Registration Dossier

Administrative data

Description of key information

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:
5 December 2012 to 19 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaCrl
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8 - 10 weeks (on Day 1)
- Weight at study initiation: 16 - 19 g (on the day prior to dosing)
- Housing: animals were group housed during acclimation period and individually housed from Day –1 in cages that conformed to the 'Code of Practice for the Housing and Care of Animals Used in Scientific Procedures' (Home Office, London, 1989)
- Diet: ad libitum
- Water: mains water, ad libitum
- Acclimation period: 8 - 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 43 - 79 %
- Air changes (per hr): 15 - 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 25, 50 100 % v/v
No. of animals per dose:
4 females per dose level
Details on study design:
PRELIMINARY SCREENING TEST
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test material, a preliminary screening test was performed with one mouse. The mouse was treated by daily application of 25 µL of the undiluted test material to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The mouse was observed daily for six days from the initiation of treatment. Any signs of toxicity or irritation during this period were recorded. Ear thickness measurements were made using a thickness gauge on Day 1 (pre-treatment), Day 3 and Day 6. Both ears were also observed for erythema and scored using the Draize scale.

MAIN TEST
- Dose selection rationale: Dose levels were selected on the basis of the preliminary screening test
- Test material formulations: formulations were freshly prepared as required using 80% v/v acetone in olive oil on Days 1, 2 and 3. The formulations were stored at room temperature, in sealed, air-tight containers prior to dosing and were used within two hours of preparation.
- Test material administration: each mouse was manually restrained with both auditory pinnae left free. The outer aspect of both pinnae of each mouse was treated by direct application of the appropriate test or control formulation (0.025 mL/pinna)
- Treatment regimen: applications of the vehicle or one of the test formulations were made to the outer aspect of the auditory pinnae, once daily on Days 1, 2 and 3. On Day 6 the mice were placed in a warming cabinet in order to dilate the peripheral blood vasculature and thus facilitate intravenous dosing. Each mouse was transferred to a cylindrical restrainer. A plastic syringe and fine gauge hypodermic needle were used to administer 0.25 mL phosphate buffered saline incorporating 20 μCi of 3HTdR into a tail vein of each mouse by slow bolus injection. After this treatment the mice were returned to their cages. Approximately five hours after intravenous injection of the 3HTdR, all mice were sacrificed by exposure to a rising concentration of carbon dioxide.
- Preparation for scintillation count: following sacrifice, the auricular lymph nodes were recovered from each animal. The nodes from mice subjected to the same treatment were pooled and suspensions of the cellular components of the lymph nodes were prepared in 5% w/v trichloroacetic acid and processed through a scintillation counter.
- Scintillation counting: all samples were submitted for scintillation counting for 10 minutes, using a 3H quench curve. All counts were corrected for background.
- Experimental observations: animals were observed twice daily for clinical signs of reaction to treatment, or other changes at the sites of application of the test material, on Days 1, 2 and 3 and once daily on Day 6. Body weights were recorded on Day 1 and on Day 6 prior to intravenous administration of 3HTdR

DATA EVALUATION
The disintegrations per minute value was transformed into a disintegrations per minute per lymph node (DLM) value by dividing by the number of sites yielding lymph nodes. The DLM value for each test group was divided by the DLM of the control group to yield the Stimulation Index (SI) for each test group. Test materials are regarded as a skin sensitiser when the maximum value of the SI is 3.0 or above.
Parameter:
SI
Remarks on result:
other: The stimulation indices were 0.8, 0.8 and 1.3 at the 25, 50 and 100 % v/v dose levels, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See table 1.

Preliminary Screening Test

No death or signs of systemic toxicity/excessive irritation were observed.

Main Test

- Mortality: all animals survived treatment

- Clinical signs: there were no clinical signs indicative of a systemic effect and the application sites remained free of irritation. Greasy fur to the back of head and neck was noted in all animals throughout the observation period.

- Bodyweights: there was no indication of a treatment related effect on body weight.

Table 1: Scintillation counts and stimulation indices

Sample identity

Number of sites yielding lymph nodes

Disintegrations per minute* (DPM)

Disintegrations per minute per node (DLM)

Stimulation Index (SI)

Scintillation fluid with

5% w/v trichloroacetic acid

--

43

--

--

Vehicle control

8

1498

187

--

Test material, 25% v/v

8

1183

148

0.8

Test material, 50% v/v

8

1206

151

0.8

Test material, 100% v/v

8

1903

238

1.3

* All scintillation counts corrected for the blank

SI =        Test group DLM value     

               Control group DLM value

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the study the test material was found not to be a skin sensitiser.
Executive summary:

The potential of the test material to cause skin sensitisation in the mouse was investigated in a GLP study which was conducted in accordance with standardised guidelines OECD 429 and EU Method B.42.

Following a preliminary screening test, the test material was applied undiluted and at concentrations of 25 and 50 % v/v in 80 % v/v acetone in olive oil. Groups of four female CBA / CaCrl mice were subjected to topical applications of vehicle or of one of the test formulations to the outer aspect of the auditory pinnae once daily on Days 1, 2 and 3. On Day 6 a 20 μCi dose of tritiated ³H-methyl thymidine was injected intravenously into each mouse. Approximately five hours later the auricular lymph nodes were recovered from each animal. The nodes from mice subjected to the same treatment were pooled and suspensions of the cellular components of the lymph nodes were prepared in 5 % w/v trichloroacetic acid and processed through a scintillation counter.

Test results are expressed in terms of Stimulation Indices, the ratios of the mean scintillation count per group obtained from the test groups relative to the corresponding mean scintillation count from controls. The threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation is 3.0. The stimulation indices for the test material were determined to be 0.8, 0.8 and 1.3 at the 25, 50 and 100 % v/v dose levels, respectively. Therefore, under the conditions of the study the test material was found not to be a skin sensitiser.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The potential of the test material to cause skin sensitisation in the mouse was investigated in a GLP study which was conducted in accordance with standardised guidelines OECD 429 and EU Method B.42.

Following a preliminary screening test, the test material was applied undiluted and at concentrations of 25 and 50% v/v in 80% v/v acetone in olive oil. Groups of four female CBA / CaCrl mice were subjected to topical applications of vehicle or of one of the test formulations to the outer aspect of the auditory pinnae once daily on Days 1, 2 and 3. On Day 6 a 20 μCi dose of tritiated3H-methyl thymidine was injected intravenously into each mouse. Approximately five hours later the auricular lymph nodes were recovered from each animal. The nodes from mice subjected to the same treatment were pooled and suspensions of the cellular components of the lymph nodes were prepared in 5% w/v trichloroacetic acid and processed through a scintillation counter.

Test results are expressed in terms of Stimulation Indices, the ratios of the mean scintillation count per group obtained from the test groups relative to the corresponding mean scintillation count from controls. The threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation is 3.0. The stimulation indices for the test material were determined to be 0.8, 0.8 and 1.3 at the 25, 50 and 100 % v/v dose levels, respectively. Therefore, under the conditions of the study the test material was found not to be a skin sensitiser.


Migrated from Short description of key information:
Not sensitising, OECD 429, EU Method B.42, Dreher 2013d

Justification for selection of skin sensitisation endpoint:
Only one study is available.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with with criteria for classification as defined in Annex I, Regulation 1272/2008, the test material did not elicit a response in the local lymph node assay and therefore does not meet the criteria for classification as a skin sensitiser.