Glycerides, C16-18 mono-, di- and tri-, hydrogenated, citrates, sodium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-10-22 to 2001-11-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

17. July 1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

EEC Directive 92/69/EEC, Annex V, Test B6, 29. December 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

OECD Guideline 429 for LLNA method was not available yet by the time the study was conducted

Test material

Test material information

Constituent 1

Reference substance name:

Glycerides, C16-18 mono-, di- and tri-, hydrogenated, citrates, sodium salts

EC Number:

294-601-7

EC Name:

Glycerides, C16-18 mono-, di- and tri-, hydrogenated, citrates, sodium salts

Cas Number:

91744-39-7

Molecular formula:

Molecular formula cannot be given as substance is a mixture.

IUPAC Name:

Glycerides, C16-18 mono-, di- and tri-, hydrogenated, citrates, sodium salts

Details on test material:

- Chemical name: glycerocitric acid-/-fatty acid ester
- Physical state: fine, ivory-coloured powder
- Analytical purity: 100 % a.i.
- Impurities (identity and concentrations): no data
- Lot/batch No.: 14 EG 13
- Expiration date of the lot/batch: March 2002
- Storage condition of test material: ambient, protected from light

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Pirbright white, HsdPoc:DH

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, 33178 Borchen, Germany
- Age at study initiation: no data
- Weight at study initiation:at arrival: 300 - 350 g; main test: males: 597 - 701 g, females. 468 - 592 g
- Housing: collective housing up to a maximum of 5 animals per cage in a battery of cages, equipped with a paper disposal system
- Diet: ad libitum, 2040 Teklad Global guinea pig diet
- Water: ad libitum, tap water as for human consumption
- Acclimation period: 26 days (range finding); 30 days (main test)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 - 70
- Air changes (per hr): about 16 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark with light on at 7:00 a.m.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: water, containing one drop of Tween 80

Induction 2

Concentration / amount:

Range finding test:
- intradermal: 5 %
- dermal: 50 %
Main test:
- intradermal induction: 5 %
- dermal induction 50 %
- challange exposure: 50 %

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: water, containing one drop of Tween 80

Challenge 2

Concentration / amount:

Range finding test:
- intradermal: 5 %
- dermal: 50 %
Main test:
- intradermal induction: 5 %
- dermal induction 50 %
- challange exposure: 50 %

No. of animals per dose:

Range finding test: 1 male and 1 female (5 % , 50 %)
Main test: 5 males and 5 females, 5 control animals (3 males, 2 females)

Details on study design:

RANGE FINDING TESTS:
Intradermal injection:
Test substance was diluted with aqua ad injectabilia (containing one drop of Tween 80) and Freund´s complete adjuvant (FCA, batch no. 20K8933, Sigma, Deisenhofen, Germany) to a final concentration of 5.0 %. Two animals were employed, skin reactions being recorded 48 h after treatment.

Dermal application:
Test substance was used 50 % in vaseline (batch no. 419496/144700, Fulka AG, Buchs, Switzerland). A closed patch exposure was effected by means of an occlusive bandage using Hill-Top Chambers (Hill Top, Cincinnati, USA), and non-irritating tape Elastoplast (Beiersdorf AG, Hamburg, Germany), which enveloped 48 h post application.


MAIN STUDY
A. INDUCTION EXPOSURE
First stage (day 0)
- No. of exposures: 3 pairs of intradermal injections (0.1 ml) in each test animal
- Test groups:
1. FCA 50 % (v/v) diluted in aqua ad injectabilia
2. test substance 5 % in aqua ad injectabilia with Tween 80
3. test substance 5 % in aqua ad injectabilia with Tween 80/FCA
- Control group:
1. FCA 50 % (v/v) diluted in aqua ad injectabilia
2. Aqua ad injectabilia with Tween 80
3. Aqua ad injectabilia with Tween 80 50 % (v/v) diluted in FCA
- Site: clipped intrascapular region on either side of the spine

Second stage (day 7):
- Pretreatment: 10 % sodium lauryl sulfate (SLS) in vaseline 24 h before application of the test article
- Concentration: 50 % test substance in vaseline
- Exposure period: 48 h
- Site: The previous injection sited were covered occlusively with a patch carrying the test substance or, in control animals, the control article
- Rationale for pretreatment: Since the 50 % test substance formulation was non-irritating after dermal application in the range finding test, the skin area was reclipped and pretreated with 10 % sodium lauryl sulfate (SLS) in vaseline 24 h before application of the test substance to induce a mild inflammation.


