Registration Dossier

Administrative data

Description of key information

There was no evidence of induction of a lymphocyte proliferative response indicative of skin sensitisation to the test substance.

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:
10 May 2007 to 11 January 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Deviations:
no
GLP compliance:
no
Remarks:
The study was not commissioned with compliance with REACH as a goal, rather the study was commissioned during early product development.
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot No. 08230AB
- Purity: 99.8%
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: The Jackson Laboratory, Bar Harbor, ME.
- Females nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known: Animals were examined to ensure good health and suitability as test subjects for use in the study.
- Age at study initiation: appromixately 7-8 weeks
- Weight at study initiation: 17-21g
- Housing: housed individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week
- Indication of any skin lesions: Animals were examined to ensure good health and suitability as test subjects for use in the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25°C
- Humidity (%): 37-61%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 / 12

- IN-LIFE DATES: From: 07 June 2007 To: 11 June 2007
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
11.3, 22.5, 33.8 and 45% in an acetone:olive oil (4:1) vehicle.
Highest concentration tested was 45% which was considered as the solubility limit in an acetone:olive oil 4:1 mixture.
No. of animals per dose:
5 females/dose
Details on study design:
Five mice per group were dosed with the test substance at concentrations of 11.3, 22.5, 33.8 and 45% in an acetone:olive oil (4:1) vehicle. The mice were dosed once daily at approximately the same time for three consecutive days. All mice were observed twice daily on dosing days immediately prior to and approximately 4-6 hours after dosing, then once daily for moribundity, mortality, and any signs of toxicity and skin reactions. Approximately 2 days after the last dose, mice were injected with the radio-label ([3H]-thymidine) and approximately 5 hours later, the auricular nodes were excised and single-cell suspensions were prepared. The cells were washed with phosphate-buffered saline to remove unbound radiolabel and then precipitated with 5% trichloroacetic acid. The total radiolabel incorporation in these precipitates was subsequently quantitated by liquid scintillation spectrometry. A single animal was dosed with less than the required volume of [3H]-thymidine (50% dose group) and this animal was not included in the analysis.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
other: 1-chloro-2,4-dinitrobenzene (DNCB)
Statistics:
The observed values of disintegrations per minutes (DPM) of acid-precipitated auricular lymph node cells were analysed using analysis of variance (ANOVA). Statistical analyses were performed using SigmaStat for windows software.
Positive control results:
2.5 µg/mL of DNCB: Proliferative response - 13024.53 DPM/lymph node; Stimulation index – 19.39
42.5% of HCA: Proliferative response - 4260.59 DPM/lymph node; Stimulation index – 6.34
Parameter:
SI
Value:
1
Test group / Remarks:
vehicle control
Parameter:
SI
Value:
1.66
Test group / Remarks:
11.3%
Parameter:
SI
Value:
1.66
Test group / Remarks:
22.5%
Parameter:
SI
Value:
1.31
Test group / Remarks:
33.8%
Key result
Parameter:
SI
Value:
1.46
Test group / Remarks:
45%
Cellular proliferation data / Observations:
Evidence of induction of T-cell proliferation was not observed with the test substance, as the stimulation index was less than three at each of the test concentrations

There were no mortalities and no signs of systemic toxicity or irritation noted for the test and control animals.
Interpretation of results:
GHS criteria not met
Conclusions:
There was no evidence of induction of a lymphocyte proliferative response indicative of skin sensitisation to the test substance.
Executive summary:

Five mice per group were dosed with the test substance at concentrations of 11.3, 22.5, 33.8 and 45% in an acetone:olive oil (4:1) vehicle. The mice were dosed once daily at approximately the same time for three consecutive days. All mice were observed twice daily on dosing days immediately prior to and approximately 4-6 hours after dosing, then once daily for moribundity, mortality, and any signs of toxicity and skin reactions. Approximately 2 days after the last dose, mice were injected with the radio-label ([3H]-thymidine) and approximately 5 hours later, the auricular nodes were excised and single-cell suspensions were prepared. The cells were washed with phosphate-buffered saline to remove unbound radiolabel and then precipitated with 5% trichloroacetic acid. The total radiolabel incorporation in these precipitates was subsequently quantitated by liquid scintillation spectrometry. 

 

There were no mortalities and no signs of systemic toxicity or irritation noted for the test and control animals. Evidence of induction of T-cell proliferation was not observed with the test substance, as the stimulation index was less than three at each of the test concentrations.

The positive control groups had significantly increased DPM values and a mean stimulation index >3.

There was no evidence of induction of a lymphocyte proliferative response indicative of skin sensitisation to the test substance.

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

Five mice per group were dosed with the test substance at concentrations of 11.3, 22.5, 33.8 and 45% in an acetone:olive oil (4:1) vehicle. The mice were dosed once daily at approximately the same time for three consecutive days. All mice were observed twice daily on dosing days immediately prior to and approximately 4-6 hours after dosing, then once daily for moribundity, mortality, and any signs of toxicity and skin reactions. Approximately 2 days after the last dose, mice were injected with the radio-label ([3H]-thymidine) and approximately 5 hours later, the auricular nodes were excised and single-cell suspensions were prepared. The cells were washed with phosphate-buffered saline to remove unbound radiolabel and then precipitated with 5% trichloroacetic acid. The total radiolabel incorporation in these precipitates was subsequently quantitated by liquid scintillation spectrometry. 

 

There were no mortalities and no signs of systemic toxicity or irritation noted for the test and control animals. Evidence of induction of T-cell proliferation was not observed with the test substance, as the stimulation index was less than three at each of the test concentrations.

 

The positive control groups had significantly increased DPM values and a mean stimulation index >3.

 

There was no evidence of induction of a lymphocyte proliferative response indicative of skin sensitisation to the test substance.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

There was no evidence of induction of a lymphocyte proliferative response indicative of skin sensitisation to the test substance.  OFPMA does not fulfil the criteria for skin sensitisation classification according to European CLP Regulation (EC) No 1272/2008 (as amended).