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EC number: 219-774-8 | CAS number: 2528-39-4
Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vivo (non-LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 12 Mar - 14 Apr 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 406 (Skin Sensitisation)
- Version / remarks:
- adopted in 1992
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Slovenska Narodna Akreditacna Sluzba, Bratislava, Slovenska Republika
- Type of study:
- guinea pig maximisation test
- Justification for non-LLNA method:
- As the test substance is likely to have surfactant properties, the default LLNA study (OECD 429) is not recommended in this case.
Test material
- Reference substance name:
- Trihexyl phosphate
- EC Number:
- 219-774-8
- EC Name:
- Trihexyl phosphate
- Cas Number:
- 2528-39-4
- Molecular formula:
- C18H39O4P
- IUPAC Name:
- trihexyl phosphate
Constituent 1
In vivo test system
Test animals
- Species:
- guinea pig
- Strain:
- Dunkin-Hartley
- Remarks:
- albino
- Sex:
- female
- Details on test animals and environmental conditions:
- TEST ANIMALS
- Source: Velaz, Czech Republic
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 10 weeks old
- Weight at study initiation: 462 – 517 g
- Housing: up to 5 animals per cage in plastic cages suspended on stainless steel racks with Lignocel S3/4 bedding (Lufa-ITL GmbH, Germany)
- Diet: laboratory food ssniff (Spezialdiäten GmbH, Germany), offered in recommended doses each day approximately at the same time (analyses were performed)
- Water: tap water (containing 0.05% ascorbic acid), ad libitum (analyses were performed)
- Acclimation period: 5 days
- Indication of any skin lesions: no
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 0.3
- Humidity (%): 58.8 ± 2.2
- Photoperiod (hrs dark / hrs light): 12/12
Study design: in vivo (non-LLNA)
Inductionopen allclose all
- Route:
- intradermal
- Vehicle:
- other: ethanol
- Concentration / amount:
- 2%
- Day(s)/duration:
- Day 0 / single injection
- Adequacy of induction:
- other: highest concentration that did not cause destructive concentration of the skin after 24 h
- Route:
- epicutaneous, occlusive
- Vehicle:
- other: ethanol
- Concentration / amount:
- 10%
- Day(s)/duration:
- Day 7 / 48 h
- Adequacy of induction:
- other: minimal irritating concentration
Challenge
- No.:
- #1
- Route:
- epicutaneous, occlusive
- Vehicle:
- other: ethanol
- Concentration / amount:
- 2%
- Day(s)/duration:
- Day 21 (treated group) and 22 (control group) / 24 h
- Adequacy of challenge:
- highest non-irritant concentration
- No. of animals per dose:
- Range finding test: 3 females
Main test:
Test group: 20 females
Negative control group: 10 females
Positive control group: 5 females - Details on study design:
- RANGE FINDING TESTS:
Three females were pre-treated intradermally with an injection of 0.1 mL of FCA mixed 1:1 with vehicle (ethanol) 24 h before the induction phase.
These animals were used to assess the primary irritation tests after intradermal injection and after topical application. One animal was treated intradermally with 6 different test substance concentrations (initially 10, 25 and 50% on the right side of the animal; on the next day 1, 2 and 8% on the left side of the animal) and two animals were treated topically (4 different test substance concentrations each: 25, 50, 75 and 100% on one animal and the next day 2, 5, 10 and 15% on the second animal). Observations of the skin for the primary irritancy were scored after 24 h according to the Draize et al. scale. At a test substance concentration of 1%, the intradermal injection caused very slight erythema (grade 1) and no edema, well-defined erythema (grade 2) and very slight oedema (grade 1) were observed at concentrations of 2% or more. After topical application no edema was observed at any concentration, while very slight erythema (grade 1) was observed at 5% and well-defined erythema (grade 2) at 10% or more. According to the results of the range finding study, the following dose levels were selected for the main study:
Induction / intradermal injection: 2%
Induction / epicutaneous application: 10%
Challenge / epicutaneous application: 2%
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous)
- Exposure period: single injection (intradermal) and 48 h (epicutaneous)
- Test groups:
Intradermal (three pairs of injections; 0.1 mL/site):
Injection 1: Freund's Complete Adjuvant (FCA) 1:1 with water
Injection 2: 2% test substance formulated in vehicle
Injection 3: 2% test substance formulated in a 1:1 mixture FCA/water
Epicutaneous:
The test substance (in the minimal irritating concentration, 10%) was spread over 2 x 4 cm piece of gauze to saturation. The patch was secured with an occlusive dressing for 48 h.
