Reaction mass of bis(2-ethylhexyl) terephthalate, butyl 2-ethylhexyl terephthalate, and dibutyl terephthalate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 July 2013 - 01 August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of bis(2-ethylhexyl) terephthalate, butyl 2-ethylhexyl terephthalate, and dibutyl terephthalate

EC Number:

946-149-3

Cas Number:

1571954-81-8

IUPAC Name:

Reaction mass of bis(2-ethylhexyl) terephthalate, butyl 2-ethylhexyl terephthalate, and dibutyl terephthalate

Test material form:

liquid

Details on test material:

Identification: GL500
Batch: 20130510
Purity: 99.81%
Expiry date: 10 May 2018
Storage Conditions: room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK.
- Females nulliparous and non-pregnant: yes
- Age at study initiation:eight to twelve weeks old.
- Weight at study initiation:15 to 23 g
- Housing:The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum):Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.
- Acclimation period:Five days


ENVIRONMENTAL CONDITIONS
The temperature and relative humidity were controlled to remain within target ranges of 19 to 25 °C and 30 to 70%, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Undiluted test item was used as well as the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4:1.

No. of animals per dose:

4 animals per dose.

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility:The mouse was treated by daily application of 25 µL of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6.
- Irritation:Local skin irritation was scored daily according to the scale
- Systemic toxicity:Any clinical signs of toxicity, if present, were also recorded. The body weight was recorded on Day 1 (prior to dosing) and on Day 6.
- Ear thickness measurements:The thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.
- Erythema scores:not specified

MAIN STUDY


ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:Skin Sensitization: Local Lymph Node Assay
- Criteria used to consider a positive response:The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3 HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3 HTdR incorporation will be classified as a "non-sensitizer."

TREATMENT PREPARATION AND ADMINISTRATION:

For the purpose of the study, the test item was used undiluted and freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced a suitable formulation at the required concentration.The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.


Groups of four mice were treated with the undiluted test item or the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.

A further group of four mice received the vehicle alone in the same manner.

Positive control substance(s):

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

Results and discussion

In vivo (LLNA)

Results

Key result

Parameter:

SI

Value:

3.61

Remarks on result:

positive indication of skin sensitisation based on QSAR/QSPR prediction

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: not specified.

DETAILS ON STIMULATION INDEX CALCULATION: Please see table in results section.


EC3 CALCULATION: The concentration of test item expected to cause a 3 fold increase in 3 HTdR incorporation (EC3 value) was calculated to be 70%.


CLINICAL OBSERVATIONS:There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHTS: Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Any other information on results incl. tables

Concentration(%v/v) in acetone/olive oil 4:1	Stimulation Index	Result
25	2.88	Negative
50	2.61	Negative
100	3.61	Positive

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test item was considered to be a sensitizer under the conditions of the test.

Executive summary:

Introduction

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

 

 

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% v/v. A further group of four animals was treated with acetone/olive oil 4:1alone.

Results

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Please see table in results section.

The concentration of test item expected to cause a 3 fold increase in3HTdR incorporation (EC3value) was calculated to be 70%.

 

 

Conclusion

The test item was considered to be a sensitizer under the conditions of the test.