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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 Oct - 29 Nov 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 22 Jul 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS, B.V., Inc., The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: 2014C Teklad Global Rodent diet by Envigo RMS Limited, UK, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

- IN-LIFE DATES: From: 24 Oct 2016 To: 29 Nov 2016

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
2.8, 5.6 and 11.1% (w/w) (equivalent to 2.5, 5 and 10% active ingredient)
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS: One female per dose was treated by daily application of 25 µL of the test item at concentrations of 11.1, 27.8 and 55.5% in acetone/olive oil 4:1 (equivalent to 10, 25 and 50% active ingredient) to the dorsal surface of each ear for three consecutive days.
- Compound solubility: The vehicle was chosen as it was suitable at the highest concentration.
- Irritation: The animals were observed once daily for local skin irritation to the application site.
- Systemic toxicity: The animals were observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6 for signs of toxicity. The body weight was recorded on Day 1 prior to dosing and on Day 6.
- Ear thickness measurements: The thickness of each ear was measured pre-dose on Day 1 and post dose on Day 3 and Day 6.
- Erythema scores: No erythema (0), very slight erythema (1), well-defined erythema (2), moderate to severe erythema (3), severe erythema to eschar formation preventing grading of erythema (4)

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by β-scintillation counting
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation is classified as a "non-sensitizer".
lf the results allowed the EC3 value could also be calculated. The EC3 value is the concentration of test item expected to cause a 3-fold increase in 3HTdR incorporation. The equation used for the calculation of EC3 is:
EC3 = c + [[(3-d)/(b-d)] x (a-c)]
a = lowest concentration giving stimulation index >3
b = actual stimulation index caused by 'a'
c = highest concentration failing to produce a stimulation index of 3
d = actual stimulation index caused by 'c'

- Other: The animals were observed for signs of toxicity twice on Day 1, 2 and 3, and once on Day 4, 5 and 6. The body weight was recorded on Day 1 prior to dosing and on Day 6 prior to termination.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of the test compound was applied to the dorsal surface of each ear of each mouse on Day 1, 2 and 3 in concentrations of 2.8, 5.6 and 11.1% (equivalent to 2.5, 5 and 10%) in acetone/olive oil. On Day 6 an injection of 250 µL phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (3H-TdR) was made into the tail vein of each experimental mouse. Five hours later, the draining auricular lymph node of each ear was excised into PBS. A single cell suspension of pooled lymph node cells was prepared per experimental group by gentle mechanical disaggregation through a 200-mesh stainless steel gauze and rinsed with PBS. The precipitates were incubated for approximately 18 h at approximately 4 °C, centrifuged, resuspended in 1 mL TCA and transferred to 10 mL scintillation fluid before β-scintillation counting.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
A study (study dates: 10 - 16 Nov 2016; study number: YY34MW) was performed to assess the sensitivity of the strain of mouse used at the testing facility to a known sensitizer and to show the sensitivity and reproducibility of the test. The positive control substance hexyl cinnamic aldehyde (25% (v/v) in acetone/olive oil 4:1) was considered to be a sensitizer under the conditions of the test (SI 5.66).

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
4.02
Test group / Remarks:
2.8% (equivalent to 2.5% w/w active ingredient)
Key result
Parameter:
SI
Value:
3.64
Test group / Remarks:
5.6% (equivalent to 5% w/w active ingredient)
Key result
Parameter:
SI
Value:
8.94
Test group / Remarks:
11.1% (equivalent to 10% w/w active ingredient)
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
The SI of the 2.5, 5 and 10% treatment group was 4.02, 3.64 and 8.94, respectively.

EC3 CALCULATION
The EC3 value could not be determined, since no concentration failed to produce a stimulation index of 3.

CLINICAL OBSERVATIONS:
Mortality or signs of systemic toxicity were not observed in all treatment groups or in the control group.

BODY WEIGHTS
Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the control group over the same period.

Any other information on results incl. tables

Table 1: Results of the measurement of ear thickness and mean ear thickness changes - preliminary screening test

Concentration (% w/w) in acetone/olice oil 4:1 Ear thickness measurement (mm)
Day 1
pre-dose
Day 3
post dose
Day 6
left right left right left right
55.5 (equivalent to 50% active ingredient) 0.22 0.21 0.24 0.23 0.30 0.29
overall mean (mm) 0.22 0.24 0.30
overall mean ear thickness change (%) na 9.30 37.21
27.8% (equivalent to 25% active ingredient) 0.23 0.23 0.28 0.28 0.40 0.36
overall mean (mm) 0.23 0.28 0.38
overall mean ear thickness change (%) na 19.565 65.217
11.1% (equivalent to 10% active ingredient) 0.21 0.23 0.23 0.24 0.22 0.24
overall mean (mm) 0.22 0.24 0.23
overall mean ear thickness change (%) na 6.82 4.55

Table 2: Results of radioactivity incorporated - main test

Concentration (% w/w) in acetone/olice oil 4:1 DPM dpm/node* SI ** Result
Vehicle 5026.26 628.28 na na
2.8 (equivalent to 2.5% w/w active ingredient) 20230.30 2528.79 4.02 Positive
5.6 (equivalent to 5% w/w active ingredient) 18292.23 2286.53 3.64 Positive
11.1 (equivalent to 10% w/w active ingredient) 44925.14 5615.64 8.94 Positive

DPM = disintegrations per minute

na = not applicable

* Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

** Stimulation Index of 3.0 or greater indicates a positive result

Applicant's summary and conclusion

Interpretation of results:
other: Cat. 1, H317 according to Regulation (EC) 1272/2008
Conclusions:
CLP: Cat. 1, H317