Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
25 June - 19 Aug 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline conform GLP study.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study already performed before implementation of LLNA Guideline
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH, Kisslegg, Germany
- Age at study initiation: 4-6 weeks
- Weight at study initiation: 341-370 g (pretest); 328-384 g (test and control group)
- Housing: individually in Makrolon type-4 cages with standard softwood bedding
- Diet (e.g. ad libitum): pelleted standard Kliba 3418 guinea pig breeding/maintenance diet (Provimi Kliba AG, Kaiseraugst, Switzerland), ad libitum
- Water (e.g. ad libitum): communitytap water, ad libitum
- Acclimation period (main study): 25. June - 22 July 2008

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 37-70 %
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES: From: 25.06 2008 To: 19.08.2008
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
Pretest I (intradermal injection): 25; 15; 10 %
Pretest II (epidermal application): 75; 50; 25; 15 %

Main study (intradermal induction): 1 %
Main study (epidermal induction): 75 %
Main study (challenge):10 %
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Pretest I (intradermal injection): 25; 15; 10 %
Pretest II (epidermal application): 75; 50; 25; 15 %

Main study (intradermal induction): 1 %
Main study (epidermal induction): 75 %
Main study (challenge):10 %
No. of animals per dose:
10 test and 5 control animals
Details on study design:
Induction
- Intradermal Injection/Test Day 1
An area of dorsal skin fron the scapular region (approx. 6x8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 mL/site) were made just within the boundaries of a 4x6 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 1 % in purified water.
3) The test item at 1 % in a 1:1 (v/v) mixture of Fraund's Complete adjuvant and physiological saline.

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 Indcution/Test Day 8
One week after the injection, the scapular area (approx. 6x8 cm) was again clipped and shaved free of hair to the application. A 2x4 cm patch of filter paper was saturated with the test item at 75 % in purified water and placed over the injcetion sites of the test animals. The amount of the tst item preparation applied was approx. 0.3 g. The patch was covered with aluminium foil and firmly secured by an plaser wrapped around the trunk of the animal and secured with impervious tape. The occlusive dressings were left in place for 48 h. The epidermal application procedure described ensured intensive contact of the test item.

The guinea pigs of the control group were treated as described above with purified water only, applied at a volume of aprox. 0.3 mL.

The reaction sites were assessed 24 and 48 h after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.

Challenge/Test Day 22
The test and control guinea pigs were challenged two weeks after the epidermal induction application and were treated in the same way.

Hair was cliped and shaved from a 5x5 cm area on the left and right flank of each guinea pig just prior to the application. Two patches (3x3 cm) of filter paper were saturated with the test item at the highest tested non-irritating concentration of 10 % (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 the test item preparation was approx. 0.2 mL and a volume of approx. 0.2 mL was used for the vehicle. The dressings were left in place for 24 h.

21 h after removal of the dressing the test sites treated with the test item were depilated as described in the epidermal pretest.

Approx. 3 h later (approx. 48 h from start of the challenge application) the skin reaction was observed and recorded according to the numerical grading system following Magnusson and Kligman grading scale.

Approx. 24 h after this observation (72 h from the start of the challenge application) was made and once again recorded.
Challenge controls:
Please refer to "Details on study design"
Positive control substance(s):
yes
Remarks:
Alpha-Hexylcinnamaldehyde
Positive control results:
Discrete/patchy to moderate/confluent erythema with or without scaling was observed in nine out of ten test animals at the 24- and 48-hour reading after the challenge treatment with Alpha-Hexylcinnamaldehyde at 1 % in PEG 300 (left shoulder). Five test animals showed discrete/patchy erythema at the 24-hour reading after treatment with Alpha-Hexylcinnamaldehyde at 01. % in PEG 300 (left flank). No skin effect was observed in the control group.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no effects
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no effects.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no effects
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no effects.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no effects
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no effects.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no effects
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no effects.
Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the result of the contact hypersensitivity maximization test in guinea pigs according to OECD 406 the test item does not have to be classified and labelled as a skin sensitizer.
Executive summary:

A read across from Reaction mass of potassium ethyl octyphosphoante and diethyl octyl phosphonate to data generated with Phosphoric acid, dodecyl ester, potassium salt was performed. For read across justification please refer to the attached document.

To assess the cutaneous allergenic potential of the test item the Maximization-Test was performed in 15 (10 test and 4 control) female albino Dunkin Hartley guinea pigs in accordance with OECD guideline 406.

