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Description of key information

In the key GLP compliant Guinea Pig Maximization Test according to OECD guideline 406, no evidence of delayed contact hypersensitivity was seen after treatment with the test item under the experimental conditions.


 


In a supporting GLP compliant study according to OECD guideline 406 (pre-guideline to OECD 429), test item was not a skin sensitizer at concentrations up to 30% and is thus designated as unlikely to be a moderate or strong skin sensitiser under the conditions of the test.


 


Conclusion: The test item was not a sensitiser in two different in vivo tests.

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:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992 (pre-guideline to OECD 429)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Young adults
- Housing: A maximum of 4 mice was housed per cage, in multiple mouse racks suitable for animals of this strain and weight range.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: For at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2
- Humidity (%): 55±15
- Air changes (per hr): At least 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From 13 November 1996 to 18 November 1996
Vehicle:
other: acetone
Concentration:
250 µL of a 1, 10 or 30% w/v test item solution
No. of animals per dose:
4 animals per dose
Details on study design:
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The criterion for a positive response is that one or more concentrations of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group. The assay is able to identify those materials that elicit moderate or greater responses in standard guinea pig tests for skin sensitisation.


TREATMENT PREPARATION AND ADMINISTRATION: The substance solutions were applied to the dorsal surface of each ear. A vehicle control group was similarly treated using acetone alone. The procedure was repeated daily for 3 consecutive days. Three days alter the third application, all the animals were injected, via the tau vein, with approximately 250 µL of phosphate buffered saline (PBS) containing approximately 20 µCi of a 2.0 Ci/mmol specific activity 3H-methyl thymidine. Approximately 5 hours later, the animals were humanely killed by inhalation of halothane vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS. A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were placed in round-bottomed centrifuge tubes and were washed three times by centrifugation with approximately 10 mL of PBS. The supematant was drawn off and discarded and approximately 3 mL of 5% w/v trichloroacetic acid (TCA) was added. After overnight precipitation at 4°C the samples were pelleted by centrifugation and the supernatant was again discarded. The cells were then resuspended in approximately 1 mL of TCA. Two days alter removal of lymph nodes, the suspensions were transferred to scintillation vials and 10 mL of scintillant (Optiphase) was added. The contents of each vial were thoroughly mixed prior to ß-scintillation counting using a Packard Tri-Carb 2500TR Liquid Scintillation Counter. Due to a high level of luminescence in one sample of the batch of studies analysed, all the samples were counted again one day later. The results from this second count were acceptable and are reported.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
Hexylcinnamaldehyde was shown to have the capacity to cause skin sensitisation at concentrations of 3 and 10% w/v in acetone.
Key result
Parameter:
SI
Value:
1.87
Test group / Remarks:
30 % (w/v)
Key result
Parameter:
SI
Value:
1.57
Test group / Remarks:
10 % (w/v)
Key result
Parameter:
SI
Value:
0.67
Test group / Remarks:
1 % (w/v)
Interpretation of results:
GHS criteria not met
Conclusions:
The test item was not a skin sensitizer at concentrations up to 30% and is thus designated as unlikely to be a moderate or strong skin sensitiser under the conditions of the test.
Executive summary:

In a GLP compliant study according to OECD guideline 406 (pre-guideline to OECD guideline 429), a sample of the test item was assessed for its skin sensitisation potential using the mouse Local Lymph Node Assay. The assay determines the level of T lymphocyte proliferation in the lymph nodes draining the site of chemical application, by measuring the amount of radiolabelled thymidine incorporated into the dividing cells. Groups of four male mice were used for this study. Approximately 25 µL of a 1, 10 or 30% w/v preparation of the test substance in acetone was applied to the dorsal surface of each ear. A vehicle control group was similarly treated using acetone alone. The procedure was repeated daily for 3 consecutive days.Three days after the third application, all the animals were injected, via the tail vein, with approximately 250 µL of phosphate buffered saline (PBS) containing approximately 20 µCi of a 2.0 Ci/mmol specific activity 3H-methyl thymidine. Approximately 5 hours later, the animals were humanely killed by inhalation of halothane vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and pooled with the nodes from the other animals in the group. A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were washed three times by centrifugation with approximately 10 mL of PBS. Approximately 3 mL of 5% w/v trichloroacetic acid (TCA) was added. After overnight precipitation at 4°C, the samples were pelleted by centrifugation and the supernatant was again discarded. The cells were then resuspended in approximately 1 mL of TCA. Two days after removal of lymph nodes, the suspensions were transferred to scintillation vials, 10 mL of scintillant (Optiphase) was added and ß-scintillation was counted. In a positive control study, hexylcinnamaldehyde was shown to have the capacity to cause skin sensitisation at concentrations of 3 and 10% w/v in acetone, confirming the validity of the protocol used for this study.The test substance induced SI values of 0.67 (1% w/v), 1.57 (10% w/v) and 1.87 (30% w/v) and was thus considered to be not sensitizing in the concentrations tested. It was thus concluded that the test item does not have the capacity to cause moderate or strong skin sensitisation.

