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Administrative data

Description of key information

LLNA, skin sensitizer Category 1A (OECD 429, GLP, K, Rel. 1)

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 to 06 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 429 without any deviation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Programme (inspected on July 05, 2016/ signed on October 28, 2016)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: Food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK), ad libitum
- Water: Mains tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: 15 changes per hour
- Photoperiod: 12 hours continuous light and 12 hours darkness
- IN-LIFE DATES: 10 May to 06 September 2016
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10%, 5% or 2.5% w/w
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Test Item preparation: For the purpose of the study, the test item was freshly prepared, as required, as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
- Preliminary Screening Test: As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using three mice, one mouse per test item concentration. The mice were treated by daily application of 25 µL of the test item at concentrations of 50%, 25% or 10% w/w in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation and clinical signs of toxicity were recorded. The body weight of each mouse was recorded on Day 1 (prior to dosing) and on Day 6. The thickness of each ear was measured using a Mitutoyo 547 300S gauge (Mitutoyo Corporation), pre dose and 1 hour post dose on Day 1, 1 hour post dose on Days 2 and 3 and on Days 4 to 6. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization. Following the Day 6 ear thickness measurements, the mice were killed by carbon dioxide asphyxiation followed by cervical separation.
- Results: Brown colored staining on the ears was noted, post dose on Days 2 and 3, in the animal treated with the test item at a concentration of 50% w/w in acetone/olive oil 4:1 with brown colored residual test item noted on the ears on Days 4 and 5. No signs of systemic toxicity or visual local skin irritation were noted. No excessive irritation, indicated by an equal to or greater than 25% increase in mean ear thickness, was noted in the animal treated with the test item at a concentration of 10% w/w in acetone/olive oil 4:1. A greater than 25% increase in mean ear thickness was noted in the animal treated with the test item at a concentration of 25% w/w in acetone/olive oil 4:1. A mean value of less than 25% (24.4%) was noted in the animal treated with the test item at a concentration of 50%, but, given natural variability within the species, this was considered to be too close to the 25% cut off value and a possible indicator of excessive irritation.
Based on this information, and after consultation with the Sponsor, the test item at concentrations of 10%, 5% and 2.5% w/w in acetone/olive oil 4:1 were selected for the main test.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitizer".

TREATMENT PREPARATION AND ADMINISTRATION:
25 µL of control or test material was applied topically on the dorsal surface of both ears using a micropipette daily for three consecutive days (Days 1-3). Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse. Five hours later animals were killed by carbon dioxide asphyxiation followed by cervical separation. The lymph nodes from each group were pooled and a single cell suspension was prepared. Cells were washed with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) for 18 h at ca. 4 °C. The pellets were recovered by centrifugation and resuspended in 1 mL of TCA and transferred to a vial containing scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non parametric Kruskal Wallis Rank Sum and Mann Whitney U test procedures were used.
Positive control results:
The positive control α Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (12.22) when tested at a concentration of 25% v/v in acetone/olive oil 4:1, thus, demonstrating the sensitivity and reliability of the test system.
Key result
Parameter:
SI
Value:
9.12
Test group / Remarks:
2.5% w/w in acetone/olive oil 4:1
Key result
Parameter:
SI
Value:
12.08
Test group / Remarks:
5% w/w in acetone/olive oil 4:1
Key result
Parameter:
SI
Value:
12.16
Test group / Remarks:
10% w/w in acetone/olive oil 4:1
Key result
Parameter:
EC3
Value:
0.6
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index for 2.5, 5 and 10% w/w in acetone/olive oil 4:1 were 9.12, 12.08 and 12.16, respectively.

EC3 CALCULATION
EC3 = 2^ {log2(c) + (3-d)/(b-d) x [log2(a) - log2(c)]}
a = 5
b = 12.08
c = 2.5
d = 9.12
(a, b) and the point with the lower stimulation index was denoted (c, d).
EC3 = 2^ {log2(2.5) + (3-9.12)/(12.08-9.12) x [log2(5) - log2(2.5)]}= 0.60
The extrapolation method is ideally intended for use when the stimulation index for the lowest concentration utilized is approaching the cut-off value of 3, although there are no actual values recommended in available literature stating what values should be considered acceptable. However, we have previously utilized the extrapolation method in similar situations and the calculated value has been comparable to the experimentally obtained value on conducting additional work.
The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (extrapolated EC3 value) was calculated to be 0.60%.

