reaction product of: saturated, monounsaturated and multiple unsaturated long-chained partly estrified alcohols of vegetable origin (Brassica napus L., Brassica rapa L., Helianthus annuus L., Glycine hispida, Gossypium hirsutum L., Cocos nucifera L., Elaeis guineensis) with O,O-diisobutyldithiophosphate and 2-ethylhexylamine and hydrogen peroxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation

Remarks:

in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug - Oct 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with recognised guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

428-630-5

EC Name:

-

Molecular formula:

Not applicable

IUPAC Name:

reaction product of Z-9-octadecen-1-ol and O,O-diisobutyl hydrogen dithiophosphate

Details on test material:

- Name of test material (as cited in study report): Becrosan 6920
- Physical state: yellowish clear liquid
- Lot/batch No.: 12447
- Expiration date of the lot/batch: December, 1998
- Storage: The test article was stored at room temperature in a closed container.

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Strain: Guinea-pig/Dunkin Hartley, Crl:(HA)BR
- Source: Charles River Wiga GmbH, 97320 Sulzfeld, Germany
- Age at study initiation: approximately 26 days
- Weight at study initiation: 282 g k 12 g (n = 15)
- Housing: The animals were housed under semi-barrier conditions with up to 5 to a cage (Makrolon@ Type 4).
- Diet (e.g. ad libitum): ALTROMIN 1322, standard diet for guinea pigs, ad libitum, Batch No. : 13 1 198/1207
- Water (e.g. ad libitum):mains tap water, ad libitum, daily change
- Acclimation period: 6 days before the start of study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 40 - 70%
- Air changes (per hr): 10 - 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light/dark cycle

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

unchanged (no vehicle)

Induction 2

Concentration / amount:

unchanged (no vehicle)

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Challenge 2

Concentration / amount:

unchanged (no vehicle)

No. of animals per dose:

Control group 5 animals
Dose group 10 animals

Details on study design:

Experimental Procedure
The study was conducted as Guinea-pig Maximisation Test (GPMT) according to Magnusson and Kligman (1). The sensitivity and reliability of the experimental technique used was checked routinely by the use of benzocaine, which is known to  have mild to moderate skin sensitisation properties (2). The results of the most recent reliability check are presented in the Appendix.
 
Pilot study
Preparation of the animals
The hair of2animals was removed from an area (approximately 2 x 4 cm) overlying the scapulae (intradermal injection), in two other animals from theflanksand the shoulder (topical application) by clipping/shaving 24 hours prior to test article administration avoiding skin abrasion.
 
Intradermal injection tolerance test
Twenty four hours after preparation of the animals, 4 pairs of intradermal injections of 0.1 ml each were administered to both animals in the shoulder region so that one of each pair lay on each side of the midline in a cranial to caudal sequence.
Injection 1: 10:90 (v/v) mixture of the test article and Paraffinum liquidum (MERCK KGaA, batch No. K2078 1674428)
Injection 2: 5:95 (v/v) mixture of the test article and Paraffinum liquidum
Injection 3: 2:98 (v/v) mixture of the test article and Paraffinum liquidum
Injection 4: 1:99 (v/v) mixture of the test article and Paraffinurn liquidum
 
Dermal tolerance test
Twenty four hours after preparation of the animals filter papers was loaded with 0.5 ml of the test article formulations and applied to the test areas.
Area 1 (left flank): undiluted test article
Area2(left shoulder): 75:25 mixture (v/v) of test article and Paraffinum liquidum
Area 3 (right shoulder):50:50mixture (v/v) of test article and Paraffinum liquidum
Area 4 (right flank): 25:75 mixture (vh) of test article and Paraffinum liquidum
 
Dose selection for main study
The application sites were examined for skin reactions at intervals of 24, 48 and 72 hours. The concentration of test article used for induction exposure was the highest to cause mild irritation. The concentration used for the challenge exposure was the highest non-irritating dose.
 
Main study
Preparation of the animals
The animals were assigned to a test group of 10 males and a control group of5males.
The hair was removed from an area (approximately 2 x 4 cm) overlying the scapulae by clipping and shaving 24 hours prior to test article administration avoiding skin abrasion.
Day 0: Induction by intradermal injection
Two pairs of intradermal injections of 0.1 ml each were administered to each animal in the shoulder region so that one of each pair lay on each side of the midline in cranial to caudal sequence.
 
