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Description of key information

LLNA, skin sensitizer Category 1B (Read-across, OECD 429, GLP, K, Rel. 2)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 06 to 28, 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 429 without some restrictions: pooled method was used instead of individual method; and ear thickness measurements were not included. However, these deviations did not affect the outcome of this study since the observed positive evidence of skin sensitisation is coherent with the composition of the UVCB tested.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 24 April 2002
Deviations:
yes
Remarks:
pooled method instead of individual method; no ear thickness measurements
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
national GLP Compliance Programme (inspected on September 02, 2006/ signed on January 19, 2007)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 17.5-22 g
- Housing: single housing. Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen). Granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen)
- Diet: pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen)
- Water: tap water, ad libitum, (Gemeindewerke, D-64380 Rossdorf)
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature:122 +/- 3°C
- Humidity: 30-70 %
- Air changes: not reported
- Photoperiod: 12 hours continuous light and 12 hours darkness

- IN-LIFE DATES: not reported
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10%, 25% or 50% w/w
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: up to 50% in acetone/olive oil (4:1 v/v)
- Irritation & systemic toxicity: To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 10, 25, 50, and 100 % on one ear each on three consecutive days. Clinical signs were recorded 24 ± 4 hours after each application. The animal treated with 50 and 100% of the test item died after the second application of the test item. Therefore, a second experiment was performed using test item concentrations of 25% on both ears of one animal and 50 % on both ears of the second animal. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.
- Ear thickness measurements: not included
- Erythema scores: not reported
The test item in the main study was assayed at 10, 25, and 50%. 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
- Name of test method: Local Lymph Node Assay - pooled method
- Criteria used to consider a positive response: A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into a volumetric flask on a tared balance and acetone:olive oil (4+1) was quantitatively added.
The preparations were made freshly before each dosing occasion.
Concentrations were in terms of material as supplied.
- 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 10, 25,and 50% (w/v) in acetone:olive oil (4+1). The application volume, 25 Ql, was spread over the entire dorsal surface (diameter 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).
- Administration of 3H-Methyl Thymidine:
3H-methyl thymidine (3HTdR) was purchased from GE Healthcare (GE Healthcare 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 Ql of 81.0 QCi/ml 3HTdR (corresponds to 20.25 QCi 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 Qm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 1 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 1ml-aliquots of 5 % trichloroacetic acid. The beta-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.
A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
Positive control results:
The positive control α Hexylcinnamaldehyde gave a Stimulation Index of greater than 3 (4.87) when tested at a concentration of 25% w/v in acetone/olive oil 4:1 and an EC-3 of 15.7 % (w/v) , thus, demonstrating the sensitivity and reliability of the test system.
Key result
Parameter:
SI
Value:
1.62
Test group / Remarks:
10% w/v in acetone/olive oil 4:1
Key result
Parameter:
SI
Value:
3.3
Test group / Remarks:
25% w/v in acetone/olive oil 4:1
Key result
Parameter:
SI
Value:
4.77
Test group / Remarks:
50% w/v in acetone/olive oil 4:1
Key result
Parameter:
EC3
Value:
22.3
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index for 10, 25 and 50% w/v in acetone/olive oil 4:1 were 1.62, 3.30 and 4.77, respectively.

EC3 CALCULATION
EC3 = (a-c) [(3-d)/(b-d)] + c = 22.3% (w/v)
a = 10
b = 1.62
c = 25
d = 3.30

VIABILITY/MORTALITY/ No deaths occurred 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 main experiment.

BODY WEIGHTS: The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Table 7.4.1/1: Grouped Disintegrations per Minute and Stimulation Index

Test item

concentration

% (w/v)

Group

Measurement

DPM

Calculation

Result

DPM-BGa)

number of

lymph nodes

DPM per

lymph nodeb)

S.I.

---

BG I

27

---

---

---

---

---

BG II

27

---

---

---

---

---

1

6405

6378

8

797.3

 

10

2

10364

10337

8

1292.1

1.62

25

3

21102

21075

8

2634.4

3.30

50

4

30476

30449

8

3806.1

4.77

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

1 = Control Group

2-4 = Test Group

S.I. = Stimulation Index

a) = The mean value was taken from the figures BG I and BG II

b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled


Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the test conditions, test material is classified as a contact sensitizer (Category 1B) according to the Regulation (EC) No. 1272/2008 and to the GHS.
Executive summary:

A study was performed to assess the skin sensitisation potential of test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP. 

