CGL 210

Endpoint summary

Description of key information

The allergenic potential of the test item was evaluated in three local lymph node assays (LLNA) and a maximization test. All studies were GLP-conform and performed according OECD guideline 429 and 406. In the course of the LLNAs, mice were exposed to the test substance at concentrations of 5-50% dissolved in acetone:olice oil. None of the three tests revealed a positive reaction to the test article. The maximization test was performed according to guidelines but with a batch of unknown purity. Positive reactions to the test article on skin were determined in this assay.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harian Netheriands B.V. Postbus6174 NL - 5960 AD Horst / The Netheriands
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 19-24g
- Housing: In groups of four in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet (e.g. ad libitum): Pelleted standard Kliba 3433, batch no. 84/02 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad libitum. Results of analyses for contaminants are archived at RCC.
- Water (e.g. ad libitum): Community tap water from Itingen, available ad libitum. Results of representative bacteriological, chemical and contaminant analyses are archived at RCC.
- Acclimation period: 04-JUN-2003 to 10-JUN-2003

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES:
Experimental Starting Date 04-JUN-2003
Experimental Completion Date 18-JUN-2003
Delivery of Animals 04-JUN-2003
Acclimatization 04-JUN-2003 to 10-JUN-2003
Treatment (epicutaneous) 11 -JUN-2003 to 13-JUN-2003
Treatment (intravenous) 16-JUN-2003
Observation 04-JUN-2003 to 16-JUN-2003
Termination 18-JUN-2003

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

5%, 10% and 25%

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Irritation: In a non-GLP conform pre-test in two mice, test item concentrations of 5 %, 10 %, 25 % and 50 % (w/w) were tested on one ear each. No irritation effects were observed at concentrations of 5 %, 10 % and 25 % after a single application.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a p-scintillation counter.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into a glass beaker on a tared Mettler balance and the vehicle (acetone:olive oil, 4:1 (v/v)) was quantitatively added. The weight/weight dilutions were prepared individually using a magnetic stirrer as homogenizer. 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. To determine the highest non-irritant and technically applicable test item concentration, a non-GLP pretest was performed in two mice with concentrations of 5 %, 10 %, 25 % and 50 % (w/w) (pretest excluded from Statement of Compliance). The test item in the main study was assayed at three consecutive concentrations. 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. Concentrations were in terms of material as supplied.

TREATMENT PROCEDURES
TOPICAL APPLICATION
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 5 %, 10 % and 25 %(w/w) in acetone:olive oil, 4:1 (v/v). The application volume, 25 pi, was spread over the entire dorsal surface ( 0 - 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). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.

ADMINISTRATION OF 3H-METHYL THYMIDINE*
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 pi of 79.5 pCi/ml 3HTdR (equal to 19.9 pCi 3HTdR) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED 3HTDR*
Approximately five hours after treatment with 3HTdR all mice were euthanized by intraperitoneal injection of VETANARCOL (Veterinaria AG, Zurich). The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 pm 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 P-scintillation counter. Similariy, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The p-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean values and standard deviations were calculated in the body weight tables.

Positive control results:

The test item ALPHA-HEXYLCINNAMALDEHYDE was found to be a sensitizer and an EC3 value of 7.08 % (w/v) was derived.

Parameter:

other: disintegrations per minute (DPM)

Value:

0

Test group / Remarks:

background I (1 ml 5 % trichloroacetic acid)

Parameter:

other: disintegrations per minute (DPM)

Value:

1

Test group / Remarks:

background II (1 ml 5 % trichloroacetic acid)

Parameter:

other: disintegrations per minute (DPM)

Value:

5 191

Test group / Remarks:

control group

Parameter:

other: disintegrations per minute (DPM)

Value:

5 848

Test group / Remarks:

test group (5%)

Parameter:

other: disintegrations per minute (DPM)

Value:

4 202

Test group / Remarks:

test group (10%)

Parameter:

other: disintegrations per minute (DPM)

Value:

10 298

Test group / Remarks:

test group (25%)

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

test group (5%)

Key result

Parameter:

SI

Value:

0.8

Test group / Remarks:

test group (10%)

Key result

Parameter:

SI

Value:

2

Test group / Remarks:

test group (25%)

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION: A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the STIMULATION INDEX. In this study STIMULATION INDICES (S.I.) of 1.1, 0.8 and 2.0 were determined with the test item at concentrations of 5 %, 10 % and 25 % (w/w) in acetone:olive oil, 4:1 (v/v). No dose-response relation was observed.