Control group
Injection 1:50:50mixture (v/v) of FCA (Sigma GmbH, Batch No. 97H8506) and Aqua pro injectione (Fresenius, batch No. FG 0 13)
 
Injection 2: Paraffinum liquidum (MERCK KGaA, batch No. K20781674428) 
 
Injection 3:50:50mixture (v/v) of Paraffinum liquidum and FCA/Aqua pro injectione (50:50, v/v)
 
 
Dose group
Injection 1: 50:50 mixture (v/v) of FCA and Aqua pro injectione
Injection 2: 2:98 mixture (v/v) of the test article and Paraffinum liquidum
Injection3:2:98 mixture (v/v) of the test article and FCA/Aqua pro injectione(50:50,v/v)
 
Day 7: Induction by dermal application
Control group
Filter papers (2 x 4 cm) were soaked with 0.5ml Paraffinum liquidum. One paper each was then placed on the skin on both sides of the body and covered with an occlusive dressing for 48 hours.
Dose group
Filter papers (2 x 4 cm) were soaked with 0.5 ml of the undiluted test article. One paper each was then placed on the skin on both sides of the body and covered with an
occlusive dressing for 48 hours.
 
Day 9: Removal of dressings
After an exposure period of 48 hours, the occlusive dressings and paper patches were removed and the test sites washed with water at approximate body temperature.
 
Day 21: Challenge by dermal application
Both right and left flanks of all animals were cleared of hair to expose areas in the middle of the trunk approximately 5x5cm.
For each animal a 0.5 ml sample of either vehicle or the 75:25 mixture (vh) of test article and Paraffnum liquidum were spread on separate 2 x 4 cm strips of gauze patch.
The control was applied to the left flank and the test article was applied to the right flank.Both treated sites were covered with an occlusive dressing for 24 hours.
 
 Day 22: Removal of dressings
After an exposure period of 24 hours, the occlusive dressings and gauze patches were removed and the test sites washed with water at approximate body temperature.
 
Days 23 and 24: Assessment of challenge reaction
Approximately 21 hours after patch removal, the challenge area was cleared of hair again. At 24 and 48 hours after patch removal (48 and 72 hours after start of challenge) the skin reaction was observed and recorded according to the OECD grading scale.

Classification
The test article was classified on the basis of the EEC Directive 93/21. According to the OECD guideline for testing (OECD 406, July 17, 1992), the test article
may be classified as non-sensitiser, if no animal shows an allergic response.

Necropsy
The animals were killed by carbon dioxide narcosis at the end of the experimental procedure, but were not subjected to any necropsy procedure.

Statistical Analysis
Body weights and skin-fold thickness were analysed using the Welch t-test. (3).

Positive control substance(s):

no

Results and discussion

Positive control results:

No data available.

In vivo (non-LLNA)

Results

Results 1

Reading:

other: After challenge

Hours after challenge:

24

Group:

negative control

Dose level:

vehicle alone

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: other: After challenge. . Hours after challenge: 24.0. Group: negative control. Dose level: vehicle alone. No with. + reactions: 0.0. Total no. in groups: 5.0.

Results 2

Reading:

other: After challenge

Hours after challenge:

24

Group:

test chemical

Dose level:

75:25 mixture (v/v) of the test article and Paraffinum liquidum

No. with + reactions:

4

Total no. in group:

10

Clinical observations:

animals showed an erythema.

Remarks on result:

other: see Remark

Remarks:

Reading: other: After challenge. . Hours after challenge: 24.0. Group: test group. Dose level: 75:25 mixture (v/v) of the test article and Paraffinum liquidum. No with. + reactions: 4.0. Total no. in groups: 10.0. Clinical observations: animals showed an erythema..

Results 3

Reading:

other: After challenge

Hours after challenge:

48

Group:

test chemical

Dose level:

75:25 mixture (v/v) of the test article and Paraffinum liquidum

No. with + reactions:

4

Total no. in group:

10

Clinical observations:

animals showed an erythema.

Remarks on result:

other: see Remark

Remarks:

Reading: other: After challenge. . Hours after challenge: 48.0. Group: test group. Dose level: 75:25 mixture (v/v) of the test article and Paraffinum liquidum. No with. + reactions: 4.0. Total no. in groups: 10.0. Clinical observations: animals showed an erythema..

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

According to the classification system for skin reactions of the EEC Directive 93/21 a test substance is classified as a sensitizer if at least 30% of animals show a positive skin reaction after using Adjuvant test. Becrosan 6920 has a skin sensitisation effect in accordance with this criterium.

