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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-01-12 to 2015-03-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Experimental study according to guideline and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Harlan Cytotest Cell Research GmbH (Harlan CCR), 64380 Rossdorf, Germany
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-chloro-4-[(prop-2-yn-1-yloxy)methyl]benzene
EC Number:
813-130-5
Cas Number:
4039-86-5
Molecular formula:
C10H9ClO
IUPAC Name:
1-chloro-4-[(prop-2-yn-1-yloxy)methyl]benzene

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V. Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: 1st pre-test: 9 - 10 weeks (beginning of treatment), 2nd pre-test: 11 -12 weeks (beginning of treatment), Main study: 8 - 9 weeks (beginning of treatment)
- Weight at study initiation: 17.7 - 21.4 g
- Housing: group, Makrolon Type II (pre-test) / III (main study), with wire mesh top
- Diet: ad libitum, 2018C Teklad Global 18% protein rodent diet
- Water: ad libitum, tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
other: unchanged (100 %) and in DMF (other concentrations)
Concentration:
5, 10 and 25 % (main test)
50 and 100 % (1. pre test)
10 and 25 % (2. pre test)
No. of animals per dose:
5
(2 animals per pre test)
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: in Dimethylformamide (DMF, 99 %)
- Irritation: ear weights, thickness and irritation scores were measured.
- Lymph node proliferation response: not evaluated

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Lymph node weight and cell count,incorporation of 3HTdR, ear weight
- Criteria used to consider a positive response: cell count index: 1.55, ear weight index: 1.1, incorporation of 3HTdR: > 3 fold

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into an appropriate container on a tared balance and DMF was added. The different test item concentrations were prepared individually. The preparations were made freshly and used within two hours before each dosing occasion. Concentrations were in terms of material as supplied.
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25 % (w/w) in DMF. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals). Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20.5 μCi of 3H-methyl thymidine (equivalent to 82 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
Positive control substance(s):
other: α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1, v/v)
Statistics:
The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation).
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations custom made statistical program ´R` Decision Tree was used. Statistical significance was set at the five per cent level (p < 0.05).
The Dean-Dixon-Test and the Grubb’s test were used for detection of possible outliers (performed with custom made statistical program ´R` Decision Tree).

Results and discussion

Positive control results:
The ear weights of the positive control group were found to be statistically significantly increased when compared to the concurrent vehicle control (index of 1.11). But as the increased ear weighs did not exceed the cut-off value for excessive irritation mentioned in the OECD guideline, this was considered not to be biologically relevant.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Vehicle Control Group (DMF): 1.00 5 % 4-Chlorobenzyl propargyl ether: 1.64 10 % 4-Chlorobenzyl propargyl ether: 1.57 25 % 4-Chlorobenzyl propargyl ether: 2.59 Vehicle Control Group for the Positive Control (AOO): 1.0 Positive Control Group: 5.88
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Mean DPM per animal (2 lymph nodes) Vehicle Control Group (DMF): 1042.4 5 % 4-Chlorobenzyl propargyl ether: 1705.0 10 % 4-Chlorobenzyl propargyl ether: 1640.8 25 % 4-Chlorobenzyl propargyl ether: 2695.2 Vehicle Control Group for the Positive Control (AOO): 1761.2 Positive Control Group: 10355.2

Any other information on results incl. tables

Test item concentration

Group Calculation

 

Mean DPM per animal (2 lymph nodes) a)

SD

S.I.

Vehicle Control Group (DMF)

1042.4

171.0

1.00

5% 4-Chlorobenzyl propargyl ether

1705.0

582.3

1.64

10% 4-Chlorobenzyl propargyl ether

1640.8

404.6

1.57

25% 4-Chlorobenzyl propargyl ether

2695.2

835.4

2.59 $

Vehicle Control Group for the Positive Control (AOO)

1761.2

762.4

1.00

Positive Control Group

10355.2

3367.8

5.88 $

a) Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals).

$ Mean DPM value for the group was according to the ANOVA (Dunnett-test) significantly higher than the corresponding control value. The p value for the analysis was 0.008 for the positive control group and 0.001 for the high dose group, respectively.

Applicant's summary and conclusion