Registration Dossier

Administrative data

Description of key information

The test material is a skin sensitizer (OECD 429 and EU Method B.42)

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:
Between 05 May 2009 and 27 May 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with recognised guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CBA/Ca (CBA/CaOlaHsd)
- Source: Harlan UK Limited, Bicester, Oxon, UK
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 15 to 23 g
- Housing: Individually housed in suspended solid floor polypropylene cages furnished with softwood flakes.
- Diet (e.g. ad libitum): 2014 Teklad global rodent diet, ad libitum
- Water (e.g. ad libitum):mains tap water, ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): at least 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light/dark cycle


IN-LIFE DATES: No data available
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10%, 25%, 50%
No. of animals per dose:
4 mice per dose.
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: the substance was considered suitable for dosing in acetone/olive oil (4:1)at a concentration of 50% as upon mixing by vortex mixer the formulation formed a solution which could be administered by micropipette.
- Irritation: None noted
- Lymph node proliferation response: Expressed as the number of radioactive disintegrations per minute per lymph nodes from each individual animal and as the ration of ^3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: “Skin Sensitisation: Local Lymph Node Assay” / “Skin Sensitisation (Local Lymph Node Assay of Commission directive 2004/73/EC”
- Criteria used to consider a positive response: The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in ^3HTdR incorporation compared to control values.


TREATMENT PREPARATION AND ADMINISTRATION:
For the purpose of the study, the test material was freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. (50%)
Determination, by analysis, of the concentration, homogeneity and stability of the test material preparations was not appropriate because it was not specified in the Study Plan and is not a requirement of the Test Guideline.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The EC3 value was interpreted using GraphPad PRISM version 3.03.
Positive control results:
Concentration v/v in acetone/olive oil 4:1: 15%
Stimulation Index: 8.34
Result: Positive

Parameter:
SI
Value:
3.14
Test group / Remarks:
10 % test item in acetone/olive oil 4:1
Parameter:
SI
Value:
7.94
Test group / Remarks:
25 % test item in acetone/olive oil 4:1
Parameter:
SI
Value:
19.03
Test group / Remarks:
50 % test item in acetone/olive oil 4:1

Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% w/w) in

Acetone/olive oil 4:1

dpm

dpm/Nodea

Stimulation Indexb

 

Result

Vehicle

9376.99

1172.12

na

na

10

29437.64

3679.71

3.14

Positive

25

74447.31

9305.91

7.94

Positive

50

178410.2

22301.28

19.03

Positive

 

Dpm = disintegrations per minutes

a = total number of lymph nodes per animals is 2

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

N/A = Not Applicable

 

Individual clinical Observations and Mortality Data

 

Conc (%v/v) in acetone/olive oil 4:1

Animal Number

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Pre-dose

Post-dose

Pre-dose

Post-dose

Pre-dose

Post-dose

Vehicle

1-1

0

0

0

0

0

0

0

0

0

1-2

0

0

0

0

0

0

0

0

0

1-3

0

0

0

0

0

0

0

0

0

1-4

0

0

0

0

0

0

0

0

0

1-5

0

0

0

0

0

0

0

0

0

10

2-1

0

0

0

0

0

0

0

0

0

2-2

0

0

0

0

0

0

0

0

0

2-3

0

0

0

0

0

0

0

0

0

2-4

0

0

0

0

0

0

0

0

0

2-5

0

0

0

0

0

0

0

0

0

25

3-1

0

0

0

0

0

0

0

0

0

3-2

0

0

0

0

0

0

0

0

0

3-3

0

0

0

0

0

0

0

0

0

3-4

0

0

0

0

0

0

0

0

0

3-5

0

0

0

0

0

0

0

0

0

50

4-1

0

0

0

0

0

0

0

0

0

4-2

0

0

0

0

0

0

0

0

0

4-3

0

0

0

0

0

0

0

0

0

4-4

0

0

0

0

0

0

0

0

0

4-5

0

0

0

0

0

0

0

0

0

 

0 = No signs of systemic toxicity

 

 

Individual Bodyweights and Bodyweight Changes

 

Conc (%v/v) in acetone/olive oil 4:1

Animal Number

Bodyweight (g)

