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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 December 2018 - 12 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 22 July 2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction products of 2,3-epoxypropyl phenyl ether and 2,2'-iminodi(ethylamine)
EC Number:
948-518-4
Molecular formula:
(C13H23N3O2 . C31H43N3O6)x
IUPAC Name:
Reaction products of 2,3-epoxypropyl phenyl ether and 2,2'-iminodi(ethylamine)
Test material form:
liquid
Details on test material:
PGE-DETA adduct
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Olin Corporation Batch: 17-09-502-04 QM 14K17-54
- Expiration date of the lot/batch: 30 October 2019
- Purity test date: Approx. 97% (including the oligomeric isomers)


The test item was placed into an appropriate container on a tared balance and DMSO was added (weight per weight).
The different test item concentrations were prepared serially.
The preparations were made freshly before each dosing occasion.

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: Envigo RMS B.V., Inc
- Females nulliparous and non-pregnant
- Age at study initiation: 1st pre-test: 10 - 11 weeks 2nd pre-test: 8 - 9 weeks 3rd & 4th pre-test: 9 - 10 weeks Main study: 12 - 13 weeks
- Weight at study initiation: 18.3 - 23.5 g
- Housing: Grouped 5 animals per solid bottom Makrolon cage containing soft wood bedding.
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days


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

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
The definitive test was performed at concentrations of 0.01, 0.05, and 0.25% due to effects seen during a series of pretest experiments that assessed a wide range of concentrations; 0.05 - 50%.
No. of animals per dose:
Pretests: 1 females (nulliparous and non-pregnant) per dose group

Main test: 5 females (nulliparous and non-pregnant) per dose group
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Dimethylsulfoxide was evaluated as vehicle and found acceptable
- Irritation: erythema observed at concentrations of 0.05% and above,
- Systemic toxicity: No systemic toxicity was observed at any concentration
- Ear thickness measurements: Increased ear thickness > 25% was observed at concentrations of 1% and above

MAIN STUDY

ANIMAL ASSIGNMENT
The animals were distributed into the test groups at random. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were identified by tail tags. In the pre-experiment, animals were identified by cage number.


- Criteria used to consider a positive response:

A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• 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.

TREATMENT PREPARATION AND ADMINISTRATION:
Test Item Administration
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 0.01, 0.05, and 0.25% in DMSO. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear once daily for three consecutive days. Three further groups of mice (two vehicle control groups and one positive control group) were treated with an equivalent volume of the relevant vehicle alone or with the positive control item at 25% (w/v).

Administration of 3H-methyl-thymidine
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20 μCi of 3H-methyl thymidine (equivalent to 79.9 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Terminal Procedure
Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.

Preparation of Single Cell Suspensions
The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for approximately 18 hours for precipitation of macromolecules.

Determination of cellular proliferation (incorporation of 3HTdR)
The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.

Observations
Clinical Observations
All animals were observed on a daily basis, including pre- and post-dose observations on days 1, 2 and 3. Any clinical signs of systemic toxicity, local skin irritation or signs of ill health during the study were recorded.



Positive control substance(s):
mercaptobenzothiazole (CAS No 149-30-4)
Statistics:
The mean values and standard deviations were calculated for body weight.

Where appropriate, the EC3 value were calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot. All calculations conducted on the DPM values and the ear thickness were performed with a validated test script of “R”, a language and environment for statistical computing and graphics. Within the program a statistical analysis conducted on the DPM values and the ear thickness to assess whether the difference was statistically significant between the test item groups and negative control group. Statistical significance was set at the five per cent level (p < 0.05). Additionally, the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. No outlier was detected. However, both biological and statistical significance will be considered together.

Results and discussion

Positive control results:
S.I. = 7.6 (positive)

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
0.7
Test group / Remarks:
Test Item: 0.01% in DMSO
Parameter:
SI
Value:
0.5
Test group / Remarks:
Test Item: 0.5% in DMSO
Parameter:
SI
Value:
0.3
Test group / Remarks:
Test Item: 0.25% in DMSO
Cellular proliferation data / Observations:
Calculation of the EC3 value
The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Viability / Mortality
No deaths occurred during the study period

Clinical Signs
No signs of systemic toxicity were observed during the study period. From days 1 to 6, the animals showed an erythema of the ear skin (Score 1). Additionally, on day 6, scaly ears and erythema was observed.

Body Weights
There was not obvious affect of treatment on body weight. Body Weights were within the range commonly recorded for animals of this strain and age.

Ear Thickness
The measured ear thickness of all animals treated was recorded prior to the 1st application, on study day 3 and prior to necropsy (day 6). A statistically relevant increase in ear thickness was not observed.


Any other information on results incl. tables

Calculation and Results of Individual Data

Test item concentration

DPM values measured

DPM−BG per animal  (2 lymph nodes)a

S.I.b

%

Group no.

Animal no.

 

---

---

BG I

16

---

 

---

---

BG II

19

---

---

0

1

1

1775

1757.5

---

1

2

2846

2828.5

---

1

3

2562

2544.5

---

1

4

3937

3919.5

---

1

5

4151

4133.5

---

0.01

2

6

2170

2152.5

0.7

2

7

1759

1741.5

0.6

2

8

1868

1850.5

0.6

2

9

2542

2524.5

0.8

2

10

3085

3067.5

1

0.05

3

11

2347

2329.5

0.8

3

12

2161

2143.5

0.7

3

13

1196

1178.5

0.4

3

14

835

817.5

0.3

3

15

837

819.5

0.3

0.25

4

16

422

404.5

0.1

4

17

867

849.5

0.3

4

18

667

649.5

0.2

4

19

952

934.5

0.3

4

20

976

958.5

0.3

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

1 = Control Group for the test item and for the positive control item
2-4 = Test Groups
S.I. = Stimulation Index
a) = values corrected for mean background value (BGI and BGII).
b) = Stimulation Indices relative to the mean of the control group (Group 1)

Applicant's summary and conclusion

Interpretation of results:
other: Not a skin sensitizer under the test conditions of this study
Conclusions:
The test item 1,2 Ethylenediamine,N-(2-Aminoethyl)-, reaction products with glycidyl Ph ether was not a skin sensitiser under the test conditions of this study.
Executive summary:

In the study the test item 1,2 Ethylenediamine,N-(2-Aminoethyl)-, reaction products with glycidyl Ph ether formulated in DMSO (dimethylsulfoxide) was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 0.01, 0.05, and 0.25%. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by four pre-experiments.

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. From days 1 to 6, the animals showed an erythema of the ear skin (Score 1). Additionally, on day 6, scaly ears and erythema was observed.

In this study Stimulation Indices (S.I.) of 0.7, 0.5, and 0.3 were determined with the test item at concentrations of 0.01, 0.05, and 0.25% in DMSO, respectively.

Conclusion: The test item 1,2 Ethylenediamine,N-(2-Aminoethyl)-, reaction products with glycidyl Ph ether was not a skin sensitiser under the test conditions of this study.

.