1,6-Bis(methoxybenzoyloxy)hexane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28th January 2002 - 1st March 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

- OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No. 406, "Skin Sensitisation" - EC Commission Directive 96/54/EC, Annex IV C, B.6: "Skin sensitisation", Official Journal of the European Communities No. L 248, 1996 - EPA OPPTS 870.2600 "Skin sensitisation", August 1998

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

13 March 2002 and 14 march 2002

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The guinea pig maximisation test results were existing data and were not commissioned for the purposes of REACH.

Test material

Test material information

Constituent 1

Reference substance name:

1,6-Bis(methoxybenzoyloxy)hexane

Cas Number:

390359-18-9

Molecular formula:

C22H26O6

IUPAC Name:

1,6-Bis(methoxybenzoyloxy)hexane

Test material form:

other: Yellowish Solid

Details on test material:

- Name of test material (as cited in study report): Setafix X 11342
- Substance type:
- Physical state: Solid
- Analytical purity: +/- 95%
- Lot/batch No.: PP02
- Expiration date of the lot/batch: 01 July 2002
- Stability under test conditions: Stable
- Storage condition of test material: Stable

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Kisslegg, Germany
- Age at study initiation: Approx. 4 weeks old
- Weight at study initiation: 309 - 323g
- Housing: Group housing of 5 animals per labelled metal cage with wire-mesh floor
- Diet (e.g. ad libitum): Free access to standard guinea pig diet, including ascorbic acid (1000 mg/kg); (Charles River Breeding and Maintenance Diet for Guinea Pigs, Altromin, Lage, Germany). Hay (B.M.I, Helmond, The Netherlands) was provided twice a week.
- Water (e.g. ad libitum): Free access to tap water
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 15 Air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluororescent light and 12 hours dark per day

IN-LIFE DATES: From: To:

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: 10% DMSO in Corn Oil

Induction 2

Concentration / amount:

MAIN STUDY:

INDUCTION:
Intradermal Induction: 2%
Epidermal Induction: 5%

CHALLENGE:
Epidermal Application: 2%

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: 10% DMSO in Corn Oil

Challenge 2

Concentration / amount:

MAIN STUDY:

INDUCTION:
Intradermal Induction: 2%
Epidermal Induction: 5%

CHALLENGE:
Epidermal Application: 2%

No. of animals per dose:

10 animals per dose

Details on study design:

PRELIMINARY IRRITATION STUDY:
A preliminary irritation study was conducted in order to select test substance concentrations to be used in the main study. The selection of concetrations was based on the following criteria:
- The concentrations are well-tolerated systemically by the animals.
- For the induction exposures: the highest possible concentration that produced mild to moderate irritation (grades 2 - 3).
- For challenge exposures: the maximum non-irritant concentration

Series of the test substance concentrations were tested. Practical feasibility of administration determined the highest starting-concentration for each route. The starting and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 20%, 10%, 5%, 2%, 1% and if needed, further lower concentrations using the same steps.
In addition, the vehicle without test substance was tested in the preliminary irritation study, to study the irritating properties of 10% DMSO in corn oil. The test system and procedures were identical to those used during the main study, unless otherwise specified. The five animals selected were between 4 and 9 weeks old. No body weights were determined.

Intradermal Injections:
A series of four test substance concentrations was used, the highest concentration being the maximum concentration that could technically be injected. Each of two animals received two different concentrations in duplicate (0.1 mL/site) in the clipped scapular region. Once animal received a single injection of 10 % DMSO in corn oil (0.1 mL/site) in the clipped scapular region. The injection sites were assessed for irritation 24 and 48 hours after treatment.

Epidermal Application:
A series of four test substance concentrations was used , the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each) per animal to the clipped flank, using Metalline patches (2 x 3 cm) mounted on Medical tape which were held in place with Microscope tape and subsequently Coban elastic bandage.
The animals receiving intradermal injections were treated with lowest concentrations and two further animals with the highest concentrations. One animal was treated with 10% DMSO in corn oil in duplicate.
After 24 hours, the dressing was removed and the skin cleaned of residual test substance using water.
The treated skin areas were assessed for irritation 24 and 48 hours after exposure.

MAIN STUDY
A. INDUCTION EXPOSURE: Experimental Animals
DAY 1
Three pairs of intradermal injections (0.1 mL/site) were made on a clipped scapular region as follows:
a) A 1:1 w/w mixture of Freund's Complete Adjuvant (Difco, U.S.A.) with water for injection (Fresenius AG, Bad Homburg, Germany)
b) The test substance at a 2% concentration.
c) A 1:1 w/w mixture of the test substance, at twice the concentration used in (b) and Freund's Complete Adjuvant.

DAY 3
The dermal reactions caused by the intradermal injections were assessed on day three for irritation

DAY 8
The scapular area between the injection sites was clipped and subsequently treated with 0.5 mL of a 5% test substance concentration using a Metalline patch (2 x 3 cm) mounted on a Medical tape, which was held in place with Microscope tape and subsequently Coban elastic bandage.

The dressing was removed after 48 hours exposure, the skin cleaned of residual test substance using water and the dermal reactions caused by the epidermal exposure were assessed for irritation.

INDUCTION EXPOSURE: Control Animals
The control animals were treated as described for the experimental animals except that, instead of the test substance, vehicle alone was administered.

B. CHALLENGE EXPOSURE: All animals (Both Experimental group & Control group)

DAY 21
One flank of all animals was clipped and treated by epidermal application of a 2% test substance concentration (0.1 mL) and the vehicle (0.1 mL, 10% DMSO in Corn Oil ), using Patch Test Plasters (Curatest®, Lohmann, Almere, The Netherlands). The patches were held in place with Micropore tape and subsequently Coban elastic bandage.
The dressing was removed after 24 hours exposure and the skin cleaned of residual test substance and vehicle using water. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.