EC3 CALCULATION: Calculation of the EC 3 value was not done because no test concentrations produced a S.I. of 3 or higher.

CLINICAL OBSERVATIONS: No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. No deaths occurred during the study period.

BODY WEIGHTS: The body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was found to be a non-sensitizer when tested at up to concentration of 25 % (w/w) in acetone:olive oil, 4:1 (v/v).

Executive summary:

In order to study a possible contact allergenic potential of the test item, three groups each of four female mice were treated daily with the test item at concentrations of 5%, 10% and 25% (w/w) 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. A control group of four mice was treated with the vehicle (acetone:olive oil, 4:1 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a p-scintillation counter.

No test item-related clinical signs were observed. All treated animals survived the scheduled study period. A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the STIMULATION INDEX (S.I.).

 

	Test item concentration % (w/w)	S.I.
Group 2	5	1.1
Group 3	10	0.8
Group 4	25	2.0
No dose-response relation was observed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Performance and Observation

To evaluate the allergenic potential of the test substance, different studies according OECD guideline 429 and 406 were performed.

 

The first study was a lymph node assay, performed according to GLP and OECD guideline 429. Three groups each of four female mice were treated daily with the test item at concentrations of 5%, 10% and 25% (w/w) 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. A control group of four mice was treated with the vehicle only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a scintillation counter. No test item-related clinical signs were observed. All treated animals survived the scheduled study period. A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of radio-labelled thymidine compared with concurrent controls, as indicated by the STIMULATION INDEX (S.I.). S.I. after exposure to 5, 10 and 25% concentration of the test item was 1.1, 0.8 and 2.0, respectively.

 

The second and third study were also lymph node assays, performed according the same protocol as given for the first test. For the second study, the test animals were exposed to 5 %, 10 % and 15 % in acetone:olive oil, 4:1. In the third study, concentrations of 10 %, 25 % and 50 % in acetone:olive oil were used. Determined S.I. were 1.9, 1.2 and 0.9 and 1.4, 1.5 and 2.8, respectively.

 

The last study, a Maximization-Test was performed in 15 (10 test and 5 control) male albino guinea pigs, in accordance with OECD Guideline 406. The intradermal induction of sensitization in the test group was performed in the nuchal region with a 5 % dilution of the test item in PEG 300 and in an emulsion of Freund's Complete Adjuvant (FCA) / physiological saline. The epidermal induction of sensitization was conducted for 48 hours under occlusion with the undiluted test item one week after the intradermal induction. Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test item at 10% in PEG 300 and PEG 300 alone under occlusive dressing. Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing. Eight out of 10 test animals showed discrete to moderate erythema at the 24-hour reading. Seven out of 10 animals were observed with discrete erythema at the 48-hour reading. No skin effect was observed in the control group.

 

Discussion

Three local lymph node assays in mice were performed with different concentrations of the test item. None of the test revealed a positive reaction to the test substance. However, in Maximization assay a positive reaction of the test substance to skin was determined. In the study report of this assay, no purity of the test substance was given. Therefore, it is possible that impurities caused the allergic reaction. Additionally, the substance consists of different isomeres with different molecular weights. The major part of the test article (99.6 %) has a molecular weight between 744 and 1142 g/mol and is therefore not membrane permeable.

The local lymph node assay (LLNA) is considered as a reliable and modern test system to determine the allergic potential of a substance. Three independent LLNA of the test item showed negative results. Thus, the substance is considered to be a non-skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

The test item is a viscous liquid and therefore not inhalable. During formulation, the substance is not exposed to high temperatures whereby development of vapour can be excluded.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. The stimulation indices in the LLNA (OECD 429) did not show a dose dependent increase. As a result, the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.