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Description of key information

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)
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
see description in the principles of method if other than guideline
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
other: - Note for Guidance on Non-Clinical Tolerance Testing of Medicinal Products, CPMP/SWP/2145/00, adopted March 1, 2001.
Qualifier:
according to
Guideline:
other: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: First round; Toxicology 212, 60-68 (2005a);
Qualifier:
according to
Guideline:
other: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round; Toxicology 212, 69-79 (2005b).
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.
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.
Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the vehicle treated ones.
Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the inter-laboratory validation of this method (Ehling et al. 2005a and 2005b).
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
Sex Female
Number of animals 42 (7 groups of 6 female animals each)
Body weight (on test day 1) 28 - 36 g
Age (on test day 1) 62 days

The animals were identified by cage label, not with ear tags.
The adaptation period has been of 5 days.

Diet: Commercial diet ssniff® R/M-H V1534 served as food (ssniff Spezialdiäten GmbH, 59494 Soest, Germany; see Appendix 3: Composition of the diet).This food was offered ad libitum. Food residue was removed. Periodic control of contaminants based on EPA/USA is conducted at least twice a year,

Housing: Before application the animals were housed in groups in MAKROLON cages (type III) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 15 cm at a room temperature of 22°C  3°C (maximum range) and a relative humidity of 55%  15% (maximum range). After application the animals were housed singly in order to prevent their licking off the test item from the ears of the other animals.
Deviations from the maximum range caused for example during cleaning procedures and change of cages are dealt with in SOPs.
The rooms were alternately lit (150 lux at approx. 1.50 m room height) and darkened for periods of 12 hours each. The air in the rooms was changed 12 - 18 times per hour.
Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned once a week.
Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted at least once a year.

Drinking water: Tap water was offered ad libitum.
Drinking water is examined according to the 'Deutsche Trinkwasserverordnung 2001' [German Regulations on drinking water 2001] by the Hamburger Wasserwerke, 20539 Hamburg, Germany, at least four times a year (see Appendix 3: Limitation for contaminants in the drinking water).
In addition, drinking water samples taken at LPT are analysed by LUFA-ITL once a year for means of bacteriological investigations according to the 'Deutsche Trink-wasserverordnung 2001, Anlage 1' [German Regulations on drinking water 2001, Addendum 1].
Vehicle:
other: acetone/olive oil (3+1, v/v)
Concentration:
1%, 10%, 25%, 50%, 100%
No. of animals per dose:
6
Details on study design:
Five concentrations of 1,4-Diethylbenzene (1%, 10%, 25% and 50%, diluted with acetone/olive oil (3+1 v/v), w/w and the undiluted test item) were tested in six female NMRI mice per group and compared to a vehicle control group. In addition, a positive control group (20% solution v/v of alfa-hexyl cinnamic aldehyde in acetone/olive oil (3+1 v/v)) was employed.
In a preliminary experiment, concentrations of 25% and 50% and the undiluted test item, employing 1 animal per concentration, were examined. As no pronounced irritating properties were observed in this preliminary experiment, the undiluted test item was chosen as top concentration in the main experiment.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The so-called stimulation (or LLN-) indices to determine the sensitising potential (this value was fixed empirically during the inter-laboratory validation of this method, for details see Ehling et al. 2005a and 2005b, page 13), 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.
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.
The LLNA method can give positive results if the substance is skin irritant. Measuring cell proliferation by radioactive or non-radioactive methods, without taking the irritating properties of test items into account, leads thus to false positive reactions.
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. 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.
The stimulation indices were calculated by dividing the average ear weight and average ear thickness on test day 4 per group of the test item treated animals by the vehicle treated ones. The cut-off threshold value for ear weight was set at 1.1.
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.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
In the main study treatment with 1,4-Diethylbenzene at concentrations of 1% or 10% did not reveal statistical significantly increased values for lymph node cell count and lymph node weight. The stimulation indices of the lymph node cell count did not exceed the threshold level of 1.4. Hence, the test item is classified as not sensitising. 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. Treatment with 1,4-Diethylbenzene at concentrations of 25% or 50% (w/w) or the undiluted test item (100%) revealed statistical significantly increased values for lymph node cell count (p ≤ 0.01 at 50% and 100%) and lymph node weight (p ≤ 0.01 at all 3 concentrations). However, ear weights were also significantly increased at all 3 concentrations pointing to strongly irritating properties on the mouse ear. The stimulation indices of the lymph node cell count (sensitising properties) and ear weight (irritation properties) exceeded the threshold levels of 1.4 or 1.1.

