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Toxicological information

Skin sensitisation

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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:
6 December 1999 to 17 January 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The guinea pig maximisation test according to OECD 406 was performed in 2000 prior to the adoption of the OECD 429 local lymph node assay in 2002.

Test material

Constituent 1
Chemical structure
Reference substance name:
Amines, C12-14-alkyl, isooctyl phosphates
EC Number:
269-119-5
EC Name:
Amines, C12-14-alkyl, isooctyl phosphates
Cas Number:
68187-67-7
Molecular formula:
C19H42NPO4 - C29H63NPO4
IUPAC Name:
Amines, C12-14-tert-alkyl, isooctyl mono phosphates
Test material form:
liquid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
Nulliparous and non preganant female Dunkin/Hartley strain guiea pigs were supplied by D Hall, Newchurch, Staffs, UK. The animals were approximately 4 to 7 weeks of age on arrival and were acclimatised for six days prior to the start of the main study. At the start of the study the animals were in the weight range of 395 to 435g.
An additional 10 animals from the same supplier were used for the preliminary investigations.
The animals on the main study were allocated without conscious bias to either control or test groups. Each animal was identified by ear tattoo number.
The guinea pigs were housed in groups of five in suspended metal cages with wire mesh floors.
Free access to drinking water and food ( a vitamin C enriched guinea pig diet, Teklad 9600 FD2 SQC) was allowed throughout the study. Hay was given three times a week.

The temperature and relative humidity were controlled to remain within target ranges of 16 to 22°C and 32 to 60%, respectively. Lighting was controlled by a time switch to give twelve hours continuous light (07.00 to 19.00) and twelve hours darkness.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: Alembicol D (a product of coconut oil)
Concentration / amount:
0.1% test item in Alembicol D
Day(s)/duration:
Intradermal injection on Day 1
Adequacy of induction:
other: Highest concentration that caused irritation but did not adversely affect the animals
Route:
epicutaneous, semiocclusive
Vehicle:
other: Alembicol D (a product of coconut oil)
Concentration / amount:
10% test item in Alembicol D
Day(s)/duration:
application for 48 hours one week after intradermal induction
Adequacy of induction:
other: Highest concentration that produced some irritation but did not adversely affect the animals
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, semiocclusive
Vehicle:
other: Alembicol D (a product of coconut oil)
Concentration / amount:
1% test item in Alembicol D
Day(s)/duration:
application for 24 hours two weeks after epicutaneous induction
Adequacy of challenge:
highest non-irritant concentration
No.:
#1
Route:
epicutaneous, semiocclusive
Vehicle:
other: Alembicol D (a product of coconut oil)
Concentration / amount:
0.5% in Alembicol D
Day(s)/duration:
application for 24 hours two weeks after epicutaneous induction
Adequacy of challenge:
not specified
No.:
#2
Route:
epicutaneous, semiocclusive
Vehicle:
other: Alembicol D (a product of coconut oil)
Concentration / amount:
1% test item in Alembicol D
Day(s)/duration:
application for 24 hours one week after the first epicutaneous challenge
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, semiocclusive
Vehicle:
other: Alembicol D (a product of coconut oil)
Concentration / amount:
0.5 % test item in Alembicol D
Day(s)/duration:
application for 24 hours one week after the first epicutaneous challenge
Adequacy of challenge:
not specified
No. of animals per dose:
Control animals : 5
Test animals : 10
Details on study design:
PRELIMINARY STUDIES:
The intradermal and topical irritancy of dilutions of the test substance was investigated to identify where possible
- concentrations of the test substance that would produce irritation suitable for the induction phase of the main study, and,
- a maximum non-irritant concentration by the topical route for the challenge phase

The animals for the topical irritancy investigations were pre-treated with an intradermal injection of Freuds complete adjuvant, 50:50 with water, approximately one week prior to the start of the preliminary investigations.

Intradermal injections - Intradermal injections (0.1ml/site) were made to the clipped flank of 2 guinea pigs, using a range of concentrations (0.1 to 10% v/v) of test material in a suitable vehicle ( Alembicol D). The resulting dermal responses were assessed approximately 24 and 72 hours later.

