6,6'-(Ethane-1,2-diylbis(azanediyl))bis(dibenzo[c,e][1,2]oxaphosphinine-6-oxide)“

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-24 - 2017-09-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

6,6'-(Ethane-1,2-diylbis(azanediyl))bis(dibenzo[c,e][1,2]oxaphosphinine-6-oxide)“

EC Number:

815-096-7

Cas Number:

1421927-53-8

Molecular formula:

C26H22N2O4P

IUPAC Name:

6,6'-(Ethane-1,2-diylbis(azanediyl))bis(dibenzo[c,e][1,2]oxaphosphinine-6-oxide)“

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Species: Mice CBA/Ca
Source: Velaz, Prague, Czech Republic
Number and Sex of Animals: 29 females
Age at First Dose: 8-10 weeks, female animals were non-pregnant and nulliparous were used
Animal Health: The health condition of animals was examined by a veterinarian before initiation of the study
Acclimation: The animals were acclimated in identical conditions as during the experiment for 5 days prior to the start of treatment. The acclimation was according to standard operation procedures.
Housing Condition: The animals were housed in polypropylene cages (5 animals per cage) suspended on stainless steel racks, in a room equipped with central air-conditioning. The room temperature was within the range of 22 ± 3°C, relative humidity was within the range of 50 - 60 %. The light regimen was set to a 12-hour light / 12-hour dark cycle The sanitation was performed according to standard operation procedures.
Diet: A laboratory food ssniff (ssniff Spezialdiäten GmbH, Germany) was served ad libitum, each day approximately at the same time.
Water: The animals received tap water for human consumption. Supply of drinking water was unlimited. The quality of drinking water is
periodically monitored (including microbiological control) and recorded; certificate of analysis is included in raw data.
Bedding: Lignocel S3/4, Lufa - ITL GmbH, Germany
Animals Identification: Each animal was marked with permanent pen on the tail. Each cage was affixed with a cage card containing pertinent animal and study information.
Justification for the Choice of Species: The CBA/Ca mice are the standard experimental rodent of choice and recommended by OECD Guideline No. 429.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

25, 50, 100%

No. of animals per dose:

5

Details on study design:

Test procedure of Pre-screen test:
All mice (2 mice/group) were observed daily for any clinical signs of toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to the termination (day 6). Both ears of each mouse were assessed for any signs of irritation. Erythema scores are shown in table 1. Ear thickness was measured by a calliper on Day 1 (pre-dose), Day 3 and Day 6. Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥25%.
Main study:
Day 1:
Each animal was identified and the body weight was recorded. To the dorsum of each ear 25μL of the appropriate dilution of the test item, or the vehicle alone was applied.
Days 2 and 3:
The application procedure carried out on day 1 was repeated.
Days 4 and 5: No treatment.
Day 6:
The body weight of each animal was recorded. 250μL of phosphate-buffered saline (PBS) containing 2 μCi (7.4 x 104 Bq) of 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine was injected into all test and control mice via the tail vein. Five hours later, the animals were sacrificed. The draining auricular lymph nodes from each ear were excised and pooled in PBS for each experimental group (pooled treatment group approach).
Preparation of cell suspensions:
Cell suspension of lymph node cells from pooled treatment groups was prepared by gentle mechanical disaggregation by glass homogenizer. Lymph node cells were centrifuged by 600g at 4C for 10 min. Suspension of cells were precipitated with 5% trichloroacetic acid (TCA) at 4C for 18-20h. Pellets were centrifuged by 2000g at 4C for 5 min., re-suspended in 1 ml TCA and transferred to gamma
counting tubes for 125I-counting.
Determination of cellular proliferation (incorporated radioactivity):
Incorporation of 125I-iododeoxyuridine was measured by 125I-counting on Automatic Gamma Counter Wizard2 (Perkin Elmer) as counts per minute (CPM). The incorporation is expressed as disintegrations per minute (DPM)/animal.
Clinical Observation:
Animals were carefully observed once daily for any clinical symptoms, either of local irritation at the application site or of the systemic toxicity. All observations were systematically recorded with individual records being maintained for each animal.
Body Weight:
The animal body weights were determined at the start of the test and at the scheduled day of euthanasia of the animals.
Calculation of results:
Results are expressed as the Stimulation Index (SI). The SI was obtained by dividing the pooled DPM for each treatment group by the incorporation of the pooled DPM vehicle control group; this yields a mean SI. A substance is regarded as sensitizer in the LLNA test when SI≥3.
EC3 value, which induce stimulation indices, is determined by linear interpolation of points on doseresponse
curve, immediately above and below of SI value, according to the equation: EC3=c+[(3-d)/(b-d)]x(a-c)
a – higher concentration, b – SI of higher concentration, c – lower concentration d – SI of lower concentration If all points are below the stimulation index of three, no EC3 value can be stated.

Positive control substance(s):

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

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.01

Test group / Remarks:

25%

Results 2

Key result

Parameter:

SI

Value:

1.33

Test group / Remarks:

50%

Results 3

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

100%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The skin sensitizing potential of EDA-DOPO was assessed using the murine local lymph node assay. Based on the results of this study, EDA-DOPO is not considered a skin sensitizer under the condition of this LLNA study.

Executive summary:

The skin sensitization potential of EDA-DOPO was evaluated by LLNA method, which basic underlying principle is that sensitizers induce a primary proliferation of lymphocytes in the auricular lymph nodes draining the site of chemical application.

In the present study, the test item was applied to the dorsum of each ear of five female mice (CBA/Ca) per group over three consecutive days, at three concentrations. All animals survived throughout the test period without showing any clinical signs. In comparison with the control group, the minor increase in lymph node weight was observed at concentrations 50 and 100%. The increase of lymph node weight was not dose dependent. A similar trend was registered in the evaluation of DPM of the

lymph nodes. Calculated SI values in treated groups remained under the value of 3, which is the threshold to consider the substance as a sensitizer. Therefore, it was not possible to calculate an EC3 value. These results demonstrate that the test item EDA-DOPO was not a skin sensitiser under the test conditions of this study.