In the preliminary screening test, two mice were treated with concentrations of 10, 25, 50, and 100 % w/v on one ear each on three consecutive days. Clinical signs were recorded 24 ± 4 hours after each application. The animal treated with 50 and 100% w/vof the test item died after the second application of the test item. Therefore, a second experiment was performed using test item concentrations of 25% w/v on both ears of one animal and 50 % w/v on both ears of the second animal. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity, therefore, the concentration of 50% w/v was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 10%, 25% or 50% w/v. A further group of five animals was treated with acetone/olive oil 4:1 alone. The results of a positive control test, using a group of four animals, performed with the known sensitizer, α‑Hexylcinnamaldehyde, at concentrations of 5%, 10% and 25% w/v in acetone/olive oil 4:1 is reported. The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of 3H-methyl Thymidine (3HTdR) by β-scintillation counting of LNC suspensions. The response was expressed as radioactive disintegrations per minute per pooled lymph node and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio), termed as Stimulation Index (SI).

Stimulation index for 10%, 25% and 50% w/v in acetone/olive oil 4:1 were 1.62, 3.30 and 4.77, respectively. The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (extrapolated EC3 value) was calculated to be 22.3 % (w/v). No sign of systemic toxicity or excessive local skin irritation were noted at the concentrations tested.

The positive control α Hexylcinnamaldehyde gave a Stimulation Index of greater than 3 (4.87) when tested at a concentration of 25% w/v in acetone/olive oil 4:1 and an EC-3 of 15.7 % (w/v) , thus, demonstrating the sensitivity and reliability of the test system.

 

Under the test conditions, test material is classified as a contact sensitizer (Category 1B) according to the Regulation (EC) No. 1272/2008 and to the GHS.

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Further information is included as attachment to Iuclid section 13]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar toxicological properties because of their structural and composition similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and source substances are both UVCB substances.

3. ANALOGUE APPROACH JUSTIFICATION
The source substance is expected to have similar toxicological profile than the target substance considering the composition similarity between the two substances. Indeed, as can be seen in Table 5 the constituents classified as Skin Sensitizer Category 1B are present in both the source and the target substance. Those constituents being present at concentrations greatly above the CLP generic concentration limits triggering classification of a mixture, the results generated on the target substance were considered doubtful and were disregarded based on several methodological deficiencies.
The design of the study performed on the source substance (OECD 429, GLP) is adequate and reliable for the purpose of the prediction based on read-across. The test material used represents the source substance as described in the hypothesis in terms of composition. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the results of the in vivo skin sensitisation study conducted with the source substance (skin sensitizer) is likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VII, 8.3.2.

4. DATA MATRIX
Cf. Iuclid section 13
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Positive control results:
The positive control α Hexylcinnamaldehyde gave a Stimulation Index of greater than 3 (4.87) when tested at a concentration of 25% w/v in acetone/olive oil 4:1 and an EC-3 of 15.7 % (w/v) , thus, demonstrating the sensitivity and reliability of the test system.
Key result
Parameter:
SI
Value:
1.62
Test group / Remarks:
10% w/v in acetone/olive oil 4:1
Key result
Parameter:
SI
Value:
3.3
Test group / Remarks:
25% w/v in acetone/olive oil 4:1
Key result
Parameter:
SI
Value:
4.77
Test group / Remarks:
50% w/v in acetone/olive oil 4:1
Key result
Parameter:
EC3
Value:
22.3
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index for 10, 25 and 50% w/v in acetone/olive oil 4:1 were 1.62, 3.30 and 4.77, respectively.

EC3 CALCULATION
EC3 = (a-c) [(3-d)/(b-d)] + c = 22.3% (w/v)
a = 10
b = 1.62
c = 25
d = 3.30

VIABILITY/MORTALITY/ No deaths occurred 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 main experiment.

BODY WEIGHTS: The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Table 7.4.1/1: Grouped Disintegrations per Minute and Stimulation Index

Test item

concentration

% (w/v)

Group

Measurement

DPM

Calculation

Result

DPM-BGa)

number of

lymph nodes

DPM per

lymph nodeb)

S.I.

---

BG I

27

---

---

---

---

---

BG II

27

---

---

---

---

---

1

6405

6378

8

797.3

 

10

2

10364

10337

8

1292.1

1.62

25

3

21102

21075

8

2634.4

3.30

50

4

30476

30449

8

3806.1

4.77

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

1 = Control Group

2-4 = Test Group

S.I. = Stimulation Index

a) = The mean value was taken from the figures BG I and BG II

b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled


Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Based on the study results on the source substance, the target substanc is classified as a contact sensitizer (Category 1B) according to the Regulation (EC) No. 1272/2008 and to the GHS.
Executive summary:

A study was performed to assess the skin sensitisation potential of the source substance material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP. 