Executive summary:

EXPERIMENTAL PROCEDURE

The study was conducted as Guinea-pig Maximisation Test (GPMT) according to Magnusson and Kligman (1). The sensitivity and reliability of the experimental technique used was checked routinely by the use of benzocaine, which is known to  have mild to moderate skin sensitisation properties (2). The results of the most recent reliability check are presented in the Appendix.

 

Pilot study

Preparation of the animals

The hair of2animals was removed from an area (approximately 2 x 4 cm) overlying the scapulae (intradermal injection), in two other animals from theflanksand the shoulder (topical application) by clipping/shaving 24 hours prior to test article administration avoiding skin abrasion.

 

Intradermal injection tolerance test

Twenty four hours after preparation of the animals, 4 pairs of intradermal injections of 0.1 ml each were administered to both animals in the shoulder region so that one of each pair lay on each side of the midline in a cranial to caudal sequence.

Injection 1: 10:90 (v/v) mixture of the test article and Paraffinum liquidum (MERCK KGaA, batch No. K2078 1674428)

Injection 2: 5:95 (v/v) mixture of the test article and Paraffinum liquidum

Injection 3: 2:98 (v/v) mixture of the test article and Paraffinum liquidum

Injection 4: 1:99 (v/v) mixture of the test article and Paraffinurn liquidum

 

Dermal tolerance test

Twenty four hours after preparation of the animals filter papers was loaded with 0.5 ml of the test article formulations and applied to the test areas.

Area 1 (left flank): undiluted test article

Area2(left shoulder): 75:25 mixture (v/v) of test article and Paraffinum liquidum

Area 3 (right shoulder):50:50mixture (v/v) of test article and Paraffinum liquidum

Area 4 (right flank): 25:75 mixture (vh) of test article and Paraffinum liquidum

 

Dose selection for main study

The application sites were examined for skin reactions at intervals of 24, 48 and 72 hours. The concentration of test article used for induction exposure was the highest to cause mild irritation. The concentration used for the challenge exposure was the highest non-irritating dose.

 

Main study

Preparation of the animals

The animals were assigned to a test group of 10 males and a control group of5males.

The hair was removed from an area (approximately 2 x 4 cm) overlying the scapulae by clipping and shaving 24 hours prior to test article administration avoiding skin abrasion.

Day 0: Induction by intradermal injection

Two pairs of intradermal injections of 0.1 ml each were administered to each animal in the shoulder region so that one of each pair lay on each side of the midline in cranial to caudal sequence.

 

• Control group

Injection 1:50:50mixture (v/v) of FCA (Sigma GmbH, Batch No. 97H8506) and Aqua pro injectione (Fresenius, batch No. FG 0 13)

 

Injection 2: Paraffinum liquidum (MERCK KGaA, batch No. K20781674428) 

 

Injection 3:50:50mixture (v/v) of Paraffinum liquidum and FCA/Aqua pro injectione (50:50, v/v)

 

 

• Dose group

Injection 1: 50:50 mixture (v/v) of FCA and Aqua pro injectione

Injection 2: 2:98 mixture (v/v) of the test article and Paraffinum liquidum

Injection3:2:98 mixture (v/v) of the test article and FCA/Aqua pro injectione(50:50,v/v)

 

Day 7: Induction by dermal application

• Control group

Filter papers (2 x 4 cm) were soaked with 0.5ml Paraffinum liquidum. One paper each was then placed on the skin on both sides of the body and covered with an occlusive dressing for 48 hours.

• Dose group

Filter papers (2 x 4 cm) were soaked with 0.5 ml of the undiluted test article. One paper each was then placed on the skin on both sides of the body and covered with an

occlusive dressing for 48 hours.

 

RESULTS

Results of the pilot study

Mortality and clinical signs

No animal died or showed clinical signs during the course of testing.

Body weight gain

Mean body weight before the test commenced: 677 g±62 g (n = 4)

Mean body weight at the end of the test: 675 g±56 g (n=4)

 

The body weight gain was in accordance with the historical data of our laboratory.

 

Skin reaction

The skin reaction after intradermal injection and topical application was recorded 24, 48 and 72 hours after application according the OECD grading scale.

The intradermal injection of all mixtures of the test article caused discrete to moderate erythema and oedema in both animals during the whole observation period. The findings at the injection site of the lowest concentration subsided to a discrete oedema 72 hours after administration.

The dermal application of the undiluted test article caused a discrete erythema in one animal over 48 hours; the 75:25 mixture (vh) in the same animal over 24 hours.

