ACETIC ACID, 2-(3-CHLORO-5-CYANOPHENOXY)-, ETHYL ESTER

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5th September 2013 to 12th November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

ethyl 2-(3-chloro-5-cyanophenoxy)acetate

EC Number:

815-596-5

Cas Number:

1613307-27-9

Molecular formula:

C11H11CINO3

IUPAC Name:

ethyl 2-(3-chloro-5-cyanophenoxy)acetate

Test material form:

solid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

EXPERIMENTAL ANIMALS
Species and strain: CBA/J mice
Source: Jackson Laboratories
600 Main Street
Bar Harbor
ME 04609,
USA
Hygienic level: standard housing conditions during the study
Justification of strain: On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 5 animals / group
Sex: Female, nulliparous, non pregnant
Age of animals at starting: 8 weeks old (age-matched, within one week)
Body weight range at starting: 19.3 – 23.6 grams (The weight variation in animals in the study did not exceed ± 20 % of the mean weight.)
Acclimatization time: At least 5 days

Husbandry
Animal health: The animals were observed prior to the start of the study to ensure that no skin lesions present on ears.
Housing / Enrichment: The animals were housed one per cage.
Cage type: suspended wire bottom cages.
Bedding: Bedding was placed beneath the cage and changed at least three times per week
Light: 12 hours light/dark cycle.
Temperature: continuously monitored
Relative humidity: continuously monitored

Food and feeding
Animals received Fresh PMI - Diet #500 ad libitum

Water supply
Animals received water ad libitum.

Identification and randomisation
The animals were identified by cage notation and indelible tail marks. The animals were assigned to test groups using a computer based random number program.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Batch No. L-005183690-000F003 25% (w/v)

No. of animals per dose:

5

Details on study design:

Dose Selection and Justification of Dose Selection
The dose selection was based on solubility. The test article was tested for solubility in Acetone Olive Oil (4:1 AOO), N, N-dimethylformamide (DMF) and Dimethyl sulfoxide (DMSO). Test article L-005183690-000F003 was found to be soluble in all vehicles at 25% (w/v).
The Study Director in consultation with the sponsor chose DMF as the vehicle.
The maximum concentration of test item in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 25% (w/v) for L-005183690-000F003 at the application site.
In the screening, all mice were observed daily for any clinical signs, either of local irritation at the application site, or systemic toxicity and for mortality.
Body weights were recorded on Day 1 immediately prior to dosing and on Day 6 prior to BrdU injection. Ear thickness was measured on Day 1 (pre-dose), Day 3 and Day 6 before sacrifice.
During the Screening Test, no mortality or signs of systemic toxicity were observed. The mice were given an intraperitoneal injection of the thymidine analog 5-bromo-2'-deoxy-uridine (BrdU)
five days following the initial dose, and five hours prior to sacrifice. At sacrifice, the auricular lymph nodes were isolated, single-cell suspensions of lymph node cells (LNC) were generated, and the LNC suspensions were analyzed by flow cytometry for BrdU incorporation and the total number of LNC.
One animal in the 25% L-005183690-000F003 dose group had test article residue at the dose sites on Day 3. This residue did not effect the ear measurements. The ear thickness values are summarized in Tables 1A and 1B. The ear thickness values were within the acceptable range. There were no indications of any irritancy at the site of application on the experimental animals.
Gross observations of the auricular lymph nodes were made, and the lymph nodes were collected (see Appendix C for lymph node location). The auricular lymph nodes were combined for each animal and single-cell suspensions were generated in RPMI-10 medium and fixed with 85% ethanol. The cell suspensions were used to determine BrdU incorporation into the lymphocyte DNA (percentage of proliferating BrdU+ Lymph Node Cells (%BrdU+ LNC)) and the total number of cells in the nodes, for each Individual animal.
Flow cytometric analyses were conducted using a FACScan flow cytometer, equipped with an Omnichrome argon laser emitting at 488 nm with 15 mW of power. For DNA and/or BrdU determinations, clumps of nuclei were excluded from analysis using gates set on integrated red fluorescence signals. The histograms generated in these experiments were analyzed using Cell Quest Software.
Proliferation of Lymphocytes (# of BrdU + cells): Measured aliquots of fixed cells were washed and resuspended In 1 N HCI containing 0.5% Triton" X-100 This acid denaturation step allows the BrdU antibody to quantitatively interact with the BrdU that has been incorporated into the cellular DNA. Following a 1-hour denaturation period. The samples were neutralized by washing with sodium tetra borate. The cell nuclei were then washed with a staining buffer (1.0% Bovine Serum Albumin, 0.03% sodium azide and 0.5% Tween"ZO in PBS) and incubated with the BrdU-specific (fluorescein conjugated) antibody. The nuclei were then washed with the staining buffer, resuspended in DPBS containing the DNA-specific dye propidlum iodide and the percentage of nuclei staining positive for BrdU (i.e., proliferating cells in "s" phase) was determined using flow cytometry.

Topical application
Groups of five CBAlJ mice were treated by a topical application of the test article (at a single concentration), Vehicle Control (DMF) or Positive Control (25% HCA) to the dorsum of each ear once daily for three consecutive days. The test substance was spread over the entire dorsal surface of the ear using a micropipette at 25 ~l/ear.

