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Diss Factsheets

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:
2012-06-28 - 2012-09-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to an internationally accepted guideline. All study parameters are based on the specific guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
During the acclimation phase of the pre-test the relative humidity in the animal room was >95% instead of 45 – 65% for several hours.
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Phenyl diamidophosphate
EC Number:
231-218-6
EC Name:
Phenyl diamidophosphate
Cas Number:
7450-69-3
Molecular formula:
C6H9N2O2P
IUPAC Name:
[(diaminophosphoryl)oxy]benzene
Test material form:
solid: crystalline
Details on test material:
Identity: PPDA
Purity: 92.4% (HPLC)
dose calculation not adjusted to purity

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Test system: Mice, CBA/CaOlaHsd
Rationale: Recognised as the recommended test system
Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
Number of animals for the pre-test: 2 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1
Age: pre-test: 11 - 12 weeks (beginning of treatment)
Main study: 8 – 9 weeks (beginning of treatment)

Identification: 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.
Acclimatisation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

Husbandry
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: group
Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
Bedding: granulated soft wood bedding, (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)
Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum (Harlan Laboratories B.V., 5960 AD Horst, Netherlands)
Water: tap water, ad libitum (Gemeindewerke, 64380 Rossdorf, Germany)
Environment: temperature 22 + 2°C, relative humidity 45-65%; (during pre-test: 45->95% for several hours), artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 2.5, 5, and 10% (w/w) in dimethyl sulfoxide. 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 was treated with an equivalent volume of the relevant vehicle alone (control animals).
No. of animals per dose:
4
Details on study design:
Administration of 3H-Methyl Thymidine
3H-methyl thymidine (3HTdR) was purchased from Hartmann Analytic, 38124 Braunschweig, Germany (specific activity, 2 Ci/mmol; concentration, 1 mCi/mL).
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline (PBS) containing 19.5 µCi of 3HTdR (equivalent to 3HTdR 78 µCi/mL) were injected into each test and control mouse via the tail vein.

Determination of Incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Sodium (Release, WDT, 30827 Garbsen, Germany).
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). 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 at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to plastic scintillation vials with 10 mL of ‘Ultima Gold’ scintillation liquid (Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany) and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a -scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany). 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 (DPM).

In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: At least once daily from experimental start to necropsy.
Body weights: In the pre-test prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.
Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6).
Ear weights: In the pre-test after sacrifice; biopsy punches were taken from each ear.
Clinical signs (local / systemic): Clinical signs (systemic toxicity or local skin irritation) were recorded at least once daily. Especially the treatment sites were observed carefully.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
see below

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
test item 2.5%
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
negative control
Key result
Parameter:
SI
Value:
0.66
Test group / Remarks:
Test item 5%
Key result
Parameter:
SI
Value:
1.02
Test group / Remarks:
test item 10 %

Any other information on results incl. tables

Calculation and Results of Individual Data

Test item concentration % (w/w)

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

17

---

---

---

---

---

BG II

16

---

---

---

---

0

1

5641

5624,5

8

703.1

1.00

2.5

2

6784

6767,5

8

845.9

1.20

5

3

3745

3728,5

8

466.1

0.66

10

4

5757

5740,5

8

717.6

1.02

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

1    =  Control Group

2-4=  Test Group

S.I. =  Stimulation Index

a)   =  The mean value was taken from the figures BG I and BG II

b)    =  Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Positive control substance: α-Hexylcinnamaldehyde

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

Positive Control Item concentration % (w/v)

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

18

---

---

---

---

---

BG II

19

---

---

---

---

0

1

3076

3058

8

382.2

1.00

5

2

2695

2677

8

224.6

0.88

10

3

4638

4620

8

577.4

1.51

25

4

11414

11396

8

1424.4

3.73

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

1    =  Control Group

2-4=  Test Group

S.I. =  Stimulation Index

a)   =  The mean value was taken from the figures BG I and BG II

b)    =  Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item PPDA was not a skin sensitiser under the test conditions of this study.
Executive summary:

In order to study a possible skin sensitising potential of PPDA, three groups each of four female mice were treated once daily with the test item at concentrations of 2.5, 5, and 10% (w/w) in dimethyl sulfoxide by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment. A control group of four mice was treated with the vehicle (dimethyl sulfoxide) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of3H-methyl thymidine measured in ab-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 1.20, 0.66 and 1.02 were determined with the test item at concentrations of 2.5, 5, and 10% (w/w) in dimethyl sulfoxide. A dose response was not observed.

The EC3 value could not be calculated, since all obtained SI´s were below the threshold value of 3.