CLINICAL OBSERVATIONS: There were no deaths. No signs of systemic toxicity or local skin irritation were noted in the test or control animals during the test.

BODY WEIGHTS: Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

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

Treatment Group

Animal Number

dpm/
Animal
a

Mean dpm/Animal
(Standard Deviation)

Stimulation Indexb

Result

Vehicle
acetone/olive oil 4:1

1-1

2024.05

2030.14 (±297.35)

na

na

1-2

2452.25

1-3

1786.59

1-4

2168.90

1-5

1718.89

Test Item
2.5w/win
acetone/olive oil 4:1

2-1

21812.20

18509.66** (±4229.97)

9.12

Positive

2-2

21499.56

2-3

11432.96

2-4

19719.48

2-5

18084.11

Test Item
5w/win
acetone/olive oil 4:1

3-1

25916.79

24524.64** (±4038.48)

12.08

Positive

3-2

19240.65

3-3

23025.72

3-4

24170.29

3-5

30269.75

Test Item
10w/win
acetone/olive oil 4:1

4-1

25703.94

24686.33** (±4871.65)

12.16

Positive

4-2

17390.84

4-3

23738.20

4-4

25685.36

4-5

30913.31

Positive Control Item
25% v/v in
acetone/olive oil 4:1

5-1

38934.00

24807.01** (±12202.13)

12.22

Positive

5-2

10588.53

5-3

17826.37

5-4

36092.09

5-5

25094.06


dpm = Disintegrations per minute

a = Total number of lymph nodes per animal is 2

b = Stimulation Index of 3.0 or greater indicates a positive result

** = Significantly different from control group p<0.01

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

Following a preliminary screening test in which no clinical signs of toxicity or excessive local irritation were noted at a concentration of10% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five 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%, 5% or 2.5% w/w. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, α‑Hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1. 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 lymph node (dpm/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 2.5, 5 and 10% w/w in acetone/olive oil 4:1 were 9.12, 12.08 and 12.16, respectively. The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (extrapolated EC3value) was calculated to be0.60%. No sign of systemic toxicity or excessive local skin irritation were noted at the concentrations of 10%, 5% or 2.5% w/w.

The positive control α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (12.22) when tested at a concentration of 25% v/v inacetone/olive oil 4:1, thus, demonstrating the sensitivity and reliability of the test system.

 

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

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

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

Since no key study was identified on the registered substance, the testing and assessment strategy, as described in ECHA R.7a Endpoint specific guidance (December 2016), was used to evaluate the skin sensitisation potential of the registered substance:

 

Element

Information

Conclusion

Comments

Existing data on physico-chemical properties

1

Is the substance a strong acid (pH≤ 2.0) or base (pH≥ 11.5), corrosive to the skin or (spontaneously) flammable in air or in contact with water or moisture at room temperature?

NO

 

Existing human data

2

Are there adequate existing human data, which provide evidence that the substance is a skin sensitiser?

NO

 

Existing animal data from sensitisation studies

3

Are there data from existing studies on skin sensitisation in laboratory animals (LLNA, GPMT, or Buehler test, EU B.42, B.50, B.51 and B.6/OECD TGs 429, 442A, 442B and 406), which provide sound conclusive evidence that the substance is a sensitiser, or non-sensitiser?

NO

 

Existing/new (Q)SAR data and read-across

4

Do “read-across” from structurally and mechanistically related substances and/or do suitable (Q)SAR predictions reliably indicate skin sensitisation potential or the absence thereof of the substance?

NO

Not applicable - UVCB substance

Existing in chemico and in vitro data

5a

Is there evidence/hypothesis of dermal bioavailability based on physico-chemical, in silico, in vitro or in vivo data?

NO

Not applicable - UVCB substance

5b

Has the substance demonstrated peptide/protein binding properties in an EU/OECD adopted in chemico test (e.g. OECD TG 442c)? (Key event 1 of the AOP), and/or
Has the substance demonstrated activation of the Nrf2-Keap1-ARE toxicity pathway in an EU/OECD adopted in vitro test (e.g. OECD TG 442d)? (Key event 2 of the AOP), and/or
Has the substance demonstrated induction of the cell surface markers (CD54 and/or CD86) on monocytic cells in a validated in vitro test, e.g. h-CLAT? (Key event 3 of the AOP).
Data from in chemico/in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by the EU and/or OECD may also be used if the provisions defined in Annex XI to the REACH Regulation are met.

