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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:
Experimenta; Starting Date: 07 Jul 2011, Experimental Completion Date; 06 Sep 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study conducted for global regulatory use

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Albino Dunkin Hartley Guinea Pig, HsdPoc: DH, SPF from Harlan Laboratories B.V., The Netherlands- Age at study initiation: 4 weeks- Weight at study initiation: Bodweight at pretest start: Pretest Groups 344.8 - 376.2 gBody weight at beginning of acclimatisation period: Test and control groups: 3126.7 - 376.7 g- Housing: In groups of up to 10 animals in stainless steel cages with standard softwood bedding- Diet (e.g. ad libitum): Teklad Global Guinea pig diet 2040C, ad libitum- Water (e.g. ad libitum): Community tap water in bottles, ad libitum.- Acclimation period: Twenty-two days for the test and control groups of the main test under standard laboratory conditions after health examination. No acclimatization for the animals of the pretest. Only animals without any visible signs of illness were used for the study.ENVIRONMENTAL CONDITIONS- Temperature (°C): continuously monitored environment with a room temperature of 22 ± 3 °C- Humidity (%): relative humidity between 30-70%,- Air changes (per hr): Air-conditioned with 10-15 air changes per hour.- Photoperiod (hrs dark / hrs light): automatically controlled light cycle of 12 hours light and 12 hours dark

