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Description of key information

LLNA: Not sensitising, (mouse, GLP study according to OECD 429, Harlan-CCR 2010)

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:
other: GLP - Guideline Study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 24 April 2002
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus, AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Mean weight (± SD) at study initiation: 20.2 ± 1.4 g
- Housing: Single caging
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (Harlan Laboratories B.V., Netherlands)
- Water (e.g. ad libitum): tap water, ad libitum,
- Acclimation period: At least 5 days prior to the start of dosing under test conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 20-65%
- Photoperiod (hrs dark / hrs light): 12h/12h
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50, and 100% (w/v)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which can be technically used was 100% of the undiluted test item. The dilutions were formulated in acetone:olive oil (4+1). Vortexing was necessary to formulate the test item.
- Irritation: To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 50% and 100% each on three consecutive days. At the tested concentrations the animals did not show any signs of local irritation as confirmed by the ear thickness and ear weight measurements. Also, animals did not show any signs of systemic toxicity.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response:

TREATMENT PREPARATION AND ADMINISTRATION:
- Topical Application
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 25, 50, and 100% (w/v) in acetone:olive oil (4+1). The application volume, 25 µl, was spread over the entire dorsal surface (8 mm) of each ear 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
Five days after the first topical application, all mice were administered with 250 µl of 79.4 µCi/ml ³HTdR (corresponds to 19.9 µCi ³HTdR per mouse) by intravenous injection via a tail vein.
- Determination of lymph node weights
For the determination of lymph node weight, after excision, the lymph nodes were pooled per animal and weighed immediately after removal using an analytical balance.
- Determination of incorporated ³HTdR
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 µm mesh size). After further processing, the level of ³HTdR incorporation was measured on a ß-scintillation counter as the number of radioactive disintegrations per minute (DPM).
- Determination of Ear Weights
After the lymph nodes have been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch. For each animal both punches were immediately weighed per animal.

- Interpretation of Raw Data
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

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.

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.
The ANOVA (Dunnett-test) was conducted on the ear and lymph node weights to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group. However, both biological and statistical significance were considered together.
Positive control results:
Positive control substance: alpha-Hexylcinnamaldehyde
Test item concentration % (w/v) S.I.
0 1.00
5 1.21
10 2.09
25 6.22
Parameter:
other: SI
Remarks on result:
other: see Remark
Remarks:
Test item concentration % (w/v) SI 0 - 25 1.48 50 1.67 100 2.28 None of the tested concentrations induced an S.I. greater than 3.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Test item concentration % (w/v) Measurement DPM 0 4785 25 7070 50 7966 100 10898
Key result
Parameter:
SI
Value:
1.48
Test group / Remarks:
25 % test group
Key result
Parameter:
SI
Value:
1.67
Test group / Remarks:
50 % test group
Key result
Parameter:
SI
Value:
2.28
Test group / Remarks:
100 % test group

Further results:

- No deaths occurred during the study period.

- No symptoms of local irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period.

- 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.

- A relevant increase in ear weights was not observed.

- A statistically significant increase in lymph node weights was observed in the groups treated with 50 and 100% test item concentration in comparison to the vehicle control group (p=0.027):

Table of Lymph Node Weights

Animal No.

Concentration
% (w/v)

Lymph Node Weights after Necropsy

 

Lymph Node Weight (mg per animal)

Mean Lymph Node Weight (mg per animal)

SD

1

0

3.90

4.45

0.61

2

4.30

3

4.27

4

5.32

5

25

5.04

5.43

0.65

6

5.80

7

4.73

8

6.13

9

50

5.91

6.11*

0.73

10

5.27

11

7.02

12

6.25

13

100

4.73

6.28*

1.10

14

6.71

15

6.36

16

7.30

* statistically significant increase vs. control group (p=0.027)

SD= Standard Deviation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Skin sensitization potential of the main constituent 3-methylbut-3 -en-1-ol (CAS 763-32-6).

In the key study for skin sensitization according to OECD 429 and GLP, i.e. a Local Lymph Node Assay according to OECD 429 and GLP, 3 -methylbut-3 -en-1 -ol was assessed for its possible contact allergenic potential (Harlan CCR 2010). 3 -methylbut-3 -en-1 –ol was dissolved in acetone:olive oil (4+1) and female mice were treated with the test item concentrations of 25, 50, and 100%. The animals did not show any signs of systemic toxicity or local irritation during the course of the study and no cases of mortality were observed. Stimulation Indices (S.I.) of 1.48, 1.67, and 2.28 were determined with the test item at concentrations of 25, 50, and 100% in acetone:olive oil (4+1), respectively. A relevant increase in ear weights was not observed. A statistically significant increase in lymph node weights was observed in the groups treated with 50 and 100% test item concentration in comparison to the vehicle control group (p=0.027). However, none of the S.I.s determined for these concentrations exceeded the threshold of 3.

Skin sensitization potential of the impurity formaldehyde (CAS 50-00-0).

The impurity formaldehyde is found to be present at concentrations up to 0.999% in the substance to be registered (3-methylbut-3 -en-1-ol or Isoprenol).

There is sufficient evidence for skin sensitizing properties of formaldehyde in guinea pig maximization tests (GPMT), in a Buehler test in guinea pigs and in mouse local lymph node assays (LLNA). Furthermore, formaldehyde is also identified as a dermal allergen in humans. Accordingly, formaldehyde is listed Annex VI to the CLP Regulation with the harmonized classification as a skin sensitizer Cat.1. Various skin sensitization data in the GPMT (see. e.g. Kimber 1991), Bühler test (see e.g. Hilton 1996), or LLNA (see e.g Hilton 1998) provide evidence of a strong skin sensitizing potential of formaldehyde. Quantitative estimates such as the EC3 values of the LLNA ranged between 0.33% and 0.96% formaldehyde.

According to current knowledge and application of the criteria given in Annex I of Regulation (EC) No. 1272/2008, the classification Skin Sens 1A, exceeding the classification given in Regulation (EC) No. 1272/2008, Annex VI, Table 3.1 is required.

 

- Kimber et. al. 1991. The murine local lymph node assay: results of an inter-laboratory trial. Toxicol Lett 55: 203-213.

- Hilton et. al. 1996. Experimental assessment of the sensitizing properties of formaldehyde. Fd Chem Toxicol 34: 571-578.

- Hilton et. al. 1998. Estimation of relative skin sensitizing potency using the local lymph node assay: a comparison of formaldehyde with glutaraldehyde. Am J Contact Dermat 9(1):29-33.

 

Conclusion:

Overall, the skins sensitization data for the impurity formaldehyde trigger a classification for the registered substance (3-methylbut-3-en-1-ol or Isoprenol) as a skin sensitizer Cat 1 (H317: May cause an allergic skin reaction).

In contrast, Isoprenol, was not a skin sensitiser under the test conditions of the performed Local Lymph Node Assay. Accordingly, the alternative grade is not to be classified as a skin sensitizer Cat.1, due to its intrinsic properties and the absence of relevant levels of the impurity formaldehyde.


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

No data available, however, no clear substance related effects were seen in the acute inhalation toxicity assay.

Justification for classification or non-classification

The present data on dermal sensitization for the main constituent 3-methylbut-3 -en-1-ol (Isoprenol) do not fulfill the criteria laid down in 1272/2008/EEC, and therefore, a non-classification is warranted. However, the skin sensitization data for the impurity formaldehyde (up to 0.999%) trigger a classification for the registered substance as a skin sensitizer Cat 1 (H317: May cause an allergic skin reaction).