Bodyweight Change (g)

Day 1

Day 6

 

 

Vehicle

1-1

20

20

0

1-2

19

19

0

1-3

21

20

-1

1-4

20

21

0

 

 

10

2-1

20

20

0

2-2

20

20

0

2-3

22

23

1

2-4

20

19

-1

 

 

25

3-1

18

17

-1

3-2

18

18

0

3-3

22

21

-1

3-4

18

17

-1

 

 

50

4-1

19

18

-1

4-2

20

19

-1

4-3

18

17

-1

4-4

21

20

-1

 

 

 

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
In a Local Lymph Node Assay in the Mouse, the substance initiated a Stimulation Index of greater than 3 at concentrations of 10, 25 and 50% and thus is considered to be a skin sensitizer under the conditions of the test.
Executive summary:

Introduction. A study was performed to assess the skin sensitization potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements OECD 429 and Method B42.

Methods. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% 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 four animals, were treated with 50 μl (25 μl per ear) of the test material as a solution in acetone/olive oil 4:1 at concentrations of 50%, 25% or 10% w/w. A further group of four animals was treated with acetone/olive oil 4:1 alone.

Results.The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (% w/w) in

acetone/olive oil 4:1

Stimulation Index

Result

 

10

3.14

Positive

25

7.94

Positive

50

19.03

Positive

 

According to the calculation the concentration of test material expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was found to be 9.39%.

Conclusion. The test material was considered to be a sensitiser under the conditions of the test.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
7 October 1992 - 22 December 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The GLP status of the study is not specified. The study report states that a “primary irritation test was conducted to determine appropriate concentrations of the test material to be used in the two phases of dosing”; however, the results of this test are not included in the study report. Therefore, it is unclear whether the challenge doses were the highest non-irritating concentrations.
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
results of the preliminary irritation test are not included in the study report. for the positive control, DNCB, 70% ethanol was used as the carrier, whereas the guideline recommends 80%
GLP compliance:
not specified
Type of study:
Buehler test
Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hazleton research Products Inc., Denver, PA
- Age at study initiation: Approximately 5-6 weeks.
- Weight at study initiation: 275-391 g.
- Housing: Individually housed in suspended stainless steel cages.
- Diet (e.g. ad libitum): Ad libitum.
- Water (e.g. ad libitum): Ad libitum.
- Acclimation period: 9 days.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 65-71ºF (18.3-21.6ºC)
- Humidity (%): 40-70%.
- Air changes (per hr): Not stated.
- Photoperiod (hrs dark / hrs ligh): 12 hours light / 12 hours dark.


IN-LIFE DATES: From: 14 October 1992 To: 20 November 1992.
Route:
epicutaneous, occlusive
Vehicle:
other: Carrier: Induction Day 0: mineral oil. Induction Days 7 and 14, Challenge and Rechallenge: peanut oil
Concentration / amount:
Induction: 50% test material in carrier (0.4 mL).
Challenge: 1% test material in carrier (0.4 mL).
Rechallenge: 1% test material in carrier (0.4 mL).
Route:
epicutaneous, occlusive
Vehicle:
other: Carrier: Induction Day 0: mineral oil. Induction Days 7 and 14, Challenge and Rechallenge: peanut oil
Concentration / amount:
Induction: 50% test material in carrier (0.4 mL).
Challenge: 1% test material in carrier (0.4 mL).
Rechallenge: 1% test material in carrier (0.4 mL).
No. of animals per dose:
20 for the treated group.
20 for the irritation control group.
10 for the positive control group.
Details on study design:
RANGE FINDING TESTS: A primary irritation test was reportedly conducted to determine the appropriate concentrations of the test material to be used in the two phases of dosing. The results of this test are not included in the study report.


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3 (Days 0, 7 and 14)
- Exposure period: Approximately 6 hours.
- Test groups: Treated.
- Irritation control group: Untreated.
- Site: Scapular region.
- Frequency of applications: Weekly.
- Concentrations: 50% test material in carrier (0.4 mL).