The intradermal induction of sensitization in the test group was performed in the nuchal region with a 1 % dilution of the test item in purified water in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline. The epidermal induction of sensitization was conducted for 48 hours under occlusion with the test item at 75 % in purified water one week after the intradermal induction. The animals of the control group were intradermally induced with purified water under occlusion.

Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test item at 10 % in purified water and purified water alone under occlusive dressing. Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing.

No toxic signs were evident in the guinea pigs of the control or test group. No deaths occurred. No skin effect/reaction was observed in the control and test group after challenge treatment with the test item at 10 % in purified water.

Based on these findings, the test item does not have to be classified and labelled as a skin sensitizer.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

To assess the cutaneous allergenic potential of Reaction mass of potassium ethyl octylphosphonate and diethyl octylphosphonate a read across was performed to Phosphoric acid, dodecyl ester, potassium salt and Reaction mass of potassium ethyl octylphosphonate and diethyl octyl phosphonate. For read across justification please refer to the attached document in IUCLID Chapter 7.4.1.

To assess the cutaneous allergenic potential ofPhosphoric acid, dodecyl ester, potassium saltthe Maximization-Test was performed in 15 (10 test and 4 control) female albino Dunkin Hartley guinea pigs in accordance with OECD guideline 406.
The intradermal induction of sensitization in the test group was performed in the nuchal region with a 1 % dilution of the test item in purified water in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline. The epidermal induction of sensitization was conducted for 48 hours under occlusion with the test item at 75 % in purified water one week after the intradermal induction. The animals of the control group were intradermally induced with purified water under occlusion. Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test item at 10 % in purified water and purified water alone under occlusive dressing. Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing.
No toxic signs were evident in the guinea pigs of the control or test group. No deaths occurred. No skin effect/reaction was observed in the control and test group after challenge treatment with the test item at 10 % in purified water. Based on these findings, the test item does not have to be classified and labelled as a skin sensitizer.

In order to study a possible skin sensitising potential of Phosphoric acid, mixed esters with butyl alcohol and ethylene glycol, three groups each of four female mice were treated once daily with the test item at concentrations of 10, 25, and 50% (w/w) in dimethylformamide by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be applied whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment. A control group of four mice was treated with the vehicle (dimethylformamide) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of3H-methyl thymidine measured in ab-scintillation counter.
The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. On day 3 and day 4,all animals treated with the test item concentrations of 10, 25, and 50% showed an erythema of the ear skin (Score 1). A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.
In this study Stimulation Indices of 1.52, 1.21, and 1.32 were determined with the test item at concentrations of 10, 25, and 50% (w/w) in dimethylformamide. A dose response was not observed. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

Migrated from Short description of key information:
To assess the cutaneous allergenic potential of Reaction mass of potassium ethyl octylphosphonate and diethyl octylphosphonate a read across was performed to Phosphoric acid, dodecyl ester, potassium salt and Reaction mass of potassium ethyl octylphosphonate and diethyl octyl phosphonate. For read across justification please refer to the attached document in IUCLID Chapter 7.4.1.

To assess the cutaneous allergenic potential of Phosphoric acid, dodecyl ester, potassium salt the Maximization-Test was performed in 15 (10 test and 4 control) female albino Dunkin Hartley guinea pigs in accordance with OECD guideline 406. No toxic signs were evident in the guinea pigs of the control or test group. No deaths occurred. No skin effect/reaction was observed in the control and test group after challenge treatment with the test item at 10 % in purified water.

In order to study a possible skin sensitising potential of Phosphoric acid, mixed esters with butyl alcohol and ethylene glycol, three groups each of four female mice were treated once daily with the test item at concentrations of 10, 25, and 50% (w/w) in dimethylformamide by topical application to the dorsum of each ear for three consecutive days. The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. On day 3 and day 4,all animals treated with the test item concentrations of 10, 25, and 50% showed an erythema of the ear skin (Score 1).
In this study Stimulation Indices of 1.52, 1.21, and 1.32 were determined with the test item at concentrations of 10, 25, and 50% (w/w) in dimethylformamide. A dose response was not observed.

Justification for selection of skin sensitisation endpoint:
Guideline conform study with structural analogue.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Based on a read across to the negative results of Guinea pig maximization test with Phosphoric acid, dodecyl ester, potassium salt and a LLNA performed with Phosphoric acid, mixed esters with butyl alcohol and ethylene glycol Reaction mass of potassium ethyl octylphosphonate and diethyl octylphosphonate does not have to be classified and labelled as a skin sensitizer. For read across justifucation please refer to IUCLID Chapter 7.4.1.


Justification for selection of respiratory sensitisation endpoint:
n.a.

Justification for classification or non-classification