1,6-dichlorohexane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-05-07 to 2012-08-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study OECD and EU guidelines were followed. No standard test system.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

adopted July 22, 2010

Deviations:

yes

Remarks:

the alternative method (see "Principles of method if other than guideline")

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Commission Regulation (EC) No. 440/2008

Deviations:

no

Principles of method if other than guideline:

The study was performed according to OECD 429, however not employing the use of radioactive labelling to measure cell proliferation, as the radioactive method proposed by the OECD guideline led to problems in various EU laboratories: such as (i) practical difficulties/complexity of the test, in particular the radiochemical steps, which sometimes resulted in loss of specimen/activity; this in turn led to variability in the results and to a poor reproducibility and (ii) radiation protection issues. However, the OECD guideline allows other endpoints for assessment of proliferation in form of lymph node cell counts and lymph node weights if justification and appropriate scientific support exist showing the validity of this method.
The alternative method used for the study employing the lymph node weight and lymph node cell count to assess proliferation has been established by an European interlaboratory validation exercise, as described in the two publications by Ehling et al. 2005a and 2005b. This method has the advantage of (i) more simplistic experimental work, (ii) less variability, (iii) better reproducibility, (iv) faster results, (v) reduced costs.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1,6-dichlorohexane

EC Number:

218-491-7

EC Name:

1,6-dichlorohexane

Cas Number:

2163-00-0

Molecular formula:

C6H12Cl2

IUPAC Name:

1,6-dichlorohexane

Test material form:

other: liquid

In vivo test system

Test animals

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: 69 days
- Weight at study initiation: 27 - 34 g
- Housing: singly in MAKROLON cages (type III)
- Diet: ad libitum (commercial diet ssniff® R/M-H V1534)
- Water: ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3°C
- Humidity (%): 55% +/- 15%
- Air changes (per hr): 12 - 18 times per hour
- Photoperiod (hrs dark / hrs light): darkened for periods of 12 hours

Study design: in vivo (LLNA)

Vehicle:

other: acetone/olive oil (3+1, v/v)

Concentration:

0%, 25%, 50%, 100%

No. of animals per dose:

6 female

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: 25 %, 50 % of test item in acetone/olive oil (3+1, v/v) and 100 %, employing 1 animal per concentration
- Irritation: No pronounced irritating properties were observed.
- Lymph node proliferation response: no differences in ear weight and ear thickness were noted

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The so-called stimulation (or LLN-) indices to determine the sensitising potential, were calculated by dividing the average absolute lymph node weight or lymph node cell counts per group of the test item treated animals by the vehicle treated ones. Thus, in case of no stimulating effect the index for the lymph node cell count is always below 1.4 (cut-off value). An index above 1.4 is considered positive.
The alternative method used for the study employing the lymph node weight and lymph node cell count to assess proliferation has been established by an European interlaboratory validation exercise, as described in the two publications by Ehling et al. 2005a and 2005b. This method has the advantage of (i) more simplistic experimental work, (ii) less variability, (iii) better reproducibility, (iv) faster results, (v) reduced costs.
By additionally measuring simple inflammatory parameters such as ear thickness or ear weight, it is possible to reliably determine the degree of response that is attributable to irritation (Vohr and Ahr, 2005 ).
Hence, in addition, the acute inflammatory skin reaction (irritating potential) was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item employing the U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control.

TREATMENT PREPARATION AND ADMINISTRATION:
The alternative method used for the study employing the lymph node weight and lymph node cell count to assess proliferation has been established by an European interlaboratory validation exercise, as described in the two publications by Ehling et al. 2005a and 2005b. This method has the advantage of (i) more simplistic experimental work, (ii) less variability, (iii) better reproducibility, (iv) faster results, (v) reduced costs.
In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

For lymph node weight significance at p ≤ 0.01 is considered positive (U-test according to MANN and WHITNEY). A possible concentration-response-relationship for the lymph node weight in order to determine a possible sensitising potential was examined by linear regression analysis employing PEARSON's correlation coefficient. Outliers would have been determined according to the Nalimov test.

Results and discussion

Positive control results:

The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The stimulation indices of the lymph node cell count did not exceed the threshold level of 1.4.

Results 2

Parameter:

SI

Remarks:

lymph node cell count

Value:

1

Test group / Remarks:

negative control

Results 3

Parameter:

SI

Remarks:

lymph node cell count

Value:

1.06

Test group / Remarks:

group 2, 25%

Results 4

Parameter:

SI

Remarks:

lymph node cell count

Value:

0.975

Test group / Remarks:

group 3, 50%

Results 5

Parameter:

SI

Remarks:

lymph node cell count

Value:

1.27

Test group / Remarks:

group 4, 100%

Results 6

Parameter:

SI

Remarks:

lymph node cell count

Value:

1.885

Test group / Remarks:

group 5, positive control

Remarks on result:

other: significantly different from control at p≤0.01

Any other information on results incl. tables

Main study results: stimulation indices (SI):

 

Parameter	Group 1, negative control	Group 2,25 %	Group 3, 50 %	Group 4,100 %	Group 5,positive control
Lymph node cell count	1.000	1.060	0.975	1.270	1.885
Lymph node weight	1.000	1.132	1.113	1.245	1.943
Ear weight	1.000	1.097	1.011	1.017	1.244
Difference of ear thickness	1.000	1.059	1.059	1.072	1.225

____ significantly different from control at p ≤ 0.01

The threshold level for the ear weight of 1.1 was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted.

No signs of local or systemic intolerance were recorded. The animal body weight was not affected by the treatment.

Applicant's summary and conclusion

Interpretation of results:

other: not classified

Remarks:

Criteria used for interpretation of results: OECD GHS

Conclusions:

1,6-Dichlorohexane at concentrations of 25% or 50% (w/w) in acetone/olive oil (3+1 v/v) or the undiluted test item did not reveal any sensitising properties in the local lymph node assay.