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Diss Factsheets

Administrative data

Description of key information

- Skin sensitisation: sensitising (GLP, OECD 429, K, rel.2)

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:
From 2004-06-10 to 2004-07-29
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Restrictions applies to this study: 1/ In an initial experiment the control group had a low DPM count, suggesting that they did not successfully inject the labelled IuDR, or had problems harvesting the lymph nodes correctly, either way it indicates some technical deficiencies. 2/ In the repeat experiment, some technical deficiencies were also observed: Control animal 103 had a DPM of 0.4, where the others were in the range of 17 to 31, which suggests that the injection of labelled IuDR was not done correctly. The average DPM excluding animal 103 is 21.0. The same may be said for animals 109 and 111 (1% group), 114, 111 and 118 (5% group). If these animals are excluded, then the averages are 22.3 and 18.3. In the 40% group there is one high animal 131, if it is excluded then the average is 48.9, which is just 2.3x control, which is less than the positive threshold of 3x control. 3/ The highest concentration used was not based on an adequate scientific rationale, no information on the effect at doses above 40% were available. However as the test material induced delayed contact hypersensitivity in the absence of irritation effects in one animal under the test conditions, this deviation was considered as acceptable. 4/ The positive control used is not a recommended reference substance for the LLNA, however as it induced significant lymphoproliferation, it was considered appropriate.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
no adequate scientific rationale on the selection of concentration although tested only up to 40%. Hydroxycitronellal is not a recommended positive control for LLNA assays.
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jackson Laboratory (Bar Harbor, ME)
- Age at study initiation: 6 to 7 weeks of age
- Weight at study initiation: 16.7-23.1 g
- Housing: individually in plastic shoebox-style cages
- Diet (e.g. ad libitum): Purina Rodent Chow 5002 ad libitum
- Water (e.g. ad libitum): City of Rayleigh tap water ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.4-25.7 °C
- Humidity (%): 44-85% (the humidity on 2004-07-13 was above the 30% to 80% range specified in the study protocol
- Air changes (per hr): no data
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: 2004-07-08 From: To: 2004-07-27
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
1%, 5%, 10%, 20% and 40%
No. of animals per dose:
5 animals/dose group. 8 control animals.
Details on study design:
RANGE FINDING TESTS: not performed

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
- The vehicle and dosing solutions were prepared on the benchtop daily prior to dosing. The dosing solution was mixed just prior to dosing. No analysis were performed by BRT.
- On days 1, 2 and 3, a dose-volume of 25 μL of the control or dosage article was applied to the dorsal of each ears, using a calibrated Finnpipette®
On day 6, mice were injected with 250 µL containing 2 µCi of [125]I-iododeoxyuridine and 10E-5 M fluorodeoxyuridine in phosphate-buffered saline (PBS). Approximately 5 hours later, the mice were euthanized by CO2 asphyxiation, and the auricular lymph nodes were excised. The nodes were dissociated using the frosted ends of glass slides. The cell suspension was washed with Hanks' balanced salt solution (HBSS) and then with PBS prior to being resuspended in 5% trichloroacetic acide (TCA) and refrigerated at approximately 4 °C. Approximately 18 hours later, the cells were centrifuged and resuspended in fresh 5% TCA. The radioactivity was measured using a gamma counter.
- Ear measurement: taken prior to dosing on days 1 and 3 using an Oditest micrometer. Percentage increase from day 1 to 3 was calculated and, if a 10% or greater increase in ear thickness occurred, the lowest concentration of test article eliciting that response (minimal irritating concentration or MIC10) was identified. When applicable the MIC10 was determined and compared to the calculated EC-3. If the MIC10 was greater than the EC-3, confounding irritation is unlikely to have affected the LLNA. If the MIC10 was less than the EC-3, the concentration of the chemical that elicited the increase may have produced an LLNA stimulation index close to 3 because of the physiology surrounding the primary irritation reaction.
Positive control substance(s):
other: Hydroxycitronellal (10% , 20% and 40%). Aldrich, lot #15718JB. 98.7% pure. Expiration date: 2006-06-25.
Statistics:
A one-sample t test was performed to test whether the individual untransformed SI value for each dose level of each compound was greater than or equal to 3. The natural log transformed DPM values for each compound were compared against vehicle with a Bartlett’s Chi-Square test for variance homogeneity. If the Bartlett’s Chi-Square results were found to be non significant, a one-way analysis of variance (ANOVA) was performed using dose (concentration). If the ANOVA was found to be significant, then a Dunnett’s test was performed using an alpha of 0.05.
If the Bartlett’s Chi-Square was found not to be significant, nonparametric analyses were conducted, specifically a Kruskall-Wallis test. If the nonparametric analysis were found to be significant, then a Jonckheere’s-Terpera test was performed for dose-dependent trends.
The data from the concentration tested was fit using a quadratic equation (a linear term and a square term of the concentration). If the quadratic term did not fit, a simple regression model was used. The concentration of chemical required to elicit an SI of 3 (EC-3) was determined from the appropriate regression equation.
Positive control results:
Mean DPM = 21.1 at 10%, 44.8 at 20% and 87.5 at 40%.
Mean SI =1.1 at 10%, 2.4 at 20% and 4.7 at 40%.
Parameter:
SI
Value:
0.8
Test group / Remarks:
1%
Parameter:
SI
Value:
0.5
Test group / Remarks:
5%
Parameter:
SI
Value:
1.3
Test group / Remarks:
10%
Parameter:
SI
Value:
1.3
Test group / Remarks:
20%
Parameter:
SI
Value:
3.8
Test group / Remarks:
40%
Key result
Parameter:
EC3
Value:
33.95
Cellular proliferation data / Observations:
Mean SI = 0.8 at 1%, 0.5 at 5%, 1.3 at 10%, 1.3 at 20% and 3.8 at 40%.
Mean DPM = 15.2 at 1%, 9.2 at 5%, 24.0 at 10%, 23.9 at 20% and 70.6 at 40%.