B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: Days 28 and 35.
- Exposure period: Approximately 6 hours.
- Test groups: Treated.
- Irritation control group: 10 animals were treated on Day 28; the remaining 10 were treated on Day 35.
- Site: Abdominal region (challenge); left flank (rechallenge).
- Concentrations: 1% test material in carrier (0.4 mL).
- Evaluation (hr after challenge): Approximately 24 and 48 hours after challenge and rechallenge.
Positive control substance(s):
yes
Remarks:
1-chlor0-2,4-dinitrobenzene (DNCB)
Positive control results:
Topical application of DNCB to the positive control group animals elicited very slight to well-defined erythema and slight oedema during the induction phase. Following the challenge phase, signs of sensitisation were observed in all ten positive control animals.
Key result
Remarks on result:
other: Based on the results of the 48-hour dermal evaluations at rechallenge, the test material does not display any sensitising potential.

Body weights and in-life observations

All animals survived to study termination and showed an overall increase in body weight during the test period. The majority of animals were free of observable abnormalities during the study. One treated animal exhibited decreased food consumption, slight emaciation, and/or little sign of stool from Day 7 to Day 16.

Induction

Induction of the treated group with the test material produced erythema ranging from very slight to well-defined in all animals. Very slight oedema was also noted in several animals. In addition, one animal exhibited desquamation.

Incidence of challenge/rechallenge dermal scores

Treated group

Day

Dermal observation

Dermal Scores

N

0

1

2

3

4

29

Erythema

Oedema

16

20

4

0

0

0

0

0

0

0

20

20

30

Erythema

Oedema

16

20

4

0

0

0

0

0

0

0

20

20

36

Erythema

Oedema

19

20

1

0

0

0

0

0

0

0

20

20

37

Erythema

Oedema

20

20

0

0

0

0

0

0

0

0

20

2

 

Irritation Control group

Day

Dermal observation

Dermal Scores

N

0

1

2

3

4

29

Erythema

Oedema

10

10

0

0

0

0

0

0

0

0

10

10

30

Erythema

Oedema

10

10

0

0

0

0

0

0

0

0

10

10

36

Erythema

Oedema

10

10

0

0

0

0

0

0

0

0

10

10

37

Erythema

Oedema

10

10

0

0

0

0

0

0

0

0

10

10

 

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Based on the results of the 48-hour dermal evaluations at rechallenge, the test material does not display any sensitising potential.
Executive summary:

The dermal sensitisation potential of the test material was tested in New Zealand White rabbits. The study method followed that described in OECD Test Guideline 406. The treated group comprised 20 females, as did the irritation control group (10 animals served as the control for the challenge application and 10 for the rechallenge application); a further 10 females were used as a positive control (DNCB) group. The test material was applied at a concentration of 50% w/w in carrier for the induction applications, and at 1% w/w in carrier for the challenge and rechallenge applications. After the challenge application, four out of the 20 animals showed very slight (barely perceptible) erythema; the rest showing no observable dermal response. After rechallenge, very slight erythema was seen in only one of the 20 treated animals. No oedema was seen in any of the animals adminstered the test material. Based on these results, the test material was concluded to show no sensitising potential. The positive control substance caused very slight to well-defined erythema and slight oedema during the induction phase, and signs of sensitisation were seen in all positive control animals after challenge, confirming the sensitivity of the test.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

This substance was tested in according to mouse local lymph node assay (OECD 429) at concentrations of 10%, 25% or 50% w/w, and the stimulation scores were positive at all concentrations. In accordance to Directive 67/548/EEC and EU CLP (Regulation (EC) No. 1272/2008), this substance was classified as a skin sensitizer.

 

This substance did not show any sensitizing potential using the Buehler method modified to use only females largely in accordance with 1992 OECD 406. Concentrations tested in the induction, challenge, and rechallenge phases were 50%, 1%, and 1%, respectively. Due to the low challenge concentration level, this study was ranked as a supporting study and was not used for classification and labeling purpose.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

An EC3 value of 9.39 % was derived following investigation of the test item via the Local Lymph Node Assay. Classification for skin sensitisation (sub-category 1B) is therefore applicable under the criteria given by Regulation (EC) No. 1272/2008 and subsequent amendments.