Main study results: stimulation indices (SI)

underlined figures: significantly increased compared to control at p ≤ 0.01

 
 Parameter  Group 1,negative control  group 6, 1%  Group 7, 10%  Group 2, 25%  Group 3, 50%  Group 4, 100%  Group 5, positive control
 Lymph node cell count  1.000  1.076  1.063  1.402  1.937  1.910  1.869
 Lymph node weight  1.000  0.942  1.000  1.192  1.577  1.865  1.923
 Ear weight  1.000  1.032  0.973  1.103  1.124  1.189  1.097
 Difference of ear thickness 1.000  1.085  1.017  1.026  1.017  1.132  1.226
Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
Under the present test conditions, 1,4-Diethylbenzene at concentrations of 1% or 10% (w/w) in acetone/olive oil (3+1, v/v) did not reveal any sensitising properties in the local lymph node assay.
Concentrations of 25% or higher did not permit an evaluation due to the irritating properties of the test item in this test system as evident from the increased ear weight and an increase in ear thickness.
Executive summary:

The purpose of this study was to determine the sensitising potential of the test item in the local lymph node assay in mice.

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 oflymph node cell counts and lymph node weightsif justification and appropriate scientific support exist showing the validity of this method.

The alternative method used for the study employing thelymph node weightandlymph nodecell countto assessproliferationhas 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.

Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the vehicle treated ones.

Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the inter-laboratory validation of this method (Ehling et al. 2005a and 2005b).

Five concentrations of the test item (1%, 10%, 25% and 50%, w/w, diluted with acetone/olive oil (3+1 v/v), and the undiluted test item)were tested in six female NMRI mice per group and compared to a vehicle control group.

In addition, a positive control group(20% solution v/v ofa-hexyl cinnamic aldehyde inacetone/olive oil (3+1 v/v)) was employed.

In the main study treatment with1,4-Diethylbenzene at concentrations of 1% or 10% did not reveal statistical significantly increased values for lymph node cell count and lymph node weight. The stimulation indices of the lymph node cell count did not exceed the threshold level of 1.4. Hence, the test item is classified as not sensitising.

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.

Treatment with 1,4-Diethylbenzene at concentrations of 25% or 50% (w/w) or the undiluted test item (100%) revealed statistical significantly increased values for lymph nodecell count (p≤ 0.01 at 50% and 100%) and lymph node weight (p≤ 0.01 at all 3 concentrations).

However, ear weights were also significantly increased at all 3 concentrations pointing to strongly irritating properties on the mouse ear. The stimulation indices of the lymph node cell count (sensitising properties) and ear weight (irritation properties) exceeded the threshold levels of 1.4 or 1.1.

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

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

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

Under the present test conditions, 1,4-Diethylbenzene at concentrations of 1% or 10% (w/w) in acetone/olive oil (3+1, v/v) did not reveal any sensitising properties in the local lymph node assay.

Concentrations of 25% or higher did not permit an evaluation due to the irritating properties of the test item in this test system as evident from the increased ear weight and an increase in ear thickness.


Migrated from Short description of key information:
The study performed with 1,4-diethylbenzene is done according to OECD429 and including deviations well described in the robust summary study, that have been validated.

Justification for selection of skin sensitisation endpoint:
One study available, GLP and scored Klimisch 1.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

As in guidance, R7a, although the LLNA does not represent a method for the specific identification of chemical respiratory allergens, there is evidence that chemical respiratory allergens will also elicit positive responses in this assay (Kimber, 1995). The interpretation is therefore that a chemical which fails to induce a positive response in the LLNA (at an appropriate test concentration) most probably lacks the potential for respiratory allergy. The effect on respiratory tissue would be also an irritating effect up from certain concentrations.

Justification for classification or non-classification

Based on the available data 1,4-diethylbenzene in not classified as sensitiser.