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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2002-06-19 to 2002-08-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Reference substance name:
Quaternary ammonium compounds, benzylbis(hydrogenated tallow alkyl)methyl, chlorides
EC Number:
263-082-9
EC Name:
Quaternary ammonium compounds, benzylbis(hydrogenated tallow alkyl)methyl, chlorides
Cas Number:
61789-73-9
IUPAC Name:
61789-73-9

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands, Kreuzelweg 53, 5961 NM Horst, PO box 6174 NL, 5960 Ad Horst, The Netherlands
- Age at study initiation: 4-5 weeks
- Weight at study initiation: 250-300 g
- Housing: During acclimatisation, animals were housed in groups of up to 10, and during the study in groups of up to 5, in suspended stainless steel cages with a grid floor.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2°C
- Humidity (%): 55±15%.
- Air changes (per hr): 15-20 air changes per hour.
- Photoperiod: 12hrs dark / 12hrs light):

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
Main study:
-Induction:
intradermal injection: 0.5% w/v in sterile water.
topical application: 10% w/v in sterile water.
-Challenge:
10% and 1% w/v in sterile water
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Main study:
-Induction:
intradermal injection: 0.5% w/v in sterile water.
topical application: 10% w/v in sterile water.
-Challenge:
10% and 1% w/v in sterile water
No. of animals per dose:
In the preliminary study: 7 animals.
In the main study, test group of 20 animals and a control group of 10.
Details on study design:
RANGE FINDING TESTS: conducted to define the concentrations to be tested in the main study

-Intradermal exposure:
Hair over the scapulae was removed using electric clippers before treatment.
Intradermal administration of 0.1 ml of the test material (TM) at increasing concentrations. Concentrations of 50%, 20%, 10%, 5%, 1% and 0.5% in sterile water were selected but only the lower concentrations of 5%, 1% and 0.5% could be injected due to the high density of the preparations.
Evalauation of the potential cutaneous reactions: 6 days later

- Epicutaneousexposure:
Hair over the scapulae was removed using electric clippers before treatment.
0.2ml of each selected concentration applied to a gauze patch measuring 2x2cm for 24 hours under occlusive dressing (2 concentrations per animal). A total of 5 concentrations (50%, 20%, 10%, 5%, 1% and 0.5% in sterile water) were tested in duplicate.
Evaluation of the potential cutaneous reactions 24 and 48 hours after patch removal.


MAIN STUDY

A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal, 1 epicutaneous
- Exposure period: epicutaneous: 48 hours

-> TEST GROUPS:
Intradermal exposure
Three injections of 0.1 mL were injected into each side of the animal as follows:
. Freund's complete adjuvant (FCA) diluted to 50% with sterile water
. TM at 0.5 % w/v in vehicle
. TM at 0.5% w/v in a 50/50 (v/v) mixture of FCA and sterile water

Epicutaneous exposure
Application of 2 x 2cm patch saturated with 0.4 ml of the TM at 10% w/v in sterile water applied to the scapular region and held in place for 48 hours using an occlusive dressing.

-> CONTROL GROUP:
Intradermal exposure
Three injections of 0.1 mL were injected into each side of the animal as follows:
. Freund's complete adjuvant (FCA) diluted to 50% with sterile water
. vehicle
. A mixture 50/50 (v/v) FCA diluted to 50% with sterile water, and vehicle

Epicutaneous exposure
Application of 2 x 2cm patch saturated with 0.4 ml of the vehicule applied to the scapular region and held in place for 48 hours using an occlusive dressing.

- Site:
Intradermal exposure
6 injections in the clipped area (2x 4cm) in the scapular region

Epicutaneous exposure
2x 2cm area over the scapulae

- Frequency of applications:
One intradermal injection and one epicutaneous application 7 days (Day 8 of the study) after on the same site.

- Duration:
Epicutaneous exposure: 48 hours


B. CHALLENGE EXPOSURE
- No. of exposures: 2 ( the animals were re-challenged at a lower concentration since a response to the first challenge was noted in a number of animals in both the control and tests groups)
- Day(s) of challenge: 22 and 29
- Exposure period: 24 hours

-> TEST GROUPS:
First challenge: 0.2 mL of the TM at 10% w/v in sterile water on the right flank and 0.2 mL of the vehicule to the left flank (occlusive epicutaneous application)
Second challenge: 0.2 mL of the TM at 1% w/v in sterile water on the right flank and 0.2 mL of the vehicule to the left flank (occlusive epicutaneous application)

-> CONTROL GROUPS:
Same treatment as test group
- Site: anterior and posterior left flank
- Evaluation (hr after challenge): 24 , 48 hours after removal of the dressing according to the method of Draize.

