bis(2-ethylhexyl) (4-hydroxy-3,5-dimethoxybenzyl)malonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-03-08 to 2006-05-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted 17 July 1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

Official Journal No. L 248 A, 30 September 1996

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

When this GPMT was performed, the LLNA was not a gold standard yet.

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

471-510-2

EC Name:

-

Cas Number:

872182-46-2

Molecular formula:

C28H46O7

IUPAC Name:

1,3-bis(2-ethylhexyl) 2-[(4-hydroxy-3,5-dimethoxyphenyl)methyl]propanedioate

Test material form:

liquid: viscous

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

-- Test system
Guinea pig, HsdPoc:DH, females
Age: about 5 weeks

--Identification and adaptation
Healthy young animals were allocated to the study groups at least 7 days before dosing to allow for acclimatization. The guinea pigs were identified by earmark and color mark.

-- Assignment
Overall 20 guinea pigs were used.
Pretest: 5 females
Group 1: negative control group (5 females) Group 2: test material group (10 females)
The mean initial body weight of the guinea pigs at the start of the study was 322 g (range from 302 to 350 g).


-- Housing and diet
The guinea pigs were housed in a 10 m2 room. Lighting was controlled by a timer to provide a 12-hour light - 12-hour dark regime.

Five guinea pigs were housed in type GM/5 (EBECO) Makrolon cages with a shelter and placed on mobile racks. The animals were kept on conventional softwood granulate as the bedding. The cages had been machine cleaned before the start of the study. The bedding was changed two times a week.

Temperature and atmospheric humidity were measured by a thermohygrograph. The room temperature within the study period was 21 to 22°C and the relative atmospheric humidity 51 to 60 %.

Diet and community tap water from Makrolon drinking bottles were available to the guinea pigs ad libitum.

The diet, Provimi Kliba 3418.0, was checked according to the specifications of the manufacturer by independent laboratories. Analysis included both qualitative and quantitative evaluation for heavy metals, aflatoxins, pesticides and antibiotics. The tap water was analyzed periodically according to the German regulations for human drinking water. The softwood granulate was analytically checked by independent laboratories.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

paraffin oil

Concentration / amount:

10 g/L with and without Freund's complete adjuvant

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Concentration / amount:

200 g/L

Day(s)/duration:

1 week after intradermal injection for 48 hours

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Concentration / amount:

50 g/L

Day(s)/duration:

2 weeks after topical induction for 24 hours.

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Control group: 5 females
Test material group: 10 females

Details on study design:

RANGE FINDING TESTS:
- intradermal injection: 0, 1, 5, 10, 25, 50 g/L given to one animal in paraffin oil
- topical applications, no pretreatment: 10, 50, 100, 200, 400 g/L given to four animals in polyethylene glycol
- topical applications, pretreatment with FCA: 1, 10, 50, 100, g/L given to four animals in polyethylene glycol

MAIN STUDY
A1. INDUCTION EXPOSURE (intradermal injection)
- No. of exposures: 6 injections of 0.1 ml each, three on each side of the spinal column)
- Test groups: 0.1 ml FCA + NaCl, 10g/L test item ad liquid paraffin, FCA + 10 g/L test item + NaCl
- Control group: 0.1 ml FCA + NaCl, 0.1 ml liquid paraffin, 0.1 ml FCA + NaCl
- Site: cranial, medial, caudal
- Frequency of applications: single
- Concentrations: 10 g/L

A2. INDUCTION EXPOSURE (topical application)
- No. of exposures: one week after injection (A1) single topical application of a filter plate soaked with either test solution, or vehicle
- Test groups: Test item (200 g/L) in PEG 400
- Control group: PEG 400
- Site: cranial, medial, caudal
- Frequency of applications: single
- Duration: 48 h, occlusive
- Concentrations: 200 g/L

B. CHALLENGE EXPOSURE
- No. of exposures: single topical occlusive application
- Day(s) of challenge: 24 h
- Exposure period: 2 weeks after topical application
- Test group: 50 g/L test item in PEG 400
- Control group: PEG 400
- Site: cranial, medial, caudal

Challenge controls:

Yes

Positive control substance(s):

no

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

0 g/L

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no signs of toxicity

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

0 g/L

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no signs of toxicity

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50 g/L

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

no signs of toxicity

Remarks on result:

no indication of skin sensitisation

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

50 g/L

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no signs of toxicity

Remarks on result:

no indication of skin sensitisation

Results 5

Key result

Group:

positive control

Remarks on result:

positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

After challenge, one animal showed positive reaction in the test material treated skin sites at the first reading but not in the second reading. This results in 10 % positive reactions at challenge. Therefore, the test material is not considered as a sensitizer for the skin. According to the EEC Directive, the test material has not to be classified as a sensitizer.

Executive summary:

Purpose

The purpose of this GPMT assay was to identify the contact allergenic potential of the test item. This study should provide a rational basis for risk assessment to the sensitising potential of the test item in man.

Study Design

The test item was evaluated for skin sensitizing properties in the guinea pig maximization test according to MAGNUSSON and KLIGMAN (1969).
5 Female guinea pigs in the negative control group (group 1) treated with the vehicles (liquid paraffin and polyethylene glycol 400) and 10 females in the test material group (group 2) were investigated.

Induction included intradermal injection of test material preparation in liquid paraffin (10 g/L with and without Freund's complete adjuvant) on experimental day 1, and topical application for 48 hours of test material preparation in polyethylene glycol 400 (200 g/L) on experimental day 8.

Challenge was performed two weeks after topical induction by topical application for 24 hours of the test material preparation in polyethylene glycol 400 (50 g/L) and readings were taken at 48 hours and 72 hours.

Results

After intradermal injection, the common signs of irritation after injection of Freund's complete adjuvant were observed. The injection sites were swollen and red, later on, scabs developed.

After challenge with polyethylene glycol 400, no erythema or edema were observed at the readings.
A single treatment was performed with the test substance  (50 g/L polyethylene glycol 400) to exclude primary irritation of the test material concentration. No positive reactions were observed in the treated areas at any reading.

After challenge with the test substance (50 g/L preparation in polyethylene glycol 400) a single positive skin reaction was observed in one animal at the first reading. Thus resulting in 10 % positive reactions after challenge.
After challenge, no positive reactions were observed in the areas treated with polyethylene glycol 400 alone at any reading.

The clinical behavior of the guinea pigs was normal during the experimental part. All animals survived the experimental part.

The body weight development corresponded to that of the animals of the vehicle group.

Reactions after challange:

Induction with	Challenge with	Positive/animals (48 h after challange)	Positive/animals (72 h after challange)	Positive/animals (overall)
test substance	test substance	1/10	0/10	1/10

Conclusion

After challenge, one animal showed positive reaction in the test material treated skin sites at the first reading but not in the second reading. This results in 10 % positive reactions at challenge. Therefore, the test material is not considered as a sensitizer for the skin.
According to the EEC Directive, the test material has not to be classified as a sensitizer.