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Description of key information

Skin sensitisation

In a Local Lymph Node Assay (LLNA) in mice (CBA/Ca) according to OECD Guideline 429 the test item T003066 was found to be a skin sensitiser and should be classified as skin sentitiser category 1B based on the calculated EC3 value ( > 5%). A precise calculation of the EC3 value was not possible since the S.I. was above the threshold of 3 even at the lowest tested concentration), according to CLP Regulation. (Wolf, 2008).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2008-09-23 to 2008-10-08
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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: 18 (2 for the pretest and 16 for the main study) female mice (nulliparous and non-pregnant, CBA/CaOlaHsd; from Harlan Laboratories B.V.
- Age at beginning of acclimatisation: 8-12 weeks
- Initial weight: 20.4 - 22.5 g
- Housing: Single housing, in Makrolon Type I cages, with wire mesh top (EHRET GmbH) and granulated soft wood bedding.
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (Harlan Laboratories GmbH)
- Water (e.g. ad libitum): tap water, ad libitum (Gemeindewerke)
- Acclimation period: under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30-70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12 (artificial light from 6.00 a.m. to 6.00 p.m.)

IN-LIFE DATES: From: 2008-09-23 To: 2008-10-08
Vehicle:
dimethylformamide
Concentration:
1, 10 and 25 % (v/v) of test article
No. of animals per dose:
4 females per group; 1 control group and 3 test groups
Details on study design:
RANGE FINDING TESTS:
Rationale for dose levels: A solubility experiment was performed. The highest test item concentration, which can be technically used was a 25% suspension in dimethylformamide. To determine the highest non-irritant test concentration or the highest technically applicable concentration, a pretest was performed in two mice. Two mice were treated with concentrations of 2.5%, 5%, 10, and 25% on one ear each on three consecutive days. Clinical signs were recored 24 +/- 4 hours after each application. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity. The test item in the main study was assayed at 1, 10 and 25%. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- 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 1, 10, and 25 % (w/w) in dimethylformamide. The application volume, 25 µl, was spread over the entire dorsal surface (diam. ca. 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).
- Criteria used to consider a positive response: A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of 3 HTdR 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.

ADMINISTRATION OF ³H-METHYL THYMIDINE (³HTdR)
- Five days after the first topical application, all mice were administered with 250 µl of 78.6 µCi/ml 3HTdR (corresponds to 19.7 µCi 3HTdR per mouse) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED ³HTdR
- Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium (Release, WDT, D-30827 Garbsen).
- 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 plastic scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid (Perkin Elmer (LAS) GmbH, D-63110 Rodgau) and thoroughly mixed.
- The level of 3HTdR incorporation was then measured on a beta-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, D-63110 Rodgau). Similarly, background 3HTdR levels were also measured in two 1 ml-aliquots of 5 % trichloroacetic acid. The beta-scintillation counter expresses 3 HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean values and standard deviations were calculated for the body weights.
Positive control results:
Background I: 43 dpm
Background II: 38 dpm
Control group 1: 2270 dpm, 2230 dpm-BG; 278.7 dpm per lymph node (8 lymph nodes)
Test group 2 (5%): 11733 dpm, 11693 dpm-BG, 1461.6 dpm per lymph node (8 lymph nodes); S.I. = 5.24
Test group 3 (10%): 16484 dpm; 16444 dpm-BG, 2055.4 dpm per lymph node (8 lymph nodes); S.I. = 7.38
Test group 4 (25%):20812 dpm; 20772 dpm-BG, 2596.4 dpm per lymph node (8 lymph nodes); S.I. = 12.45

The EC3 value was estimated to be < 5%. A precise calculation of the EC3 value was not possible since the S.I. was above the threshold of 3 even at the lowest tested concentration. The obtained values are within the expected range and the positive control experiment is considered fully valid.
Parameter:
SI
Value:
2.94
Test group / Remarks:
test group 4 (25%)
Remarks on result:
other:
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
- Background I: 21 dpm - Background II: 23 dpm - Control group 1: 2163 dpm, 2141 dpm-BG*; 267.6 dpm per lymph node** (8 lymph nodes) - Test group 2 (1%): 4335 dpm, 4313 dpm-BG*, 539.1 dpm per lymph node** (8 lymph nodes) - Test group 3 (10%): 6932 dpm; 6910 dpm-BG*, 863.8 dpm per lymph node** (8 lymph nodes) - Test group 4 (25%): 6310 dpm; 6288 dpm-BG*, 786.0 dpm per lymph node** (8 lymph nodes) * The mean value was taken from the figures of background (BG) I and BG II ** Since the lymph nodes of the animals of a dose group were pooled, DPM/nodewas determined by dividing the measured value by the number of lymph nodespooled
Parameter:
SI
Value:
2.01
Test group / Remarks:
test group 2 (1%)
Parameter:
SI
Value:
3.23
Test group / Remarks:
test groud 3 (10%)
Parameter:
EC3
Value:
8.3

- Viability/mortality: no deaths occured during the study period.

- Clinical signs: 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 the treatment with ³HTdR, was within the range commonly recorded for animals of this strain and age.

Interpretation of results:
sensitising
Conclusions:
The test item RT003066 was found to be a skin sensitiser under the described conditions and should be classified as skin sentitizer category 1B based on the calculated EC3 value, according to CLP Regulation.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In a key LLNA study by Wolf (2008), three experimental groups of four female CBA/J mice were treated with test item concentrations of 1, 10 or 25% v/v for three consecutive days, by topical application on the dorsal surface of each ear lobe (left and right). Four vehicle control animals were similarly treated, but with vehicle alone (Methyl ethyl ketone).

Five days after the first topical application, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

Mean DPM/animal values for the experimental groups treated with test item concentrations 1, 10 and 25% were 4335, 6932 and 6310 DPM, respectively. Since the lymph nodes of the animals of a dose group were pooled, DPM/nodewas determined by dividing the measured value by the number of lymph nodes pooled. The SI values calculated for the test item concentrations 1, 10, and 25 % were 2.01, 3.23 and 2.94, respectively.

A precise calculation of the EC3 value was not possible since the S.I. was above the threshold of 3 even at the lowest tested concentration), according to CLP Regulation.

Based on the results the test item T003066 was found to be a skin sensitiser and should be classified as skin sentitiser category 1B based on the calculated EC3 value (8.3 %), according to CLP Regulation.

An in vitro or in chemico skin sensitisation study was waived based on the justification that adequate data from an in vivo skin sensitisation study (initiated before October 11th 2016) were available.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitisation:

Based on the results of the LLNA study in female mice (CBA/Ca), the test item should be classified as skin sentitizer category 1B according to the criteria of the CLP regulation (EC) No 1272/2008.