NO

(at the initiation of the dossier, no test was available and the in vitro testing was not the REACH standard requirement)

5c

Are there data from (a) non-validated in vitro test(s), which provide evidence that the substance may be a skin sensitiser?

NO

 

Weight-of- Evidence analysis

6

The “elements” described above may be arranged as appropriate. Taking all existing and relevant data (elements 1-5) into account, is there sufficient information to meet the information requirement of Section 8.3 of Annex VII and to make a decision on whether classification and labelling are warranted?

NO

 

Generation of new non-animal data

7a

Does the substance demonstrate peptide/protein binding properties in an EU/OECD adopted in chemico test (e.g. B. 59/OECD TG 442c)? (Key event 1 of the AOP)
In chemico test methods that have been validated and are considered scientifically valid but are not yet adopted by the EU and/or OECD may also be used if the provisions defined in Annex XI to the REACH Regulation are met.

NO

(at the initiation of the dossier, no test was available and the in vitro testing was not the REACH standard requirement)

7b

Does the substance demonstrate activation of the Nrf2-Keap1-ARE toxicity pathway in an EU/OECD adopted in vitro test (e.g. B.60/OECD TG 442d)? (Key event 2 of the AOP)
In vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by the EU and/or OECD may also be used if the provisions defined in Annex XI to the REACH Regulation are met.

NO

(at the initiation of the dossier, no test was available and the in vitro testing was not the REACH standard requirement)

7c

Does the substance demonstrate induction of the cell surface markers (CD54 and/or CD86) of monocytic cells in a validated in vitro test (e.g. h-CLAT)? (Key event 3 of the AOP)
In vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by the EU and/or OECD may also be used if the provisions defined in Annex XI to the REACH Regulation are met.

NO

(at the initiation of the dossier, no test was available and the in vitro testing was not the REACH standard requirement)

7d

Is any additional testing/generation of data considered necessary in order to conclude on classification, or e.g. to explain the inconsistent data obtained in previous elements or to address the Key event 4 of the AOP (T-cell proliferation) with an in vitro test?

NO

(at the initiation of the dossier, no test was available and the in vitro testing was not the REACH standard requirement)

Weight-of-Evidence analysis

8

The “elements” described above may be arranged as appropriate. Taking all existing and relevant data (elements 1-7) into account, is there sufficient information to meet the respective information requirement of Section 8.3 of Annex VII and to make a decision on whether classification and labelling are warranted?

NO

 

Generation of new in vivo data for sensitisation as a last resort (Annex VII to the REACH Regulation)

8b

Does the substance demonstrate sensitising properties in an EU/OECD adopted in vivo test, the LLNA (EU B.42/OECD TG 429)?

YES

=> A LLNA assay was initiated: Skin sensitizer Categor 1A (EC3 = 0,60 %)

Therefore, a new LLNA study was performed according to the OECD test guideline No. 429 and in compliance with GLP (Envigo, 2017, rel.1) [NB: at the time of test performance, i.e. before 11 October 2016, in vitro testing was not the REACH default requirement].

 

Following a preliminary screening test in which no clinical signs of toxicity or excessive local irritation were noted at a concentration of 10% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five 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%, 5% or 2.5% w/w. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, α‑Hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1.

Stimulation index for 2.5, 5 and 10% w/w in acetone/olive oil 4:1 were 9.12, 12.08 and 12.16, respectively. The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (extrapolated EC3value) was calculated to be 0.60%. No sign of systemic toxicity or excessive local skin irritation were noted at the concentrations of 10%, 5% or 2.5% w/w.

The positive control α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (12.22) when tested at a concentration of 25% v/v in acetone/olive oil 4:1, thus, demonstrating the sensitivity and reliability of the test system.

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

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. 1A, H317 (May cause an allergic skin reaction) according to the Regulation (EC) No. 1272/2008 (CLP), since EC3 is < 2% (0.60%).