 500 mg/kg bw/day.

- In the one-generation study (Huntingon 2001a) mice were treated by gavage at dosages of 50, 150 or 600 mg/kg bw/day. After eight weeks of treatment males and females from within the same treatment groups were paired. Treatment was continued during mating period, pregnancy, parturition until weaning of the offspring. The treatment was well tolerated by the F0 generation. There were no treatment-related findings throughout the study. The substance did not affect mating performance or fertility of the parental animal, or the ability of the females to successfully rear a litter to weaning. Treatment of the parental animals did not have any detrimental effects on the condition, survival and development to weaning of the offspring. Body weight relative liver weights for males and females were 20% greater at 600 mg/kg bw/day than the Control values, and histopathological examination revealed a high incidence of centrilobular hepathocytic hypertrophy. Because the type of liver induction is adaptive, histopathology is considered non-adverse and peroxisome proliferation is excluded (see result of the preliminary study) the effect is considered adaptive. Males at 600 mg/kg bw/day also showed increased thyroid weights, with the bodyweight relative group mean value 19% greater than Control. In these males T3levels at termination appeared to be slightly elevated, and T4levels appeared to be slightly reduced when compared to Control and other groups. This may be an effect of treatment. Liver weights were also increased at 150 mg/kg bw/day for the females and to a lesser extent the males, although no microscopic changes were seen in the liver at this dosage. There were no findings at necropsy of the offspring that could be attributed to the treatment of the F0 parents. In conclusion a combination of slight to moderate liver and thyroid findings in adult mice was observed at 600 mg/kg bw/day. Therefore the NOAEL was considered to be at 150 mg/kg bw/day. The NOAEL for reproductive function and survival, growth and development of the offspring to weaning in the context of the study was 600 mg/kg bw/day.

Literature data on the toxicity of phenolic antioxidants, human experience with BHT and the mice studies the test substance clearly demonstrate that the strong stimulation of the thyroid in repeated dose rat studies by phenolic antioxidants is due to a rat specific mechanism and not relevant for the human hazard and risk assessments. Therefore, and in line with Commission Directive 93/21/EEC, labelling with R48/22 is considered inappropriate.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data on structural analogue substance are reliable and suitable for classification purposes under Regulation No 1272/2008. Literature data on the toxicity of phenolic antioxidants, human experience with BHT and the mice studies the test substance demonstrate that the strong stimulation of the thyroid in repeated dose rat studies by phenolic antioxidants is due to a rat specific mechanism and not relevant for the human hazard and risk assessments. Therefore, labelling with STOT RE is considered inappropriate and the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No 1272/2008, as amended for the eighth time by Regulation (EU) No 2016/218.