In the preliminary screening test, two mice were treated with concentrations of 10, 25, 50, and 100 % w/v on one ear each on three consecutive days. Clinical signs were recorded 24 ± 4 hours after each application. The animal treated with 50 and 100% w/vof the test item died after the second application of the test item. Therefore, a second experiment was performed using test item concentrations of 25% w/v on both ears of one animal and 50 % w/v on both ears of the second animal. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity, therefore, the concentration of 50% w/v was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 10%, 25% or 50% w/v. A further group of five animals was treated with acetone/olive oil 4:1 alone. The results of a positive control test, using a group of four animals, performed with the known sensitizer, α‑Hexylcinnamaldehyde, at concentrations of 5%, 10% and 25% w/v in acetone/olive oil 4:1 is reported. The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of 3H-methyl Thymidine (3HTdR) by β-scintillation counting of LNC suspensions. The response was expressed as radioactive disintegrations per minute per pooled lymph node and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio), termed as Stimulation Index (SI).

Stimulation index for 10%, 25% and 50% w/v in acetone/olive oil 4:1 were 1.62, 3.30 and 4.77, respectively. The concentration of the source substance expected to cause a 3 fold increase in 3HTdR incorporation (extrapolated EC3 value) was calculated to be 22.3 % (w/v). No sign of systemic toxicity or excessive local skin irritation were noted at the concentrations tested.

The positive control α Hexylcinnamaldehyde gave a Stimulation Index of greater than 3 (4.87) when tested at a concentration of 25% w/v in acetone/olive oil 4:1 and an EC-3 of 15.7 % (w/v) , thus, demonstrating the sensitivity and reliability of the test system.

 

Based on the study results on the source substance, the target substance is classified as a contact sensitizer (Category 1B) according to the Regulation (EC) No. 1272/2008 and to the GHS.

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A study was identified on the target substance (Evik, 2001, rel.3). This Magnusson & Kligman maximisation study (GPMT) was performed according to OECD Guideline 406 and in compliance with GLP. Under the test conditions, test substance is not a skin sensitiser. However, the dose selection for topical application (induction and challenge) is not adequate: the maximum concentration was 6% although the substance is not a skin irritant, and no sign of irritation were observed in the pilot assay at this concentration (maximum concentration tested). Therefore the concentrations should have been higher. This deviation has an impact on the reliability of the results and the conclusion cannot be used for classification purpose.

The non-adequacy of the study results is supported by the composision of the target substance. With more than 14% (as a minimum) of its constituents already classified as Skin sensitiser category 1B, the target substance should be classified as a skin sensitiser based on the CLP generic classification limits of ingredients of a mixture classified as skin sensitisers triggering classification of a mixture.

Therefore, a read-across approach was used to cover this endpoint. Based on composition similarity between the source and the target substances (see Iuclid section 13 for read-across justification), it was considered appropriate to use the available data on the source substance for the target substance.

A key study was identified on the source substance (RCC, 2008, rel.2). This LLNA was conducted according to the OECD test guideline No 429 and in compliance with GLP. Stimulation index for 10%, 25% and 50% w/v in acetone/olive oil 4:1 were 1.62, 3.30 and 4.77, respectively. The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (extrapolated EC3 value) was calculated to be 22.3 % (w/v). No sign of systemic toxicity or excessive local skin irritation were noted at the concentrations tested. The positive control α Hexylcinnamaldehyde gave a Stimulation Index of greater than 3 (4.87) when tested at a concentration of 25% w/v in acetone/olive oil 4:1 and an EC-3 of 15.7 % (w/v) , thus, demonstrating the sensitivity and reliability of the test system. Under the test conditions, test material is a skin sensitizer. In this study, some deviations from the OECD Guideline No. 429 can be noted: pooled method was used instead of individual method; and ear thickness measurements were not included. However, these deviations did not affect the outcome of this study since the observed positive evidence of skin sensitisation is coherent with the composition of the UVCB tested (constituents classified as skin sensitizer Category 1B and present at concentration above the CLP classification treshold).

Based on the available data, the target substance is considered to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available data, the substance is classified as Skin Sens. 1B, H317 (May cause an allergic skin reaction) according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS, since EC3 is > 2% (22.3%).