- No. of exposures: 1
- Day(s) of challenge: Day 21 after induction exposure
- Exposure period: 24 hours
- Test groups: 10 animals (females)

- Control group: 5 animals (females)
- Site: One flank of each animal
- Concentrations: 2% test substance concentration (0.1 mL) and the vehicle (0.1 mL, 10% DMSO in Corn Oil )
- Evaluation (hr after challenge): 24 and 48 hours after removal of the dressing.

Challenge controls:

One flank of each control animals was clipped and treated by epidermal application of the vehicle (0.1 mL, 10% DMSO in Corn Oil ), using Patch Test Plasters (Curatest®, Lohmann, Almere, The Netherlands). The patches were held in place with Micropore tape and subsequently Coban elastic bandage.

The dressing was removed after 24 hours exposure and the skin cleaned of residual vehicle using water. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.

Positive control substance(s):

yes

Remarks:

ALPHA-HEXYLCINNAMICALDEHYDE, TECH. 85%

Results and discussion

Positive control results:

A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by NOTOX. In this study, performed August - October 2001, females of the albino Dunkin Hartley guinea pig (from Charles River Deutschland, Kisslegg, Germany) were checked for the sensitivity to ALPHA-HEXYLCINNAMICALDEHYDE, TECH. 85%. The females were approx. 4 weeks old at commencement of the study. The sudy was based on EPA OPPTS 870.2600 guideline; OECD Guideline No. 406, the EC Directive 96/54/EC, Part B.6 and on the method described in "Allergic Contact Dermatitis in the Guinea Pig: Identification of Contact Allergens" Magnusson and Kligman, 1970. ALPHA-HEXYLCINNAMICALDEHYDE, TECH. 85% (CAS No. 101-86-0) was fabricated under lot no. 01016AQ (Aldrich Chemicals Co., Germany).

Test substance concentrations selected for this study were:
Intradermal induction: A 20 % solution in water (Milli-U, w/w). Epidermal induction: undiluted.
First Challenge: a 5 % solution in water (w/w). Second challenge: a 10 15 and a 20 % solution.

CONCLUSION:
The skin reactions observed at the challenge phase in two experimental animals in response to the 10 % test substance concentration and in four experimental animals in response to the 20% concentration were indicative of sensitisation, taking into account the intensity and the duration of the response in the control animals. These results lead to a sensitisation rate of 40 per cent to the 20 % concentration. From these results, it was concluded that the female guinea pig of the albino Dunkin Hartley strain is an appropriate animal model for the performance of studies designed to evaluate the sensitising potential of a substance in aMaximisation type of test.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.1 ml (2 % concentration)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0.1 ml (2 % concentration). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.1 ml ( 2% concentration)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0.1 ml ( 2% concentration). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0.1 ml (vehicle)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0.1 ml (vehicle). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0.1 ml (vehicle)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0.1 ml (vehicle). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Results 5

Group:

positive control

Dose level:

20%

No. with + reactions:

4

Total no. in group:

4

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

PRELIMINARY IRRITATION STUDY

The results of the intradermal injections and epidermal exposures for the selection of suitable test substance concentrations and vehicle for the main study are described in Table 1.

Based on the results, the tst substance concentrations selected for the main study were 2 % concentration for the intradermal induction and a 5 % concentration for the epidermal induction.

A 2 % test substance concentration was selected for the challenge phase. In addition, 10% DMSO in corn oil was considered a suitable vehicle.

MAIN STUDY

Induction Phase

The skin effects caused by the intradermal injections and epidermal exposure during the induction phase are given in Table 2.

Challenge Phase

No skin reactions were evident after the challenge exposure in the experimental and control animals (see Table 3).

Toxicity / Mortality

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Body Weights

Body weights and body gain of experimental animals remained in the same range as controls over the study period (see Table 4).

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on these results and according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC), SETAFIX X 11342 does not have to be classified and has no obligatory labelling requirements for sensitisation by skin contact.

Executive summary:

The purpose of this study "Assessment for Contact Hypersensitivity to SETAFIX X 11342 in the Albino Guinea Pig (maximisation Test)" was to evaluate whether the test substance induces contact hypersensitivity in guinea pigs after intradermal and epidermal exposure of the animals uner the conditions described in the study report. The study should provide a rational basis for risk assessment in man.

The sudy was carried out based on the guidelines described in: EC Directive 96/54/EC, Part B.6 "Skin Sensitisation", OECD No. 406, "Skin Sensitisation" and EPA OPPTS 870.2600 "Skin Sensitisation", August 1998 and based on the method described by Magnusson and Kligman, "Allergic Contact Dermatitis in the Guinea Pig - Identification of Contact Allergens".

Test substance concentrations selected for the main study were based on the results of a preliminary study.

In the main study, ten experimental animals were intradermally injected with a 2 % concentration and epidermally exposed to a 5 % concentration. Five control animals were similarly treated, but with vehicle alone (10% DMSO in corn oil).

Two weeks after the epidermal application all animals were challnged with a 2 % test substance concentration and vehicle.

No skin reactions were evident after the challenge exposure in the experimental and control animals.

There was no evidence that SETAFIX X 11342 had caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in the challenge phase.

This results indicates a sensitisation rate of 0 per cent.

Based on these results and according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Guideliens in Commission Directive 93/21/EEC), SETAFIX X 11342 does not have to be classified and has no obligatory labelling requiremnets for sensitisation by skin contact.