Ethylene bis[3,3-bis(3-tert-butyl-4-hydroxyphenyl)butyrate]

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Nov - 7 Dec 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline conform GLP study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst/The Netherlands
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 19.6 +/- 0.9 g
- Housing: single in Macrolon Type I cages with wire mesh top with granulated soft wood bedding
- Diet (e.g. ad libitum): pelleted standard diet (Harlan Winkelmann GmbH, Borchen, Germany), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days prior to the start of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1 Dec To: 7 Dec 2004

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

7.5, 15, 30%

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
To determine the highest non-irritant and technically applicable test item concentration, a pre-test was performed in two mice with concnetrations of 5.0, 10.0, 20.0 and 30.0% (w/v) dissolved in acetone:olive oil, 4:1 (v/v). The preparations were made freshly before each dosing occasion. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: The sensitivity and reliability of the experimental technique employed was assessed by use of a
substance which is known to have skin sensitisation properties in CBA/CaOlaHsd mice (α-Hexylcinnamaldehyde in acetone:olive oil, 4:1 )

TREATMENT PREPARATION AND ADMINISTRATION:
25 µl was spread to the entire dorsal surface of each ear of each mouse once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the the relevant vehicle alone (control animals). On day 6 an injection of 250 µl phosphate buffered saline (PBS) containing 19.6 µCi of 3H-methyl thymidine (3H-TdR) was made into the tail vein of each experimental mouse. Five hours later, the draining Auricular lymph node of each ear was excised and pooled per group (8 nodes per group) into PBS. A single cell suspension of lymph node cells was prepared from each group. After washing two times with phosphate buffered saline the lymph node cells were resuspended in 5 % trichloroacetic acid and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid and transferred to plastic scintillation vials. The level of 3HTdR incorporation was then measured on a ß-scintillation counter.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean values and standard deviations were calculated for the body weights.
A statistical analysis was conducted for assessment of the dose response relationship, and the EC3 value was calculated according to the equation

EC3 = (a-c) [(3-d)/(b-d)] + c

where EC3 is the estimated concentration of the test item requird to produce a 3-fold increase in draining lymph node cell proliferative activity (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed by use of Hexylcinnaldehyde which is known to have skin sensitisation properties in CBA/CaOlaHsd mice.
The EC3 value was calculated to be 9.9%.

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

CG1 /test item concentration: 0 % (w/w); 4 animals

Key result

Parameter:

SI

Value:

0.75

Test group / Remarks:

TG 2 / test item concentration 7.5 % (w/w); 4 animals

Remarks on result:

other:

Remarks:

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Key result

Parameter:

SI

Value:

0.73

Test group / Remarks:

TG 3/test item concentration 15 % (w/w); 4 animals

Remarks on result:

other:

Remarks:

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Key result

Parameter:

SI

Value:

1.07

Test group / Remarks:

TG 4/test item concetration 30 % (w/w); 4 animals

Remarks on result:

other:

Remarks:

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Table 1: Calculation and Results of Individual Data

Vehicle: acetone:olive oil (4+1 v/v)

Test item concentration % (w/w)	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
---	BG I	4.8	---	---	---	---
---	BG II	0.0	---	---	---	---
0	CG 1	8271.0	8268.6	8	1033.6	1.00
7.5	TG 2	6212.9	6210.5	8	776.3	0.75
15.0	TG 3	6037.2	6034.8	8	754.4	0.73
30.0	TG 4	8879.9	8877.5	8	1109.7	1.07

BG =  Background (1 ml 5% trichloroacetic acid) in duplicate

CG 1    =  Control Group

TG 2-4=  Test Group

S.I. =  Stimulation Index

^a)   =  The mean value was taken from the figures BG I and BG II

^b)    =  Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test itemHostanox O 3 was found to be not a skin sensitiser under the described conditions.

Executive summary:

The sensitization potential was investigated according to OECD Guideline 429.

The test item concentrations choosen for the LLNA test were 7.5, 15.0 and 30.0 %.

No deaths occurred during the study period. The animals treated with 7.5 and 15 % of the test itemdid not show any clinical signs during the course of the study. At the highest dose of 30 % the ear skin of all four animals was swollen and inflamed after the second and third application.

In this study Stimulation Indices of 0.75, 0.73 and 1.07 were determined with the test item at concentrations of 7.5, 15.0 and 30.0 % (w/v) in Acetone:Olive oil, 4:1 (v/v). Hostanox O 3 was found to be not a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

No deaths occurred during the study period. The animals treated with 7.5 and 15 % of the test itemdid not show any clinical signs during the course of the study. At the highest dose of 30 % the ear skin of all four animals was swollen and inflamed after the second and third application.

Migrated from Short description of key information:
The sensitization potential of Ethylene-bis[3,3-bis(3-tert.-butyl-4-hydroxyphenyl)butyrate] was investigated according to OECD Guideline 429. The test item concentrations chosen for the LLNA test were 7.5, 15, 30 %.

Justification for selection of skin sensitisation endpoint:
Guideline conform GLP study.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Ethylene-bis[3,3-bis(3-tert.-butyl-4-hydroxyphenyl)butyrate] did not show skin sensitizing properties in the Local Lymph Node Assay according to the current OECD guidelines. Allergic skin reactions or case reports of acute contact dermatitis to Ethylene-bis[3,3-bis(3-tert.-butyl-4-hydroxyphenyl)butyrate] have not been described in the literature. Therefore, the substance does not have to be classified as a skin sensitizer.