Registration Dossier

Administrative data

Description of key information

Read-across:

OECD 426; GLP; LLNA in four female mice; concentrations: 3.5%, 7%, 14%; SI below 2.0; no sensitizer

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Remarks:
RCC Cytotest Cell Research GmbH
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands
- Age at study initiation: 7 weeks (beginning of acclimatization)
- Housing: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen) with granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen), housed individually
- Diet (e.g. ad libitum): pelleted standard diet (Harlan Winkelmann GmbH, D-33178 Borchen), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: Under test conditions after health examination. Only animals without any visible signs of illness will be used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
dimethyl sulphoxide
Concentration:
0% (vehicle control), 3.5%, 7% and 14% (based a non-GLP local toxicity pre-test, 14% was the highest technically applicable concentration in the chosen vehicle)
No. of animals per dose:
4 females
Details on study design:
RANGE FINDING TESTS:
- Irritation: In a non-GLP conform pre-test in two mice, test item concentrations of 1.75, 3.5, 7 and 14 % (w/v) were tested on one ear each. No irritation effects were observed at these concentrations after a single application.

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 3.5, 7 and 14% (w/v) in DMSO. The application volume, 25 μl, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
3H-methyl thymidine (3HTdR) was purchased from Amersham International (Amersham product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were administered with 250 μl of 82 μCi/ml 3HTdR (corresponds to 20.5 μCi 3HTdR per mouse) by intravenous injection via a tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Na-thiopental (Trapanal, Altana, D-78467 Konstanz).
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 μm 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. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

- Criteria used to consider a positive response:
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:
The test item was placed into a volumetric flask glass beaker on a tared balance and the vehicle (DMSO) was quantitatively added. The test item concentrations were prepared individually. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer. The preparations were made freshly before each dosing occasion.
Positive control substance(s):
other: α-Hexylcinnamaldehyde in acetone:olive oil, 4:1 (v/v)
Statistics:
The mean values and standard deviations were calculated in the body weight tables.
Parameter:
SI
Value:
>= 1.3 - <= 2
Test group / Remarks:
3.5%, 7%, 14%
Parameter:
other: disintegrations per minute (DPM)
Value:
>= 430 - <= 863
Test group / Remarks:
control, 3.5%, 7%, 14%

No deaths occurred during the study period. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. Due to the intense red colour of the test item local irritation reactions such as ear redness could not be detected. 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.

Calculation Result
Test item concentration Group Measurement DPM DPM-BG a) number of lymph nodes DPM/lymph node b) S.I.
-- BG I 23.6 -- -- -- --
-- BG II 34.2 -- -- -- --
-- CG 1 3467.18 3438.3 8 429.8
3.5 % (w/v) TG 2 4733.81 4704.9 8 588.1 1.4
7 % (w/v) TG 3 4615.95 4587.1 8 573.4 1.3
14 % (w/v) TG 4 6932.97 6904.1 8 863 2

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Group

S.I. = Stimulation Index

a) = The mean value was taken from the figures BG I and BG II

b) = Since the lymph nodes of the animals of a dose group were pooled,

DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was found not to be a skin sensitiser.
Executive summary:

In GLP compliant study following OECD guideline 429 the test item dissolved in DMSO was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed using test item concentrations of 3.5, 7 and 14%. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. Due to the intense red colour of the test item local irritation reactions such as ear redness could not be detected. In this study Stimulation Indices (S.I.) of 1.4, 1.3 and 2.0 were determined with the test item at concentrations of 3.5, 7 and 14% (w/v) in DMSO, respectively. The test item was not a skin sensitiser in this assay.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Justification for type of information:
see attached justification
Reason / purpose:
read-across source
Parameter:
SI
Value:
>= 1.3 - <= 2
Test group / Remarks:
3.5%, 7%, 14%
Remarks on result:
other: results obtained from read-across
Parameter:
other: disintegrations per minute (DPM)
Value:
>= 430 - <= 863
Test group / Remarks:
control, 3.5%, 7%, 14%
Remarks on result:
other: results obtained from read-across
Cellular proliferation data / Observations:
SI for test item concentrations 3.5%, 7%, and 14% were 1.4, 1.3, and 2.0, respectively.
DPM for test item concentrations 3.5%, 7%, and 14% were 588.1, 573.4, and 863, respectively. Control DPM was 429.8.
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results obtained from a LLNA study performed with the read-across substance, the target substance was not assumed to be a skin sensitizer.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

No sensitization studies are available for the test substance. However, four GLP-compliant Local Lymph Node Assays have been conducted according to OECD guideline 429 with four category members (see attached category justification). This data is used to evaluate the test article’s potential to cause skin sensitization.

 

In the first study performed with the read-across substance, groups of four mice per dose group were treated with the test substance dissolved in DMSO once daily for three consecutive days. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. Due to the intense red color of the test item local irritation reactions such as ear redness could not be detected. In this study Stimulation Indices (S.I.) of 1.4, 1.3 and 2.0 were determined with the test item at concentrations of 3.5, 7 and 14% (w/v) in DMSO, respectively. The test item was not a skin sensitizer in this assay (CCR, 2005).

 

In the second study performed with another category member, three groups each of five female mice were treated daily with the test item in acetone/olive oil (4/1, v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. All treated animals survived the scheduled study period. Neither clinical / local signs nor other findings were observed in any animals of the control group. After the first topical application, the residual test item was found at both dosing sites in all mice of the test item groups (Groups 2-4), persisting for the remainder of the in-life phase of the study. In this study Stimulation Indices of 1.2, 1.2 and 0.8 were determined with the test item at concentrations of 5%, 10 % and 25 % in acetone/olive oil (4/1, v/v), respectively. Based on this result, the test article was found to be a non-sensitizer when tested up to the highest applicable concentration of 25 % in acetone/olive oil (4/1, v/v) (RCC, 2006).

 

In the study performed with the third read across compound, groups of 6 female CBA/Ca mice each were treated with a 30% w/w preparation of the test substance in AOO 4:1 (mixture of acetone:olive oil Ph.Eur./DAB in a ratio 4:1 parts by volume) or with the vehicle alone. No signs of systemic toxicity were noticed. On the2nd and 3rd day of application and on the day of lymph node removal slight black discoloration of the ears were noticed in all animals of the test group. The test substance did not induce a statistically significant or biologically relevant response of the auricular lymph nodes when applied as a 30% preparation (highest applicable concentration) in AOO 4:1. The limited, but statistically significant increase in ear weight indicates some irritation of the ear at this concentration. In conclusion, the test article does not have a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen (BASF, 2005).

 

In a fourth study with another category member groups of four male CBA/Ca mice per dose level were treated with a preparation of the test substance in propylene glycol or with the vehicle alone. Each test animal was applied with 25 µL of the test substance preparation to the dorsum of both ears for three consecutive days. Lymph node response was evaluated by measuring the proliferative capacity of the prepared lymph node cells. The application of the test substance at concentrations of 3%, 10% and 30% w/v in propylene glycol resulted in an increase in isotope incorporation which was less than 3-fold at all three concentrations (SI indices were: 1.16 (3%); 1.61 (10%); 1.31 (30%)). Therefore, the test substance is considered to be not sensitizing (CTL, 1999).

 

Conclusion: No data generated with the test article are available. But according to the category approach, the data available for other category members is used to assess the sensitization potential of the test article. Based on the available and reliable data obtained in Local Lymph Node Assays with four category members, no classification for sensitization is warranted. The SI indices for the tested substances were all below 3. Therefore, the test article is characterized as not sensitizing based on this read across/category approach.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the data, classification for sensitization is not warranted under Regulation (EC) No.1272/2008.