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

Skin sensitisation in vivo, RL1


The dermal sensitising potential of the test item was investigated according to one of the methods recommended in the OECD Guidelines No. 406, "Skin Sensitisation", 1992 and the EEC Guideline "EEC 92/69 part 6B", 1992. The delayed contact hypersensitivity test used was the Guinea Pig Maximization Test described by B. Magnusson and A. M. Kligman. Thirty animals divided into a test group of 20 animals and a negative control group of 10 animals were included in the study. The study comprised an induction and a challenge phase. The animals in the test group were induced with the test article whereas the animals in the control group were induced with Arachis oil for intradermal induction and Ethanol/diethylphthlate 1:1 for dermal induction. The induction procedure included intradermal injections and a closed patch topical application one week apart. All animals were challenged by a closed patch topical application of the test article for 24 hours. The challenge procedure included a closed patch topical treatment of the test article an the flank 3 weeks after the intradermal injection. The skin reactions were evaluated 24 and 48 hours after the challenge application. A 6% (w/w) test article concentration in Arachis oil was used for the intradermal induction. A 50% (w/w) test article concentration in Ethanol/diethylphthlate 1:1 was used for the topical induction and a 25% (w/w) test article concentration in Ethanol/diethylphthlate 1:1 was used for the challenge application. Under these experimental conditions no evidence of delayed contact hypersensitivity was seen after treatment with the test item.


 


Skin sensitisation in vivo, RL2


In a GLP compliant study according to OECD guideline 406 (pre-guideline to OECD guideline 429), a sample of the test item was assessed for its skin sensitisation potential using the mouse Local Lymph Node Assay. The assay determines the level of T lymphocyte proliferation in the lymph nodes draining the site of chemical application, by measuring the amount of radiolabelled thymidine incorporated into the dividing cells. Groups of four male mice were used for this study. Approximately 25 µL of a 1, 10 or 30% w/v preparation of the test substance in acetone was applied to the dorsal surface of each ear. A vehicle control group was similarly treated using acetone alone. The procedure was repeated daily for 3 consecutive days.Three days after the third application, all the animals were injected, via the tail vein, with approximately 250 µL of phosphate buffered saline (PBS) containing approximately 20 µCi of a 2.0 Ci/mmol specific activity 3H-methyl thymidine. Approximately 5 hours later, the animals were humanely killed by inhalation of halothane vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and pooled with the nodes from the other animals in the group. A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were washed three times by centrifugation with approximately 10 mL of PBS. Approximately 3 mL of 5% w/v trichloroacetic acid (TCA) was added. After overnight precipitation at 4°C, the samples were pelleted by centrifugation and the supernatant was again discarded. The cells were then resuspended in approximately 1 mL of TCA. Two days after removal of lymph nodes, the suspensions were transferred to scintillation vials, 10 mL of scintillant (Optiphase) was added and ß-scintillation was counted. In a positive control study, hexylcinnamaldehyde was shown to have the capacity to cause skin sensitisation at concentrations of 3 and 10% w/v in acetone, confirming the validity of the protocol used for this study.The test substance induced SI values of 0.67 (1% w/v), 1.57 (10% w/v) and 1.87 (30% w/v) and was thus considered to be not sensitizing in the concentrations tested. It was thus concluded that the test item does not have the capacity to cause moderate or strong skin sensitisation.


 


 


Conclusion: The test item was not a sensitiser in two different in vivo tests.

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 test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered to be not classified for skin sensitisation under Regulation (EC) No 1272/2008, as amended for fifteenth time in Regulation (EU) No 2020/1182.