Bis(2-chloroethyl) ether

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 02-oct-2015 to 18-mar-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

temperature range was 18.0-25.9 °C instead of 22 ± 3 °C and the actual relative humidity range was 31-72 % instead of 30-70 %

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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:

Bis(2-chloroethyl) ether

EC Number:

203-870-1

EC Name:

Bis(2-chloroethyl) ether

Cas Number:

111-44-4

Molecular formula:

C4H8Cl2O

IUPAC Name:

1-chloro-2-(2-chloroethoxy)ethane

Test material form:

liquid

Specific details on test material used for the study:

TEST MATERIAL
- Name (as cited): 2,2`- Dichlorodiethyl ether
- Purity: 99.76%

SOURCE OF TEST MATERIAL
- Batch No.: 20150706
- Expiration date of the lot/batch: 01 June 2016
- Purity test date: 28 August 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled Room Temperature (15-25°C, below 70 RH%), protected from light

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories S.r.l.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks old (age-matched, within one week)
- Weight at study initiation: 20.0-21.7g (The weight variation in animals in the study did not exceed ± 20 % of the mean weight)
- Housing: Group caging / mice were provided with glass tunnel-tubes
- Diet: ssniff® SM Rat/Mouse ad libitum
- Water: ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.0 – 25.9 °C
- Humidity (%): 31 - 72 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Preliminary Irritation/Toxicity Test:
Item concentrations were 100 % (undiluted) and 50 % (w/v) in AOO

Main Test:
Item concentrations were 50, 25 and 10 % (w/v) in AOO

No. of animals per dose:

Preliminary Irritation/Toxicity Test: 2 animals/dose
Main Test: 4 animals/dose

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: 10.6 g/L in water at 25°C
- Irritation: No irritation
- Systemic toxicity: Both animals in the 100 % (undiluted) dose group were found dead on Day 2. Animals in the 50% dose group were symptom-free.
- Ear thickness measurements: No change at day 3
- Erythema scores: ES score = 0

MAIN STUDY
No mortality or signs of systemic toxicity. No indications of any irritancy at the site of application. No treatment related effects on the mean body weight changes. Body weight loss of 5.1% was observed for one animal (No. 13) in the 10 % (w/v) dose group but this was considered as animal variability and not related to test item.

ANIMAL ASSIGNMENT AND TREATMENT
- Animal assignment: The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.
- Criteria used to consider a positive response: Stimulation index

TREATMENT PREPARATION AND ADMINISTRATION:
- Justification of dose selection: The maximum concentration of test item in an acceptable solvent was established according to OECD guideline 429 and was found to be 100 % (undiluted). During the Preliminary Irritation / Toxicity Test mortality was observed. Both animals in the 100 % (undiluted) dose group were found dead on Day 2. The 50 % (w/v) dose was selected as top dose for the main test as no mortality occur, no marked body weight loss (>5%) and no increased ear thickness values were detected, and draining auricular lymph nodes were normal at this dose.

Positive control substance(s):

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

Results and discussion

Positive control results:

No mortality or signs of toxicity were observed. A significant lymphoproliferative response (stimulation index value of 8.6) was noted. The DPN values observed for the vehicle and positive control substance in this experiment were within the historical control range.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

50% (w/v) in AOO

Results 2

Parameter:

SI

Value:

1

Test group / Remarks:

25% (w/v) i n AOO

Results 3

Parameter:

SI

Value:

1.3

Test group / Remarks:

10% (w/v) in AOO

Cellular proliferation data / Observations:

CLINICAL OBSERVATIONS
No mortality or signs of systemic toxicity were observed during the main study. There were no indications of any irritancy at the site of application.

BODY WEIGHTS
No treatment related effects were observed on the mean body weight changes. Body weight loss of 5.1% was observed for one animal (No. 13) in the 10 % (w/v) dose group but this was considered as animal variability and not related to test item.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present assay, 2,2`-Dichlorodiethyl ether, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

Executive summary:

The aim of this study was to determine the skin sensitisation potential of 2,2`-Dichlorodiethyl ether following dermal exposure. The study was performed according to OECD Test Guideline No. 429 and EC No 440/2008 Guideline B.42.

Based on the results of the Preliminary Compatibility Test and on the recommendations of the OECD Guideline, the test item was formulated in acetone:olive oil 4:1 (v:v) mixture (abbreviated as AOO). The Preliminary Irritation / Toxicity Test was performed in CBA/CaOlaHsd mice using two doses: 100 % (undiluted) and 50 % (w/v) in AOO. Based on the observations recorded in the preliminary test, the 50 % (w/v) was selected as top dose for the main test.

The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

No mortality or signs of systemic toxicity were observed during the study. No treatment related effects were observed on the mean body weight changes. The stimulation index values were 1.1, 1.0 and 1.3 at concentrations of 50, 25 and 10 % (w/v), respectively. The result of the positive control substance α-Hexylcinnamaldehyde (HCA) showed a significant lymphoproliferative response (stimulation index value of 8.6), which confirmed the validity of the assay.

In conclusion, under the conditions of the present assay 2,2`-Dichlorodiethyl ether, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.