CELLULAR PROLIFERATION DATA

DETAILS ON STIMULATION INDEX CALCULATION

EC3 CALCULATION

CLINICAL OBSERVATIONS:

BODY WEIGHTS

Table 7.4.1/1: Results of LLNA

Treatment

Animal #

Individual animal DPM

Mean DPM ± SD

SI

Vehicle (AOO)

1

30.8

18.4 ± 8.4

 

2

18.7

3

0.4

4

20.9

5

17.6

6

18.4

7

21.9

8

18.7

ST 06 C 04 - 1 %

1

6.3

15.2 ± 10.7

0.8

2

29.0

3

3.0

4

17.1

5

20.7

ST 06 C 04 - 5 %

1

0.0

9.2 ± 8.6

0.5

2

3.2

3

17.6

4

18.9

5

6.0

ST 06 C 04 - 10 %

1

13.1

24.0 ± 14.4

1.3

2

49.3

3

20.2

4

20.7

5

16.9

ST 06 C 04 - 20 %

1

27.0

23.9 ± 6.6

1.3

2

28.3

3

16.1

4

17.7

5

30.6

ST 06 C 04 - 40 %

1

27.1

70.6 ± 50.5

3.8

2

54.4

3

157.9

4

62.5

5

51.4

Hydroxycitronellal – 10%

1

19.4

21.1 ± 11.5

1.1

2

22.5

3

39.9

4

12.8

5

10.8

Hydroxycitronellal – 20%

1

37.4

44.8 ± 21.3

2.4

2

41.9

3

24.7

4

81.1

5

38.9

Hydroxycitronellal – 40%

1

66.2

87.5 ± 42.0

4.7

2

54.3

3

117.1

4

146.4

5

53.6

 

In an initial LLNA, the vehicle (AOO) values were low (less than 10 DPM), resulting in artificially high SI values for the test and control articles.

In the repeat analysis, none of the treated mice experienced visible irritation or other adverse toxic effects.

Both the control, hydroxycitronellal, and the test material had SI value greater than 3 at the 40% concentration, but neither value was found to be statistically significant.

The calculated EC-3 values were 24.03% for hydroxycitronellal and 33.95% for the test material. The EC-3 potency values were determined to be 6.008 µg/cm² for hydroxycitronella and 8.488 µg/cm² for the test material. Calculation of the potency value was assumed a conversion of 1 mL = 1 g and was based on an exposure area of 1 cm² per mouse ear and the application of 25 µL.

None of the treatment groups had 10% or greater increases in mean ear thickness from day 1 to 3, thus there was no irritation reaction to potentially affect the LLNA simulation indices.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test material induced delayed contact hypersensitivity in this Local Lymph Node Assay.
Executive summary:

In a dermal sensitization study performed similarly to the OECD test guideline No. 429 and in compliance with GLP, the test material was tested in female CBA/J mice using the Local Lymph Node Assay.

 

Forty eight mice were allocated to nine groups:

- Five treated groups of five animals receiving the test material diluted in acetone/olive oil (4/1) at the concentration of 1%, 5%, 10%, 20% or 40%

- One negative control group of height animals receiving the vehicle

- Three positive control groups of five animals receiving the Hydroxycitronellal diluted in acetone/olive oil (4/1), at the concentration of 10%, 20% or 25%.

During the induction phase, 25 µL of the test item, the vehicle or the positive control was applied to the dorsum of each ear for 3 consecutive days (days 1, 2 and 3). The animals were allowed to rest without dosing on days 4 and 5. On day 6, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of 125I-iododeoxyuridine and fluorodeoxyuridine.

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1 and 3.

 

The positive control used, Hydroxycitronellal, presented a Stimulation index of 4.7 at 40%. Although it is not a recommended reference substance for the LLNA, the positive control gave acceptable positive results and the study was considered as valid.