Topical application - Patches of Whatmen NO. 3 paper (20 x 20mm) were saturated (volume approximately 0.2ml / patch) with a range of concentrations (0.005% v/v to as supplied) of test material in vehicle (Alembicol D) and applied to the clipped and shaved flanks of 8 guinea pigs. The patches were covered by a strip of 'Blenderm' and secured by 'Elastoplast' wound round the trunk and fixed with 'Sleek' impervious plastic adhesive tape. The dressings were removed after an exposure of approximately 24 hours and the reaction sites assessed for erythema and oedema. Further examination of the sites was carried out approximately 24 and 48 hours after removal of the dressings.

MAIN STUDY:
Control group: 5 female guinea pigs
Test Group : 10 female guinea pigs

INDUCTION PHASE
TEST GROUP:
Intradermal Induction:
Test Group :
- 2 ID: Freund's Complete Adjuvant diluted with an equal volume of water for irrigation
- 2 ID: test item at 0.1% v/v in Alembicol D
- 2 ID: test item at 0.1% v/v in a 50 : 50 mixture of Freund's Complete Adjuvant and in Alembicol D.

Topical Induction:
One week after the injections the same 40 x 60 mm interscapular area was clipped and shaved free of hair. A 20 x 40 mm patch of Whatman No 3 paper was saturated with approximately 0.4ml of test material, 10% v/v in AlembIcol D. The patch was placed on the skin of the test animals and covered by a length of impermeable plastic adhesive tape. This in turn was secured by elastic adhesive bandage wound round the torso and fixed with 'Sleekimperviuos plastic adhesive tape.

CONTROL ANIMALS
During the induction phase, the control animals were treated similarly to the test animals with the exception that the test substance was omitted from the intradermal injections and topical application.

CHALLENGE PHASE: CONTROL AND TEST ANIMALS
The control and test animals were challenged topically two weeks after the topical induction application using the test material at 1% and 0.5% v/v in Alembicol D.
Hair was removed by clipping and then shaving from an area on the left flank of each guinea pig. A 20 x 20 mm patch of Whatman No 3 paper was saturated with approximately 0.2ml of test material., 1% v/v in Alembicaol D and applied to an anterior site on the flank. Test material 0.5% v/v in Alembicol D was applied in a similar manner to the posterior site. The patches were sealed to the flank for 24 hours under strips on 'Blenderm' secured with 'Elastoplast' wound round the trunk and fixed with 'Sleek'

A second challenge was made one week later. The method employed was the similar with the exception that the test substance 1 and 0.5% v/v was applied to the left flank of all the control and test animals.

OBSERVATIONS
Mortality and clinical signs were recorded daily. The bodyweight of each animal was recorded on the day of intradermal injections and on the last day that observations were made of the dermal response to the challenge application.

DERMAL RESPONSES
The dermal reactions resulting from intradermal induction and topical application on the preliminary study, and topical application at the challenge were assessed.
Challenge controls:
During the induction phase, the control animals were treated similarly to the test animals with the exception that the test substance was omitted from the intradermal injections and topical application.
The control animals were challenged topically two weeks after the topical induction application using the test material at 1% and 0.5% v/v in Alembicol D
Positive control substance(s):
yes
Remarks:
Hexyl cinnamic aldehyde conducted between 7 June and 10 July 1999.