B. CHALLENGE EXPOSURE (day 21)
- Day(s) of challenge: 14 days after second stage of induction
- Exposure period: 24 h
- Exposure: occlusive: closed patch exposure was effected by means of an occlusive bandage using Hill-Top Chambers (Hill Top, Cincinnati, USA), and non-irritating tape Elastoplast (Beiersdorf AG, Hamburg, Germany), which enveloped the whole animal´s trunk)
- Site: 5 x 5 cm clipped skin area on each flank. Test substance was applied to the left flank, and the control article to the right flank
- Volume of test substance and control article: 0.5 g
- Concentrations: 50 %
- Control group: control article: petrolatum
- Evaluation (hr after challenge): after 24 and 48 h
- Classification system for skin reactions: according to Draize


OTHER:
- The animals were weighed before treatment and at the end of the study.

Positve control (reliability check):
- The sensitivity of the test system and the reliability of the experimental technique was assessed at least every six month. 4-aminobenzoic acid ethyl ester (benzocaine, Lot-no.: 128H1235, concentration 10 and 50 % in vaseline) is known to induce skin sensitisation in guinea pigs. The test was conducted as a Magnusson und Kligman test according to OECD 406, 17 July 1992 and to the EEC Directive 92/69/EEC, 31 July 1992.

Positive control substance(s):

yes

Remarks:

4-aminobenzoic acid ethyl ester (benzocaine) periodically tested

Results and discussion

Positive control results:

Periodically checks of the sensitivity of the test system and the reliability of the experimental technique performed. According to the maximisation scheme of Magnusson and Kligman, the positive control article 4-aminobenzoic acid ethyl ester (benzocaine) was classified as a mild sensitiser at the concentration of 10 % , referring to an independent experiment (22 August to 15 September 2001)

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 %

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: 50 %. 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:

50 %

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: 50 %. 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 %

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 %. 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 %

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 %. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: none.

Results 5

Reading:

other: 1st and 2nd reading

Group:

positive control

Remarks on result:

not measured/tested

Any other information on results incl. tables

Range finding test:

Intradermal injection: No skin reactions were observed after intradermal injection of the test substance at a concentraton of 5 %.

Dermal application: No skin reactions were observed after dermal application of the test substance at a concentraton of 50 %.

Main test:

There were no skin reactions after the dermal application in the induction phase. No allergic skin reactions were observed in the test animals after the challange exposure 24 and 48 h after patch removal. No findings were observed in control animals.

The sensitisation rate was determined 24 and 48 h after patch removal and was 0 % in the test animal in each case.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

No allergic skin reactions occurred in test or control animals 24 and 48 h after the end of the challenge procedure. The sensitisation rate was 0 %

Executive summary:

In a dermal sensitization study with CAS 91744-39-7 (100 % a.i.), 10 Pirbright white guinea pigs (5 males, 5 females) were tested using the MAXIMATION TEST method of Magnusson und Kligman according to OECD Guideline 406. The sensitivity of the test system was periodically assessed with the positive control 4-Aminobenzoic acid ethyl ester (benzocaine).

Concentrations used for intradermal and dermal induction were 5 % in water and 50 % in vaseline, respectively. Since the 50 % test substance formulation was non-irritating after dermal application in the range finding test, the skin area was pretreated with 10 % sodium lauryl sulfate in vaseline 24 h before dermal induction treatment to induce a mild inflammation. Challenge exposure concentration was 50 % in vaseline.

No allergic skin reactions occurred in test or control animals 24 and 48 h after the end of the challenge procedure. The sensitisation rate was 0 %.

In this study, the test substance is not a dermal sensitizer.