- Control group:
Intradermal (three pairs of injections, 0.1 mL/site):
Injection 1: FCA 1:1 with water
Injection 2: undiluted vehicle
Injection 3: 50% w/v formulation of the vehicle in a 1:1 mixture FCA/water
Epicutaneous:
The vehicle was applied in the same manner as the test item in the treated group and held in contact by an occlusive dressing for 48 h.
- Site: shoulder region
- Frequency of applications: once (intradermal induction on Day 0 and epicutaneous application on Day 7)
- Concentrations: 2% (intradermal) and 10% (epicutaneous)
B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day of challenge: 21 (treated group), 22 (control group)
- Exposure period: 24 h
- Test groups: test substance and vehicle
- Control group: test substance and vehicle
- Site: flanks, previously unexposed area
- Concentrations: 2% of test substance (epicutaneous application)
- Evaluation: 24 and 48 h after removing the patches
OTHER:
The animals were inspected daily during the study for clinical symptoms. Individual weights of animals were determined shortly before the test substance was applied and at the end of the study. - Challenge controls:
- the control group is actually a challenge control
- Positive control substance(s):
- yes
- Remarks:
- α-hexylcinnamaldehyde (in concurrent study, reliability check)
Results and discussion
- Positive control results:
- The reliability of experimental technique used was performed with five animals, using α-hexylcinnamaldehyde (positive control) as a part of the study.
The same procedure as on treated and control animals were used. A pilot study was not performed due to the fact that the used substance (α-hexylcinnamaldehyde) is well known. The applied concentrations were chosen based on literature and previous studies.
Induction / Intradermal injection: 0.3%
Induction / epicutaneous application: 100% (undiluted)
Challenge / epicutaneous application: 100% (undiluted)
Well defined erythema (grade 2) was seen 24 h after removal of the patches in 2/5 animals of the positive control group, and slight erythema (grade 1) for the remaining 3/5 animals. This result represents skin sensitization reaction in 100% of tested animals. Slight erythema (grade 1) was still visible 48 h after removal of the patches in 4/5 animals. Edema was not observed in any of the animals. Thus the reliability of the experimental technique could be shown.
In vivo (non-LLNA)
Resultsopen allclose all
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- negative control
- Dose level:
- intradermal induction: 0%; challenge: 2%
- No. with + reactions:
- 0
- Total no. in group:
- 10
- Remarks on result:
- no indication of skin sensitisation
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- test chemical
- Dose level:
- intradermal induction: 2% challenge: 2%
- No. with + reactions:
- 5
- Total no. in group:
- 20
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- positive control
- Dose level:
- intradermal induction: 0.3%, challenge: 100%
- No. with + reactions:
- 5
- Total no. in group:
- 5
- Remarks on result:
- positive indication of skin sensitisation
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- negative control
- Dose level:
- intradermal induction: 0% challenge: 2%
- No. with + reactions:
- 0
- Total no. in group:
- 10
- Remarks on result:
- no indication of skin sensitisation
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- test chemical
- Dose level:
- intradermal induction: 2% challenge: 2%
- No. with + reactions:
- 2
- Total no. in group:
- 20
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- positive control
- Dose level:
- intradermal induction: 0.3%, challenge: 100%
- No. with + reactions:
- 4
- Total no. in group:
- 5
- Remarks on result:
- positive indication of skin sensitisation
Any other information on results incl. tables
Table 3: Skin reaction in Range finding study
Animal No. |
Test substance concentration (%) |
Erythema |
Edema |
Route |
1 |
50 |
2 |
1 |
intradermal |
25 |
2 |
1 |
||
10 |
2 |
1 |
||
8 |
2 |
1 |
||
2 |
2 |
1 |
||
1 |
1 |
0 |
||
2 |
100 |
2 |
0 |
epicutaneous application |
75 |
2 |
0 |
||
50 |
2 |
0 |
||
25 |
2 |
0 |
||
3 |
15 |
2 |
0 |
epicutaneous application |
10 |
2 |
0 |
||
5 |
1 |
0 |
||
2 |
0 |
0 |
Table 4: Skin response after challenge (treated with 2% of the test substance) and body weight change
Animal No. |
Group |
Reaction scores |
Body weight change/ gain (g)** |
|||
24 h* |
48 h* |
|||||
Erythema |
Edema |
Erythema |
Edema |
|||
1 |
Control group |
0 |
0 |
0 |
0 |
36 |
2 |
0 |
0 |
0 |
0 |
52 |
|
3 |
0 |
0 |
0 |
0 |
36 |
|
4 |
0 |
0 |
0 |
0 |
86 |
|
5 |
0 |
0 |
0 |
0 |
54 |
|
6 |
0 |
0 |
0 |
0 |
46 |
|
7 |
0 |
0 |
0 |
0 |
62 |
|
8 |
0 |
0 |
0 |
0 |
76 |
|
9 |
0 |
0 |
0 |
0 |
90 |
|
10 |
0 |
0 |
0 |
0 |
48 |
|
11 |
Treatment group |
0 |
0 |
0 |
0 |
66 |
12 |
0 |
0 |
0 |
0 |
38 |
|
13 |
0 |
0 |
0 |
0 |
30 |
|
14 |
0 |
0 |
0 |
0 |
64 |
|
15 |
0 |
0 |
0 |
0 |
74 |
|
16 |
0 |
0 |
0 |
0 |
68 |
|
17 |
1 |
0 |
1 |
0 |
38 |
|
18 |
0 |
0 |
0 |
0 |
72 |
|
19 |
0 |
0 |
0 |
0 |
10 |
|
20 |
0 |
0 |
0 |
0 |
62 |
|
21 |
1 |
0 |
0 |
0 |
70 |
|
22 |
1 |
0 |
0 |
0 |
46 |
|
23 |
0 |
0 |
0 |
0 |
38 |
|
24 |
1 |
0 |
1 |
0 |
32 |
|
25 |
0 |
0 |
0 |
0 |
56 |
|
26 |
0 |
0 |
0 |
0 |
54 |
|
27 |
0 |
0 |
0 |
0 |
70 |
|
28 |
1 |
0 |
0 |
0 |
84 |
|
29 |
0 |
0 |
0 |
0 |
6 |
|
30 |
0 |
0 |
0 |
0 |
42 |
|
* = time after removal of the patches
** = before test substance application and at end of the study
Table 5: Skin response and body weight change after treatment with undiluted α-hexylcinnamaldehyde (positive control)
Animal No. |
Reaction scores |
Body weight change/ gain (g)** |
|||
24 h* |
48 h* |
||||
Erythema |
Edema |
Erythema |
Edema |
||
1 |
1 |
0 |
1 |
0 |
18 |
2 |
1 |
0 |
0 |
0 |
48 |
3 |
2 |
0 |
1 |
0 |
48 |
4 |
1 |
0 |
1 |
0 |
60 |
5 |
2 |
0 |
1 |
0 |
32 |
* = time after removal of the patches
** = before test substance application and at end of the study
No clinical signs and no deaths in any of the groups were observed. The body weights of all animals were increasing during the study.
Applicant's summary and conclusion
- Interpretation of results:
- other: CLP/ EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
- Conclusions:
- In this guinea pig maximisation test (GPMT) 25% of the animals revealed a positive response after challenge at an intradermal induction concentration of 2%. Thus, the substance is not considered a skin sensitiser according to Regulation (EC) No. 1272/2008.
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