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Description of key information

Local lymph node assay: not a skin sensitiser

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 May - 11 Jun 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
23 Jul 2010
Deviations:
yes
Remarks:
ear thickness measurements in pre-test not performed
GLP compliance:
yes (incl. QA statement)
Remarks:
Hessisches Ministerium für Umwelt, ländlichen Raum und Verbraucherschutz, Wiesbaden, Germany
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Dehydroacetic acid
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands, Horst, The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 20.2 ± 0.5
- Housing: Individual in Makrolon Type I with wire mesh top and granulated soft wood bedding
- Diet: pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
propylene glycol
Concentration:
5, 10 and 20% (w/v)
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The highest test item concentration, which can be technically used was a 20 % suspension in propylene glycol.
- Irritation: To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 2.5, 5, 10, and 20 % on one ear each on three consecutive days. Clinical signs were recorded 24 ± 4 hours after each application. At the tested concentrations the animals did not show any signs of irritation.
- Systemic toxicity: During the pre-test, no systemic toxicity could be observed.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by β-scintillation.
- Criteria used to consider a positive response: The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
25 µl of the test compound was applied to the entire dorsal surface of each ear of each mouse. The application was repeated on days 2 and 3. On day 6 an injection of 250 µl phosphate buffered saline (PBS) containing 19.7 µCi of 3H-methylthymidine (3H-TdR) 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 lymph node cells was prepared from each mouse and pooled per group (8 nodes per group).The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group).Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 um mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations of the body weights were calculated.
Positive control results:
The positive control substance hexyl cinnamic aldehyde (25% (w/v) induced positive reactions and a SI of 4.87 could be determined, thus meeting the reliability criteria for the LLNA.
Key result
Parameter:
SI
Value:
1.07
Test group / Remarks:
5% (w/v)
Key result
Parameter:
SI
Value:
2.1
Test group / Remarks:
10% (w/v)
Key result
Parameter:
SI
Value:
2.36
Test group / Remarks:
20% (w/v)
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Please refer to table 1 under "any further information on results".

EC3 CALCULATION
The EC3 value could not be calculated, since all SI values were below 3.

CLINICAL OBSERVATIONS
No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Table 1 Calculation and results of individual data

Test item concentration % (w/v) Measurement DPM Calculation Result
DPM-BG number of lymph nodes DPM per lymph node SI
BG I 35 -
BG II 33 -
Vehicle control 3816 3782 8 472.8 -
5 4098 4064 8 508.0 1.07
10 7963 7929 8 991.1 2.10
25 8959 8925 8 1115.6 2.36

BG = Background (1 mL 5% trichloracetic acid) in duplicate

Vehicle = propylene glycol

SI = Stimulation index

Table 2 Individual body weights

Animal No. Dose group Initial Weight (g) weight prior to treatment with 3HTdR (g)
Individual Mean± SD Individual Mean± SD
1 1 19.9 20.3 ± 0.9 21.2 21.2 ± 1.1
2 1 19.3 19.8
3 1 21.3 21.2
4 1 20.8 22.4
5 2 18.6 19.4 ± 1.1 20.3 21.1 ± 1.2
6 2 18.9 19.9
7 2 21.0 22.7
8 2 19.0 21.3
9 3 20.9 20.3 ± 0.5 21.0 20.6 ± 0.5
10 3 20.4 19.8
11 3 20.0 20.6
12 3 19.8 20.9
13 4 21.5 20.8 ± 1.5 22.1 21.0 ± 1.4
14 4 22.4 22.2
15 4 18.9 19.6
16 4 20.3 20.0
Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008
Conclusions:
The test material was not a skin sensitiser.
CLP: not classified
Executive summary:

In a LLNA three groups each of four female mice were treated daily with the test item at concentrations of 5, 10, and 20% (w/v) in propylene glycol by topical application to the dorsum of each ear lobe (left and

right) for three consecutive days. A control group of four mice was treated with the vehicle (propylene glycol) only. All treated animals survived the scheduled study period and no signs of toxicity were

observed. The test material was found not to be a skin sensitiser.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Positive control results:
The positive control substance hexyl cinnamic aldehyde (25% (w/v) induced positive reactions and a SI of 4.87 could be determined, thus meeting the reliability criteria for the LLNA.
Key result
Parameter:
SI
Value:
1.07
Test group / Remarks:
5% (w/v)
Key result
Parameter:
SI
Value:
2.1
Test group / Remarks:
10% (w/v)
Key result
Parameter:
SI
Value:
2.36
Test group / Remarks:
20% (w/v)
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Please refer to table 1 under "any further information on results".

EC3 CALCULATION
The EC3 value could not be calculated, since all SI values were below 3.

CLINICAL OBSERVATIONS
No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Table 1 Calculation and results of individual data

Test item concentration % (w/v) Measurement DPM Calculation Result
DPM-BG number of lymph nodes DPM per lymph node SI
BG I 35 -
BG II 33 -
Vehicle control 3816 3782 8 472.8 -
5 4098 4064 8 508.0 1.07
10 7963 7929 8 991.1 2.10
25 8959 8925 8 1115.6 2.36

BG = Background (1 mL 5% trichloracetic acid) in duplicate

Vehicle = propylene glycol

SI = Stimulation index

Table 2 Individual body weights

Animal No. Dose group Initial Weight (g) weight prior to treatment with 3HTdR (g)
Individual Mean± SD Individual Mean± SD
1 1 19.9 20.3 ± 0.9 21.2 21.2 ± 1.1
2 1 19.3 19.8
3 1 21.3 21.2
4 1 20.8 22.4
5 2 18.6 19.4 ± 1.1 20.3 21.1 ± 1.2
6 2 18.9 19.9
7 2 21.0 22.7
8 2 19.0 21.3
9 3 20.9 20.3 ± 0.5 21.0 20.6 ± 0.5
10 3 20.4 19.8
11 3 20.0 20.6
12 3 19.8 20.9
13 4 21.5 20.8 ± 1.5 22.1 21.0 ± 1.4
14 4 22.4 22.2
15 4 18.9 19.6
16 4 20.3 20.0
Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008
Conclusions:
CLP: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Local lymph node assay: not a skin sensitiser: no classification warranted