Results and discussion

Positive control results:
All 10 test animals gave a positive sensitisation response.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
1%
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
Well defined or slight erythema with slight oedema observed in 2/10 animals. Dryness and sloughing of epidermis observed in another 1/10 animal.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0.5%
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
Slight erythema and slight oedema observed in 2/10 animals
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
1%
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
Slight erythema and slight oedema observed in 2/10 animals. Dryness and sloughing of epidermis observed in another 1/10 animal.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0.5%
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
Slight erythema and slight oedema observed in 2/10 animals
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
1%
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
Slight erythema and slight oedema and thickening, dryness and sloughing of the epidermis observed in 2/10 animals.
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
0.5%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
1%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
0.5%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
1%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Dryness and sloughing of epidermis in some animals
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
0.5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Dryness and sloughing of epidermis in some animals
Key result
Reading:
rechallenge
Hours after challenge:
72
Group:
test chemical
Dose level:
1%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Dryness and sloughing of epidermis in some animals
Key result
Reading:
rechallenge
Hours after challenge:
72
Group:
test chemical
Dose level:
0.5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Dryness and sloughing of the epidermis in one animal. Thickening dryness and sloughing of the epidermis in one animal.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
1%
No. with + reactions:
1
Total no. in group:
5
Clinical observations:
Slight erythema observed in 1/5 animals
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0.5%
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
1%
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0.5%
No. with + reactions:
0
Total no. in group:
5
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
1%
No. with + reactions:
0
Total no. in group:
5
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
0.5%
No. with + reactions:
0
Total no. in group:
5
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
1%
No. with + reactions:
1
Total no. in group:
5
Clinical observations:
Well defined erythema, slight oedema and thickness, dryness and sloughing of epidermis observed in 1/5 animals
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
0.5%
No. with + reactions:
1
Total no. in group:
5
Clinical observations:
slight erythema observed in 1/5 animals

Any other information on results incl. tables

Preliminary Screening Test

Please see the attached background material for the results of the preliminary investigations.

Main Test

There were no deaths or signs of systemic toxicity noted in the test or control group animals during the test.

Intradermal injections - Necrosis was observed at intradermal sites receiving Freuds Complete Adjuvant in test and control animals. Slight irritation was seen in test animals receiving the test substance 0.1% in Alembicol D and slight irritation was observed in control animals receiving Alembicol D.

Topical application - Moderate erythema with blanching of the dose site was observed in test animals following topical application of the test substancce 10% v/vin Alembicol D. Slight irritation was seen in the control guinea pigs.

Challenge - First challenge dermal reactions were more marked and persistent in 2 test animals compared to the control animals, therefore these 2 animals gave a positive response. The remaining 8 test animals gave negative responses. A second challenge was conducted to confirm reproducibility.

.

Second challenge - Dermal reactions of similar persistence and severity were observed for control and test animals.

For both challenges, dermal reactions were observed in the control group and as the responses seen in the first challenge were not reproducible in the same test anmals in the second challenge, the dermal reactions were considered to represent irritation rather than sensitisation.

Please see attached background information for results.

Results for the negative control rechallenge readings at 48 and 72 hours is given in the attached background information.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be a non-sensitiser under the conditions of the test.
Executive summary:

Introduction. 

A study was performed to assess the skin sensitisation potential of the test item in the guinea pig. The study was performed in compliance with the OECD Guideline for the Testing of Chemicals No. 406 "Skin Sensitisation (adopted 17 July 1992), Method B6 of Commission Directive 96/54/EC an d EPA Health Effects Test Guidelines OPPTS 870.2600 "Skin Sensitisation" EPA 712 -C-98 -197, August 1998.

Methods

Following a preliminary screening test guinea pigs were dosed by intradermal injection (0.1% v/v in Alembicol D) and topical application (10% V/V in Alembicol D). This was followed approximately 2 weeks later by a topical challenge application of 1 and 0.5% v/v in Alembicol D. A second challenge application was made one week later.

Results

Following the first challenge dermal reactions were more marked and persistent in two test animals compared to the control animals, suggesting a positive response in these two animals. The remaining eight animals gave a negative response.

Following the second challenge dermal reactions for test animals were of similar persistence and severity to those of the control animals.

For both challenges dermal reactions were observed in the control group and as the responses seen in the first challenge were not reproducible in the same test animals in the second challenge, the dermal reactions were considered to represent irritation rather than sensitisation.

Conclusion

The test item did not produce evidence of skin sensitisation in any of the ten test animals.

The test item does not meet the criteria for classification according to the Classification, Labelling and Packaging Regulation (CLP)