Benzethonium chloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

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.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

the study was performed in 1995, before other alternative methods were available; at that time the maximisation test was the preferred test method to determine the skin sensitisation potential of chemicals

Test material

Test material information

Constituent 1

Reference substance name:

Benzethonium chloride

EC Number:

204-479-9

EC Name:

Benzethonium chloride

Cas Number:

121-54-0

Molecular formula:

C27H42NO2.Cl

IUPAC Name:

benzyldimethyl(2-{2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethoxy}ethyl)azanium chloride

Test material form:

solid: compact

Specific details on test material used for the study:

TEST MATERIAL
- Sponsor’s identification: P4123
- Batch number: 3072-S
- Date received: 28 June 1995
- Purity: 99.5%
- Description: white powder
- Stability: considered to be stable under conditions of shipment, storage, and use in this study.
- Storage Conditions: room temperature in the dark
- Expiration date: no data

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: David Hall Limited, Burton-on-Trent, Staffordshire, UK
- Age at study initiation: 8-12 weeks old
- Weight at study initiation: 341-415 g
- Housing: housed singly or in pairs in solid-floor polypropylene cages furnished with woodflakes
- Diet (e.g. ad libitum): Guinea Pig FD1 Diet, Special Diets Services Limited, Witham, Essex, UK
- Water (e.g. ad libitum): tap water
- Acclimation period: 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-21°C
- Humidity (%): 46-70%
- Air changes (per hr): 15 changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

water

Concentration / amount:

0.05% w/v

Day(s)/duration:

24 and 48 hours

Adequacy of induction:

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

Induction 2

Route:

intradermal

Vehicle:

other:

Concentration / amount:

0.05% w/v in a mixture of Freund's Complete Adjuvant plus distilled water (1:1)

Day(s)/duration:

24 and 48 hours

Adequacy of induction:

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

Induction 3

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

2% w/w

Day(s)/duration:

24 and 48 hours

Adequacy of induction:

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

Challenge

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

0.5% and 0.2%

Day(s)/duration:

24 and 48 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

A group of thirty guinea pigs was used for the main study, twenty test and ten control animals.

Details on study design:

INDUCTION:

Induction of the Test Animals:
Shortly before treatment on Day 0, the hair was removed from an area approximately 40mm x 60mm on the shoulder region of each animal with veterinary clippers. A row of three injections (0.1 ml each) was made on each side of the mid-line. The injections were:
a) Freund's Complete Adjuvant plus distilled water in the ratio 1:1
b) a 0.05% w/v formulation of the test material in distilled water
c) a 0.05% w/v formulation of the test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water.
Approximately 24 and 48 hours after intradermal injection, the degree of erythema at the test material injection sites (ie. injection site b) as evaluated according to the scale acc. Draize. One week later (Day 7), the same area on the shoulder region used previously for intradermal injections was clipped again and treated with a topical application of the test material formulation. A filter paper patch (WHATMAN No.4: approximate size 40 mm x 20 mm), saturated with the test material formulation (2% w/w in distilled water) as a thick, even layer was applied to the prepared skin and held in place with a strip of surgical adhesive tape (BLEN DERM: approximate size 5Omm x 30mm) covered with an overlapping length of aluminium foil. The patch and foil were further secured with a strip of elastic adhesive bandage (ELASTOPLAST: approximate size 250mm x 35mm) wound in a double layer around the torso of each animal. This occlusive dressing was kept in place for 48 hours.


Challenge
Shortly before treatment on Day 21, an area of approximately 50 mm x 70 mm on both flanks of each animal, was clipped free of hair with veterinary clippersA square filter paper patch (WHAT MAN No.4: approximate size 20 mm x 20 mm), saturated with the test material formulation at the maximum non-irritant concentration (0.5% w/w in distilled water) was applied to the shorn right flank of each animal and was held in place with a strip of surgical adhesive tape (BLEN DERM: approximate size 40 mm x 50 mm). To ensure that the maximum non-irritant concentration was used at challenge, the test material at a concentration of 0.2% w/w in distilled water was similarly applied to a skin site on the left shorn flank. The patches were occluded with an overlapping length of aluminium foil and secured with a strip of elastic adhesive bandage (ELASTOPLAST: approximate size 250 mm x 75 mm) wound in a double layer around the torso of each animal. After 24 hours, the dressing was carefully cut using blunt-tipped scissors, removed and discarded. The challenge sites were swabbedwith cotton wool soaked in distilled water to remove residual material. The position of the treatment sites was identified by using a black indelible marker-pen. Prior to the 24-hour observation the flanks were clipped using veterinary clippers to remove regrown hair.

Challenge controls:

animals treaded with vehicle only

Positive control substance(s):

yes

Remarks:

2-Mercaptobenzothiazole; Ethyl 4-aminobenzoate; 2,4-Dinitrochlorobenzene; 2,4-Dinitrochlorobenzene

Results and discussion

Positive control results:

The strain used in these laboratories has been shown to produce satisfactory sensitisation responses using known positive sensitisers (see above). The results of the study are believed to be of value in predicting the likely contact sensitisation potential of the test material to man:
- 2-Mercaptobenzothiazole: sensitization rate 80%
- Ethyl 4-aminobenzoate: sensitization rate 39%
- 2,4-Dinitrochlorobenzene: sensitization rate 100%

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.2 & 0.5%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0.2 & 0.5%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.2 & 0.5%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0.2 & 0.5%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

Skin Reactions Observed After Intradermal Induction:

Well-defined to severe erythema was noted at the intradermal induction sites of all test group animals at the 24 and 48-hour observations. Very slight erythema was noted at the intradermal induction sites of two control group animals at the 24-hour observation. No skin reactions were noted at the intradermal induction sites of control group animals at the 48 -hour observation.

Skin Reactions Observed After Topical Induction:

Very slight to well-defined erythema was noted at the induction sites of all test group animals at the 1-hour observation. Very slight erythema was noted at the induction sites of four test group animals at the 24-hour observation. Other skin reactions noted were bleeding from intradermal induction sites and hardened dark brown/black coloured scab. No skin reactions were noted at the treatment sites of control group animals at the 1 and 24-hour observations.

Skin Reactions Observed After Topical Challenge:

No skin reactions were noted at the challenge sites of the test or control group animals at the 24 or 48-hour observations.

Body weights:

Bodyweight gains of guinea pigs in the test group, between Day 0 and Day 24, were comparable to those observed in the control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test material produced a 0% (0/20) sensitisation rate and was classified as a non-sensitiser to guinea pig skin. The test material did not meet the criteria for
classification as a sensitiser according to GHS-criteria.

Executive summary:

The study was performed in compliance with OECD Guidelines for the Testing of Chemicals No. 406 "Skin Sensitisation" (adopted 17 July 1992) and Method B.6 of Commission Directive 92/69/EC to assess the contact sensitisation potential of the test material. Twenty test and ten control animals were used for the main study. Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as follows:

- Intradermal Induction: 0.05% w/v in distilled water

- Topical Induction: 2% w/v in distilled water

- Topical Challenge: 0.5 and 0.2% w/v in distilled water

The test material produced a 0% (0/20) sensitisation rate and was classified as a non-sensitiser to guinea pig skin. The test material did not meet the criteria for classification as a sensitiser according to GHS-criteria.