Cyclohexane, 1,4-bis(ethoxymethyl)-

Skin sensitisation

Administrative data

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 study according to current guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan CCR

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

(1r,4r)-1,4-bis(ethoxymethyl)cyclohexane; (1s,4s)-1,4-bis(ethoxymethyl)cyclohexane

EC Number:

700-868-7

Cas Number:

54889-63-3

Molecular formula:

C12 H24 O2

IUPAC Name:

(1r,4r)-1,4-bis(ethoxymethyl)cyclohexane; (1s,4s)-1,4-bis(ethoxymethyl)cyclohexane

Details on test material:

- Name of test material (as cited in study report): Cyclohexane, 1,4-bis(ethoxymethyl)-
- Physical state: colorless, clear liquid
- Analytical purity: >99.6 corrected area-% determined by Capillary GC
- Lot/batch No.: 10474-11-115
- Expiration date of the lot/batch: 26 July 2014
- Storage condition of test material: at room temperature
- Homogeneity: The test item appeared to be homogeneous

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: CBA/CaOlaHsd; Harlan Laboratories B.V., The Netherlands.
- Age: pre-test: 10 - 11 weeks (beginning of treatment), main study: 8 - 9 weeks (beginning of treatment)
- Weight at study initiation: 17.9 g - 21.8 g
- Housing: animals belonging to the same experimental group were kept in one cage
- Diet: ad libitum
- Water: tap water, ad libitum
- Acclimation period: least 5 days

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

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

25, 50 and 100%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
A solubility experiment was performed and the highest test item concentration, which could be technically used, was 100% of the undiluted test item. To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals.
Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 50 and 100%. This pre-experiment was cancelled and the animals euthanized approximately 4 hours after the first application, due to strong excitement observed in both animals.
Therefore, a second pre-test was performed and two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5 and 10% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6.
On day 3, the animal treated with 10% test item concentration showed an erythema of the ear skin (score 1) and high activity, excessive scratching and cleaning behaviour even 3 hours after the third application. Increase in ear weight for the animal treated with 5% and 10% was 12.4 and 23.6%, respectively compared to historical vehicle values. The animal treated with 5% test item concentration did not show any signs of local skin irritation or systemic toxicity.
Thus, the test item in the main study was assayed at 1, 2 and 5%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
• 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.
In addition, an index was calculated for the lymph node weight and – cell count as well as for the ear weight by dividing the mean values of the test item treated groups by the mean of the vehicle treated group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response and the cut-off value for the ear weight index regarding a positive response (ear irritation) was reported to be 1.1. However, these cut-off values mentioned in the respective papers have been determined using a different strain of mice and can thus not be implicitly adopted.


TREATMENT PREPARATION AND ADMINISTRATION:
The test item 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 1, 2 and 5% in MEK. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (Ø 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).

Positive control substance(s):

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

Statistics:

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. A One-Way-Analysis-of-Variance was used as a statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05).
The Dean-Dixon-Test was used for detection of possible outliers.
However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 25% TS: 1.38 50% TS: 1.97 100% TS: 2.28

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Vehicle control: 677.8 (+/- 236.7) 25% TS: 935.4 (+/- 325.0) 50% TS: 1336.2 (+/- 390.0) 100% TS: 1548.6 (+/- 334.5)

Any other information on results incl. tables

- Viability / Mortality: No deaths occurred during the study period.

- Clinical Signs: On day 2 and day 3, all animals treated with the test item concentrations of 25, 50, and 100% burrowed themselves into the bedding immediately after the application of the test item. On day 4 and day 5 the animals treated with 100% of the test item showed an erythema of the ear skin (Score 1).

- Body Weights: All body weights were within the range commonly recorded for animals of this strain and age.

- Lymph Node Weights and Cell Counts: A statistically significant increase in lymph node weights and –cell counts was observed in the test item treated groups in comparison to the vehicle control group (for lymph node cell counts: statistically significant for all treatment groups, for lymph node weights: statistically significant for mid and high dose group, respectively). For BALB/c mice, a cutoff value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count exceeded this threshold in all dose groups.

- Ear Weights: The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A statistically significant increase in ear weights was observed in all treatment groups. Furthermore, the cut-off-value (1.1) of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was reached or slightly exceeded in all dose groups (1.10– 1.16), thus indicating a slightly irritant property of the test item.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The present data on dermal sensitization do not fulfill the criteria laid down in 67/548/EEC and regulation (EU) 1272/2008, and therefore, a non-classification is warranted.