Positive control substance(s):

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

Results and discussion

In vivo (LLNA)

Results

Parameter:

SI

Value:

1.2

Test group / Remarks:

L-005183690-000F003

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The results of the proliferation assay are summarized in Table 2, Appendix E.

DETAILS ON STIMULATION INDEX CALCULATION
Calculation of Stimulation Index (SI): For each animal, lymph node cell proliferation as measured by the number of Proliferating Lymphocytes was determined by flow cytometry and the mean ±SD was then calculated for each group. The number of Proliferating Lymphocytes in each animal was then divided by the mean number of Proliferating Lymphocytes in the Vehicle Control group. This 'Test I Control Ratio" is the "Stimulation Index" (SI) and was calculated for each animal according to Equation 3 below:
Equation 3:
# of Proliferating Lymphocytes per animal / Mean # of Proliferating Lymphocytes of Vehicle Control group = Stimulation Index (SI) per animal
The mean Sl and standard deviation (SO) were calculated for each group.

CLINICAL OBSERVATIONS:
No mortality or signs of systemic toxicity were observed during the study. All animals survived the in-life phase of the study and were observed to be normal. There were no difficulties in administration of these test articles or with their adherences to the dosed ears. One animal in the 25% L-005183690-000F003 dose group had test article residue at the dose sites on Day 3. This residue did not effect the ear measurements. Ear thickness measurements and individual animal observations indicated that neither of the test article treatments resulted in dermal irritation.

BODY WEIGHT
Body weight changes were normal.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Topical application of test article L-005183690-000F003 at 25% (w{v) in DMF resulted in an SI value of less than 3.0 (SI < 3.0). Therefore, the test article is not a dermal sensitizer in the Screening local lymph Node Assay in Mice (LLNA).

Executive summary:

Objective: To screen for possible sensitizing potential of topically applied test articles, The flow cytometry-modified LLNA protocol on which this screen is based is designed to be an alternative assay for the Buehler Guinea Pig Sensitization Assay defined in the NIH report, "The Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Contact Dermatitis Potential of Chemicals/Compounds", NIH No, 99-4494, 1999, and the LLNA as defined in EPA OPPTS 8702600, Final GUideline (March 2003), and OECD Guideline for the Testing of Chemicals No, 429. revised July 2010.

 

Method Synopsis: The test article was tested for solubility in Acetone:Olive Oil (4: 1, AOO), N,N-Dimethylformamide (DMF) and Dimethyl sulfoxide (DMSO). Test article L-005183690-000F003 was found to be soluble in all vehicles at 25% (w/v). The Study Director, in consultation with the Sponsor, chose DMF as the vehicle. A single concentration of the test article was tested: 25% (w/v) L-005183690-000F003. For the test article, one group of five healthy female CBNJ mice was treated by topical application to the dorsum of each ear, once daily for three consecutive days. A Vehicle Control group of five mice was treated with N,N-Dimethylformamide (DMF), and another group of five mice was treated with the Positive Control, alpha-Hexylcinnamaldehyde. 85% in DMF (25% HCA), in the exact same manner. The Vehicle Control and Positive Control groups were shared with one other study (MB# 13-22020.26), The mice were given an intraperitoneal injection of the thymidine analog 5-bromo-2'-deoxy-uridine (BrdU) five days following the initial dose, and five hours prior to sacrifice, At sacrifice, the auricular lymph nodes were isolated, single-cell suspensions of lymph node cells (LNC) were generated, and the LNC suspensions were analyzed by flow cytometry for BrdU incorporation and the total number of LNC. The amount of proliferating (#BrdU+) LNC was determined as a measure of the proliferative response of the local lymph node. The stimulation index (SI) was calculated by dividing the proliferative response (#BrdU+ LNC) of each test article-treated animal by the mean proliferative response of the Vehicle Control group The mean SI ± S,D, was calculated for each group from the individual animal data. Test article groups that yielded an SI ≥ 3 were characterized as sensitizing substances. 

 

Summary: All animals survived the in-life phase of the study and were observed to be normal. There were no difficulties in administration of these test articles or with their adherences to the dosed ears. One animal in the 25% L-005183690-000F003 dose group had test article residue at the dose sites on Day 3. One animal in the 10% L-004950413-000K005 dose group had test article residue at the dose sites on Day 2, and all animals in that dose group had residue on Days 3 and 4 This residue did not effect the ear measurements, Body weight changes were normal. Ear thickness measurements and individual animal observations indicated that neither of the test article treatments resulted in dermal irritation.

 

The SI of the Positive Control group, 25% HCA, was 5.8. The group SI values for the test article were as follows:

L-005183690-000F003 25% (w/v)  - 1.2
L-004950413-000K005 10% (w/v) - 1.4

Conclusion: Topical application of test article L-005183690-000F003 at 25% (w/v)  in DMF resulted in an SI values less than 30 (SI < 3.0). Therefore, the test article is not a dermal sensitizer in the Screening Local Lymph Node Assay in Mice (LLNA).