Challenge controls:
Yes - see above.
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamalaldehyde

Study design: in vivo (LLNA)

Statistics:
None

Results and discussion

Positive control results:
Regular checks are performed at the laboratory with the positive control substance alpha-hexylcinnamalaldehyde. The most recent reliability check achieved a 100% response in the test group and 0% response in the control group at challenge.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
challenged with Test material 10% w/v in sterile water
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
7 and 1 animals with erythema grade 1 and 2 respectively.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: challenged with Test material 10% w/v in sterile water. No with. + reactions: 8.0. Total no. in groups: 10.0. Clinical observations: 7 and 1 animals with erythema grade 1 and 2 respectively..
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
challenged with Test material 10% w/v in sterile water
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
5 and 1 animals with erythema grade 1 and 2 respectively.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: challenged with Test material 10% w/v in sterile water. No with. + reactions: 6.0. Total no. in groups: 10.0. Clinical observations: 5 and 1 animals with erythema grade 1 and 2 respectively..
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
challenged with Test material 10% w/v in sterile water
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
15 and 5 animals with erythema grade 1 and 2 respectively.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: challenged with Test material 10% w/v in sterile water. No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: 15 and 5 animals with erythema grade 1 and 2 respectively..
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
challenged with Test material 10% w/v in sterile water
No. with + reactions:
13
Total no. in group:
20
Clinical observations:
10 and 3 animals with erythema grade 1 and 2 respectively. Desquamation was noted in 6 animals.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: challenged with Test material 10% w/v in sterile water. No with. + reactions: 13.0. Total no. in groups: 20.0. Clinical observations: 10 and 3 animals with erythema grade 1 and 2 respectively. Desquamation was noted in 6 animals..
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
challenged with Test material 1% w/v in sterile water
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: challenged with Test material 1% w/v in sterile water. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
challenged with Test material 1% w/v in sterile water
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: challenged with Test material 1% w/v in sterile water. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
challenged with Test material 1% w/v in sterile water
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: challenged with Test material 1% w/v in sterile water. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
challenged with Test material 1% w/v in sterile water
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: challenged with Test material 1% w/v in sterile water. No with. + reactions: 0.0. Total no. in groups: 20.0.

Any other information on results incl. tables

The preliminary study indicated that the test material injected at 0.5% concentration was reasonably well tolerated. A concentration of 10% was selected for the topical induction as it was judged to be well tolerated and non-irritant. A response to treatment was noted in a number of animals in both the control and test groups so a re-challenge at the lower concentration of 1% was performed.

Table 1: Challenge responses

 

Group

Treatment

Incidence of response (%) at challenge

24 hours

48 hours

First challenge

Control

10% Test material

80%

60%

Vehicle

0%

0%

Test

10% Test material

100%

65%

Vehicle

0%

0%

Second challenge

Control

1% Test material

0%

0%

Vehicle

0%

0%

Test

1% Test material

0%

0%

Vehicle

0%

0%

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
There was no reaction observed at challenge with 1% test material concentration.Under these experimental conditions and according to the criteria laid down in CLP (Reg 1272/2008/EC) and directive 67/548/EEC, the substance does not induce delayed contact hypersensitivity and is not classified.
Executive summary:

The potential of the substance to induce delayed contact hypersensitivity was assessed in guinea pigs according to OECD guideline 406 and in compliance with the principles of the Good Laboratory practice regulations.

The induction phase was realized both by intradermal route on day 1 (Test material 0.5 % w/v in sterile water) and by cutaneous route on day 8 (Test material 10% w/v in sterile water ) in 2 groups of guinea pigs: 10 females for control group and 20 females for treated group. The challenge phase was realized on day 22 by cutaneous application of the test material at 10% w/v in sterile water. The cutaneous reactions were scored 24 and 48 after the challenge phase. As a response to treatment was noted in a number of animals in both the control and test groups, a re-challenge at the lower concentration of 1% was performed on day 29.

The first challenge application with 10% w/v of the test material in sterile water resulted in very slight to well defined erythema in the majority of animals of both test and control groups (6/10 and 13/20 at the 48-hour reading respectively). No reaction was noted to the vehicle alone. The presence of reaction in both test and control group animals indicated an irritant response to treatment rather than sensitisation. This was investigated by repeating the challenge with a lower concentration of 1% one week later. On this occasion, no response was observed in any animal of the test or control groups following 24 hours topical exposure. Again, no reaction to the vehicle alone was observed.

It was concluded that the substance does not elicit a sensitisation response in the guinea pig.