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
water
Remarks:
(see details on study design for justification).
Concentration / amount:
Main Study:Intradermal induction: 10% dilution of test item in purified water and in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline.Epidermal induction: 100% (neat)Challenge - Epidermal application of the test item at 75% in purified water and purified water alone
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
(see details on study design for justification).
Concentration / amount:
Main Study:Intradermal induction: 10% dilution of test item in purified water and in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline.Epidermal induction: 100% (neat)Challenge - Epidermal application of the test item at 75% in purified water and purified water alone
No. of animals per dose:
Main study:Test group: 10 malesControl group: 5 males
Details on study design:
VEHICLE:The vehicle was selected based on preliminary solubility testing, which was performed before the study initiation date. The test item was prepared at a concentration of 75% (w/w) in purified water, which was well soluble and resulted in a colorless liquid. However, the undiluted test item (100%) was used as highest concentration in the epidermal pretest.For the intradermal pretest, the test item was prepared at a concentration of 75% in FCA/physiological saline, resulting in a white emulsion. This preparation went through a needle of 0.5 x 16 mm. Therefore, this test item concentration in purified water was used as highest concentration in the intradermal pretest.AUXILIARY COMPOUNDS:FCA - Freund's Adjuvant - completePhysiological Saline - Natrium chloratum 0.9%PREPARATION OF DOSE FORMULATIONS:The test item and vehicle or auxiliary compounds were placed into a glass beaker on a tared Mettler balance and a weight/weight dilution was prepared. Homogeneity of the test item preparation was ensured and maintained during preparation and treatment using a magnetic stirrer or a spatula (for the neat sticky liquid test item during the epidermal pretest and epidermal induction). The preparations were made prior to each dosing.All dose concentrations were corrected for water and ethylene glycol content.SELECTION OF TEST ITEM CONCENTRATION FOR MAIN STUDY:Intradermal Induction:The test item prepared at 10% (used for the intradermal induction) was the highest suitable concentration, causing mild skin irritation during the pretest.Epidermal Induction:The neat test item at (100%) was the highest technically applicable concentration, causing mild skin irritation during the pretest.Epidermal ChallengeThe test item at 75% was the highest tested concentration which did not cause any skin irritating reactions.To determine the different concentrations, intradermal and epidermal pretests were performed as described below.RATIONALE:The application form and dose was used to detect a possible allergenic potential of the test item applied.OBSERVATIONS:Viability / Mortality: Daily from delivery of the animals to the termination of the test.Clinical Signs: Daily from delivery of the animals to the termination of test.Local signs: Skin responses were graded during the pretest, induction and challenge periodBody Weights: At delivery/acclimatization start, at test item treatment day in the pretest, at the end of the pretest, at test day 1 (day of treatment) and at the termination of the study.PATHOLOGY:Necropsy:No necropsy was performed on all surviving animals. The control and test animals were euthanized at the end of the test period by intraperitoneal injection of pentobarbitone at a dose of 2.0 mL/kg of 162 mg/mL sodium pentobarbitone and discarded. The intradermal pretest II animal was euthanized as described above at the treatment start of the main study. The intradermal pretest I and epidermal animals were euthanized at the delivery of the intradermal pretest II animal.TREATMENT METHOD:The animal's fur was shaved with a fine clipper blade just prior to exposure. Intradermal injections or closed patches were applied to the animals as follows:0.12 mL/site for the intradermal administrations or 0.24 to 0.36 mL or 0.24 to 0.36 g on a patch of filter paper for the epidermal administrations of test item.All applications without test item were 0.1 ml intradermally and 0.2 to 0.3 ml epidermally.The patch was covered by a strip of aluminium foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressing was left in place for 24 (epidermal pretest and epidermal challenge) or 48 hours (epidermal induction).Identical patching method was used for the epidermal pretest, epidermal induction and epidermal challenge.PRETEST:The pretest was performed during the acclimatization period of the animals for the main test.The test item concentrations described below were selected during a preliminary solubility testing which was performed before the study initiation date.Intradermal injections:Four intradermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of one guinea pig (no. 37). Five days later, three intradermal injections (0.1 mL/site) were made into the clipped flank of the same guinea pig at concentrations of A = 75%, B = 50% and C = 25% of the test item in purified water.Dermal reactions were assessed 24 hours later.At this stage of procedure, no test item concentration causing mild-to-moderate skin irritation could be chosen. Therefore, a second intradermal pretest animal (No. 55) was treated in the same manner as described with the animal No. 37, just the concentrations were changed. The animal was first treated, 6 days prior to the test item injections, with four intradermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline made into the shaved neck. Thereafter, it was treated intradermally with the test item at 10, 3 and 1% in purified water.Based on the results, a test item concentration of 10% was selected for intradermal induction in the main test.Epidermal Applications:Four intradermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of Freund's CompleteAdjuvant/physiological saline were made into the shaved neck of two guinea pigs (nos. 38 - 39). Five days later, four patches of filter paper (3 x 3 cm) were saturated with the test item at D = 100% (neat test item), E = 75%, F = 50%, G = 25% and applied to the shaved flanks of the same guinea pigs. The volume of test item preparation applied was approximately 0.24 mL for the test item concentrations of 75%, 50% and 25% and an amount of approximately 0.24 g was applied for the neat test item (sticky liquid). The occlusive dressings were left in place for 24 hours.The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.The allocation of the different test item dilutions to the sites (D, E, F, G) on the two animals was alternated in order to minimize site-to-site variation in responsiveness.Based on the results the test item concentration selected for epidermal induction and challenge in the main test was 100% (neat) and 75%, respectively.MAIN STUDY:INDUCTION:Intradermal injection performed on Test Day 1:An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.12 mL/site for test item and 0.1 mL/site for other applications) were made just within the boundaries of a 4 x 6 cm area in the clipped region as follows: Test Group:1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.2) The test item at 10% in purified water.3) The test item at 10% in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.Epidermal induction performed on Test Day 8:One week after the intradermal induction, the scapular area (approximately 6 x 8 cm) was again shaved prior to epidermal induction. A 2 x 4 cm patch of filter paper was saturated with the neat test item at 100% and placed over the injection sites of the test animals. The amount of the sticky liquid test item applied (with a spatula) was approximately 0.36 g. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressings were left in place for 48 hours. The epidermal application procedure described ensured intensive contact of the test item.The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and KligmanCHALLENGEPerformed on Test Day 22The test and control guinea pigs were challenged two weeks after epidermal induction and treated in the same way.Two patches (3 x 3 cm) of filter paper were saturated with the test item at the highest tested non-irritating concentration of 75% in purified water (applied to the left flank) and the vehicle only (purified water applied to the right flank) using the same method as for the epidermal application.The volume of test item preparation and vehicle applied was approximately 0.24 mL and 0.2 mL respectively. The dressings were left in place for 24 hours.
Challenge controls:
Induction:Intradermal:Control Group: 1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.2) Purified water.3) 1:1 (w/w) mixture of purified water in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.Epidermal:The guinea pigs were treated as for the test group with purified water only, applied at a volume of approximately 0.3 mL.Challenge:The test and control guinea pigs were challenged two weeks after epidermal induction and treated in the same way.Two patches (3 x 3 cm) of filter paper were saturated with the test item at the highest tested non-irritating concentration of 75% in purified water (applied to the left flank) and the vehicle only (purified water applied to the right flank) using the same method as for the epidermal application. The volume of test item preparation and vehicle applied was approximately 0.2 mL. The dressings were left in place for 24 hours.

