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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:
The study was conducted between 05 April 2017 and 25 April 2017
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Specific details on test material used for the study:
Identification: Diammonium phthalate
Batch: 7172002
Purity: 99.3%
Physical state, appearance: White crystalline powder
Expiry Date: August 2017
Storage Conditions: At room temperature, protected from moisture

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS:
Source: Envigo RMS B.V., / 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 (beginning of treatment): Pre-test: 10 - 11 weeks; Main study: 8 - 9 weeks
Body weight (at start of experiment): Main study: 16.7 to 20.7 g
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.
Acclimation: 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.

ENVIRONMENTAL CONDITIONS:
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
Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 ± 2°C
relative humidity approx. 45-65% (except for deviation)
artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
other: ethanol/water (3+7, v/v)
Concentration:
0, 5, 10, and 25% (w/v).
No. of animals per dose:
4 females per dose
Details on study design:
TEST ITEM PREPARATION:
VEHICLE AND DOSE SELECTION:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration which could be technically used was a 25% solution in ethanol/water (3+7, v/v). Vortexing was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved by the use of other vehicles.

To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals . Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 25% once daily each on three consecutive days.

Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6.

At the tested concentrations the animals did not show any signs of systemic toxicity. On days 4 and 5, the animals showed an erythema of the ear skin (Score 1).

Thus, the test item in the main study was assayed at 5, 10, and 25%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

TEST ITEM PREPARATION:
The test item was placed into a volumetric flask on a tared balance and ethanol/water (3+7, v/v) was quantitatively added (w/v).
The different test item concentrations were prepared serially.
The preparations were made freshly before each dosing occasion.

EXPERIMENTAL DESIGN AND STUDY CONDUCT
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 5, 10, and 25% in ethanol/water (3+7, v/v). 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 (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

Administration of 3H-methyl-thymidine:
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20.4 μCi of 3H-methyl thymidine (equivalent to 81.4 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 for each experimental 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.

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 (DPM).

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.

Determination of Ear Thickness:
In the pre-test, the ear thickness was determined prior to the first application of the test item (day 1), on day 3, and on day 6 prior to sacrifice using a micrometer.

In the pre-test, after the lymph nodes had been excised, both ears of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed per animal using an analytical balance.

Determination of Body Weights:
The body weights were recorded on day 1 (prior to dosing) and prior to sacrifice (pre-test) or prior to treatment with 3HTdR (main experiment)

DATA EVALUATION:
Interpretation of raw data:
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/lymph node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

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.















Positive control substance(s):
other: α-Hexylcinnamaldehyde

Results and discussion

Positive control results:
The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1 v/v) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in October 2016.

Test item concentration %: 0
Stimulation Index (S.I.): 1.00

Test item concentration %: 5
Stimulation Index (S.I.): 1.50

Test item concentration %: 10
Stimulation Index (S.I.) 3.84

Test item concentration %: 25%
Stimulation Index (S.I.): 11.76

EC3 calculated as 8.2% (w/v)

The positive control substance was shown to be a skin sensitiser and was within historical control data values.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1
Test group / Remarks:
Control Group
Remarks on result:
other: non-sensitising
Parameter:
SI
Value:
0.89
Test group / Remarks:
5% test concentration
Remarks on result:
other: non-sensitising
Parameter:
SI
Value:
0.98
Test group / Remarks:
10% test concentration
Remarks on result:
other: non-sensitising
Parameter:
SI
Value:
1.2
Test group / Remarks:
25% test concentration
Remarks on result:
other: non-sensitising
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA:
See table in any other information on results incl. tables section.

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

CLINICAL OBSERVATIONS:
No deaths occurred during the study period. No signs of systemic toxicity were observed during the study period. On day 3, the animals showed a transient erythema of the ear skin (Score 1).

BODY WEIGHTS:
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Any other information on results incl. tables

Calculation and Results of Individual Data

Vehicle: ethanol/water (3+7, v/v)

Test item concentration %

Group

Measurement, DPM

Calculation

Result

DPM-BGa)

Number of lymph nodes

DPM per lymph nodeb)

S.I.

-

BG I

14

-

-

-

-

-

BG II

16

-

-

-

-

0

1

5307

5292

8

661.5

1.00

5

2

4748

4733

8

591.6

0.89

10

3

5207

5192

8

649.0

0.98

25

4

6386

6371

8

796.4

1.20

1 = Control Group

2-4 = Test Group

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:
GHS criteria not met
Conclusions:
The test item Diammonium phthalate was not a skin sensitiser under the test conditions of this study.
Executive summary:

In the study the test item Diammonium phthalate formulated in ethanol/water (3+7, v/v) was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 25% (w/v). The highest concentration tested was the highest concentration that could technically be achieved.

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. On day 3, the animals showed a transient erythema of the ear skin (Score 1).

I

n this study Stimulation Indices (S.I.) of 0.89, 0.98, and 1.20 were determined with the test item at concentrations of 5, 10, and 25% (w/v) in ethanol/water (3+7, v/v), respectively.

The test item Diammonium phthalate was not a skin sensitiser under the test conditions of this study.