On the base of these results, the 2: 98 mixture (v/v) of the test article and Paraffinum

liquidum was used for intradermal injection. The undiluted test article was used for

topical induction and the 75:25 mixture (vh) of the test article and Paraffinum liquidum for challenge.

 

Results of the main study

Mortality and clinical signs

No animal died or showed clinical signs during the course of testing.

 

Body weight gain

Table 1 shows the mean values for body weight before the test commenced and at the end of the test and the results of the statistical analysis.

The mean body weight and the body weight gain were not affected by the treatment. Table 1: Body weight gain in the skin sensitisation test with Becrosan 6920

 

Skin reaction

• Induction period

Afterintradermal injectionof Paraffinurn liquidum the contra animals showed no skin reaction. The injection of the50:50mixture (v/v) FCA/ Aqua pro injectione and of mixture Paraffinum liquidum andFCA/Aqua pro injectione induced, as expected, discrete swelling and erythema (grade 1) in all animals. Only one animal showed a moderate.erythema at one application site.

All animals treated with the 2:98 mixture (v/v) of the test article and Paraffinum liquidum and the 2:98 mixture (v/v) of the test article and FCA/Aqua pro injectione(50:50,v/v) reacted with a discrete erythema and a discrete oedema. These symptoms subsided by the 7th day of the test. The healing process at the application sites of FCA mixtures was going on until the end of the test, in some cases with erosion and formation of scab.

Fortopical induction,the undiluted test article was applied under occlusive conditions for 48 hours; the controls got an occlusive dressing with Paraffinum liquidum.

After removal of the occlusive dressing on day 9 no skin reaction was recorded in control animals. Eight dose group animals showed a discrete erythema over 24 hours.

 

• Challenge

The challenge was carried out with the75:25mixture (v/v) of the test article and Paraffinum liquidum on day 2 1 of investigation.

In the control group no skin reaction was recorded on days 23 and 24.

Four dose group animals showed a discrete erythema, one animal a moderate erythema (only on day 23) on days 23 and 24.

The skin-fold thickness was increased in the dose group animals after the challenge procedure on days 23 and 24, statistically significant only on day 24. But the difference between left and right flank showed a statistically significant increase of the skin fold thickness on days 23 and 24.

The mean values are summarised in Table 2.

 

CONCLUSION

The skin sensitisation potential of the test articleBecrosan 6920was investigated in the Guinea-pig Maximisation Test with the Dunkin Hartley albino strain, whose sensitivity to benzocaine has been demonstrated.

In the courseoftesting, no clinical signs were observed. The body weight gain of treated animals was not affected.

The choice of doses was based on the results of the pilot study.

The animals showed a very homogeneous reaction to the application of the test article.

The intradermal injection of the2:98mixture (v/v) of the test article and Paraffinum liquidum alone and in combination with sensitisation potentiating FCA/Aqua pro injectione(50:50,v/v) lead to discrete erythema and discrete oedema. The same pattern was seen at injection site 1 (FCA/Aqua pro injectione) and3(Paraffinum liquidum and FCA/Aqua pro injectione) in the control animals.

Topical induction was attempted with the undiluted test article on day 7. After removal of the occlusive dressing on day 9 no skin reaction was recorded in control animals.Adiscrete erythema was recorded in eight animals of the dose group.

After challenge with the75:25mixture (v/v) of the test article and Paraffinum liquidum 5 of 10 dose group animals showed an erythema. Oedema formation proved by measurement of skin-fold thickness was statistically significant increased.

According to the classification system for skin reactions of the EEC Directive 93/21 a test substance is classified as a sensitizer if at least 30% of animals show a positive skin reaction after using Adjuvant test.Becrosan 6920 has a skin sensitisation effectin accordance with this criterium.

SUMMARY

The skin sensitisation potential ofBecrosan 6920was tested in the Guinea-pig Maximisation Test (E.E.C. Guideline B.6. / OECD guideline 406).

A2:98 (v/v) mixture of the test article and Paraffinum liquidum was used for intradermal induction. The dermal induction was carried out with the undiluted test article on day 7 of investigation, and the challenge was done with the 75:25 (vh) mixture of the test article and Paraffinum liquidum on day 2 1.

The test article was a discrete skin irritant with and without Freund's adjuvant after intradermal injection. The dermal induction induced discrete erythema. Challenge patch test reactions were evaluated according the OECD grading scale.Asensitisation reaction was recorded in 5 of 10 animals on days 23 and 24. The skin fold thickness was statistically significant increased. The test article was classified on the basis of the EEC Directive 93/21 as askin sensitiserin guinea-pigs.