Study design: in vivo (LLNA)

Positive control substance(s):
other: alpha-hexylcinnamaldehyde
Statistics:
Descriptive statistics (means and standard deviations) were calculated for body weights. No inferential statistics were used.

Results and discussion

Positive control results:
The results from positive control test confirm alpha-hexylcinnamaldehyde as skin sensitizer, as it produced allergic contact dermatitis in >30% of the test animals.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
75% (test item in purified water)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
75% (test item in purified water)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Purified water only
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Purified water only
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
other: Control group
Dose level:
75% (test item in purified water)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Control group
Dose level:
75% (test item in purified water)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
other: Control group
Dose level:
Purified water only
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Control group
Dose level:
Purified water only
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None

Any other information on results incl. tables

Observations:

Viability/Mortality/Macroscopic Findings

No intercurrent deaths occurred during the course of the study, hence no necropsies were performed.

Clinical Signs

No clinical signs were recorded throughout the entire observation period.

Body Weights

The body weight of the animals was within the range commonly recorded for animals of this strain and age.

Skin Reactions in the Intradermal Induction

Expected common findings were observed in the test and control groups after the different injections using FCA intradermally. These findings consisted of erythema, oedema, necrotizing dermatitis, encrustation and exfoliation of encrustation. No detailed description of the skin reactions is given in the report as these FCA effects are well known.

Skin Reactions in the Epidermal Induction

See Tables 1 and 2 (attached background material).

Control Group:

No local skin reactions were observed in the control animals when treated with purified water alone.

Test Group:

No local skin reactions were observed in the test animals when treated with the neat test item (applied at 100%).

Skin Reactions in the Challenge

See Tables 3 to 6 (attached background material)

Control Group:

No local skin reactions were observed in the control animals when treated with purified water alone or when treated with the test item applied at 75% in purified water.

Test Group:

No local skin reactions were observed in the test animals when treated with purified water alone or when treated with the test item applied at 75% in purified water.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the findings in an adjuvant sensitisation test (M&K-test) in guinea pigs and in accordance to Regulation (EC) No 1272/2008, the test item does not have to be classified and labeled as a skin sensitizer.
Executive summary:

In order to assess the cutaneous allergenic potential of Polyphosphoric acids, esters with triethanolamine, sodium salts, the Maximization-Test was performed in 15 (10 test and 5 control) male albino Dunkin Hartley guinea pigs, in accordance with OECD Guideline No. 406 and the Commission Regulation (EC) No 440/2008, B.6.

The intradermal induction of sensitisation in the test group was performed in the nuchal region with a 10% dilution of the test item in purified water and in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline. The epidermal induction of sensitisation was conducted for 48 hours under occlusion with the test item at 100% (neat, applied as delivered) one week after the intradermal induction. The animals of the control group were intradermally induced with purified water and FCA/physiological saline,

Two weeks after epidermal induction the test and control animals were challenged by epidermal application of the test item at 75% in purified water and purified water alone under occlusive dressing.

Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing.

Results - Skin Reactions after the Challenge Procedure

 

After 24 hours

Positive/Total

% Positive of Total

After 48 hours

Positive/Total

% Positive of Total

Control Group

 

 

Test item, 75% in purified water (left flank)

0/5

0%

0/5

0%

Purified water only (right flank)

0/5

0%

0/5

0%

 

 

 

Test Group

 

 

Test item, 75% in purified water (left flank)

0/10

0%

0/10

0%

Purified water only (right flank)

0/10

0%

0/10

0%

No intercurrent deaths occurred during the course of the study.

No toxic signs were evident in the guinea pigs of the control or test group.

No local skin effects were observed in the guinea pigs of the control or test group.

Based on the findings in an adjuvant sensitisation test (M&K-test) in guinea pigs and in accordance to Regulation (EC) No 1272/2008, the test item does not have to be classified and labeled as a skin sensitizer.