 

No mortality related to treatment and no clinical signs were observed during the study. No cutaneous reactions and no notable increase in ear thickness were observed in the animals of the treated groups.

 

A significant lymphoproliferation (SI = 3.8) was noted at the concentration of 40%. This SI value doesn’t achieve statistical significance. The EC3value for the test material was calculated to be 33.95%.

However, some restrictions applies to this study:

1/ In an initial experiment the control group had a low DPM count, suggesting that they did not successfully inject the labelled IuDR, or had problems harvesting the lymph nodes correctly, either way it indicates some technical deficiencies.  

2/ In the repeat experiment, some technical deficiencies were also observed: Control animal 103 had a DPM of 0.4, where the others were in the range of 17 to 31, which suggests that the injection of labelled IuDR was not done correctly. The average DPM excluding animal 103 is 21.0. The same may be said for animals 109 and 111 (1% group), 114, 111 and 118 (5% group). If these animals are excluded, then the averages are 22.3 and 18.3. In the 40% group there is one high animal 131, if it is excluded then the average is 48.9, which is just 2.3x control, which is less than the positive threshold of 3x control.

3/ The highest concentration used was not based on an adequate scientific rationale, no information on the effect at doses above 40% were available. However as the test material induced delayed contact hypersensitivity in the absence of irritation effects in one animal under the test conditions, this deviation was considered as acceptable.

4/ The positive control used is not a recommended reference substance for the LLNA, however as it induced significant lymphoproliferation, it was considered appropriate.

Overall there is a question mark against the technical quality of the study and perhaps it can be said to be unreliable, although a weak effect is also a possibility. As a worst-case approach, it is concluded that under the test conditions, the test material is classified as a dermal sensitizer in the Local Lymph Node Assay according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

 

 

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

In a dermal sensitisation study performed similarly to the OECD test guideline No. 429 and in compliance with GLP, the test material was tested in female CBA/J mice using the Local Lymph Node Assay.

 

Forty eight mice were allocated to nine groups:

- Five treated groups of five animals receiving the test material diluted in acetone/olive oil (4/1) at the concentration of 1%, 5%, 10%, 20% or 40%

- One negative control group of height animals receiving the vehicle

- Three positive control groups of five animals receiving the Hydroxycitronellal diluted in acetone/olive oil (4/1), at the concentration of 10%, 20% or 25%.

During the induction phase, 25 µL of the test item, the vehicle or the positive control was applied to the dorsum of each ear for 3 consecutive days (days 1, 2 and 3). The animals were allowed to rest without dosing on days 4 and 5. On day 6, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of 125I-iododeoxyuridine and fluorodeoxyuridine.

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1 and 3.

 

The positive control used, Hydroxycitronellal, presented a Stimulation index of 4.7 at 40%. Although it is not a recommended reference substance for the LLNA, the positive control gave acceptable positive results and the study was considered as valid.

 

No mortality related to treatment and no clinical signs were observed during the study. No cutaneous reactions and no notable increase in ear thickness were observed in the animals of the treated groups.

 

A significant lymphoproliferation (SI = 3.8) was noted at the concentration of 40%. This SI value doesn’t achieve statistical significance. The EC3value for the test material was calculated to be 33.95%.

However, some restrictions applies to this study:

1/ In an initial experiment the control group had a low DPM count, suggesting that they did not successfully inject the labelled IuDR, or had problems harvesting the lymph nodes correctly, either way it indicates some technical deficiencies.  

2/ In the repeat experiment, some technical deficiencies were also observed: Control animal 103 had a DPM of 0.4, where the others were in the range of 17 to 31, which suggests that the injection of labelled IuDR was not done correctly. The average DPM excluding animal 103 is 21.0. The same may be said for animals 109 and 111 (1% group), 114, 111 and 118 (5% group). If these animals are excluded, then the averages are 22.3 and 18.3. In the 40% group there is one high animal 131, if it is excluded then the average is 48.9, which is just 2.3x control, which is less than the positive threshold of 3x control.

3/ The highest concentration used was not based on an adequate scientific rationale, no information on the effect at doses above 40% were available. However as the test material induced delayed contact hypersensitivity in the absence of irritation effects in one animal under the test conditions, this deviation was considered as acceptable.

4/ The positive control used is not a recommended reference substance for the LLNA, however as it induced significant lymphoproliferation, it was considered appropriate.

Overall there is a question mark against the technical quality of the study and perhaps it can be said to be unreliable, although a weak effect is also a possibility. As a worst-case approach, it is concluded that under the test conditions, the test material is classified as a dermal sensitiser in the Local Lymph Node Assay according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

 

 

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonised classification according to the Regulation (EC) No. 1272/208 (CLP).

Self-classification:

As a worst-case approach, the substance is classified as a skin sensitiser [Skin Sens. 1B (H317: May cause an allergic skin reaction)] according to the CLP and the GHS.

No information is available regarding respiratory sensitisation.