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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:
2017-09-20 to 2017-10-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 2010-07-22
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008-05-30
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003-03
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2015-06-05
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
State of aggregation at room temperature: white powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature; protected from light; dry in closed containers

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/OlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo (formed partially from Harlan in September 2015), 5800 AN Venray, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: The animals were derived from a controlled full-barrier maintained breeding system (SPF).
- Age at study initiation: 8-9 weeks
- Weight at study initiation: Prescreen test:18.3 - 21 g, Main Study: 18.3 - 22.5 g
- Housing: The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
- Diet (ad libitum): Altromin 1324 maintenance diet for rats and mice
- Water ( ad libitum): Tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at least five days
- Indication of any skin lesions: No skin lesions observed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Relative Humidity (%): 55 ± 10%
- Air changes (per hr): at least 10 x / hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
12.5%, 25% and 50% (w/v)
Dose selection based on the results observed in the prescreen test: the maximum technically applicable concentration of the test item in the vehicle was
found to be 50% in AOO (4:1 (v/v) acetone / olive oil)
No. of animals per dose:
5 female mice per dose
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The maximum technically applicable concentration was found to be 50% in AOO (4:1 (v/v) acetone / olive oil)

- A prescreen test was performed which was conducted under the same conditions as the main study, except there was no assessment of lymph node proliferation and fewer animals per dose group were used.
- The mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site.
- Body weights were recorded pre-test and prior to termination (day 6).
- Both ears were observed for erythema and scored.
- Ear thickness measurements were performed on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6 using a digital calliper. Excessive local irritation was indicated by an erythema score ≥ 3 and/or ear swelling of ≥ 25%. In addition to a 25% increase in ear thickness, a statistically significant increase in the treated mice compared to control mice was used to identify irritants in the LLNA.
- Four animals were treated by topical application on three consecutive days at the following concentrations to the entire dorsal surface of each ear:
Two animals were treated with a test item concentration of 50% (suspended in AOO)
Two animals were treated with a test item concentration of 25% (suspended in AOO)
One animal was treated with 100% AOO and served as negative control.
- Before the first application, approximately 48 hours after the first application and shortly before sacrificing the thickness of both ears of all animals was measured. During this period also all clinical signs were recorded. Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge).
Results:
- Neither signs of systemic toxicity nor signs of excessive irritation at any application site could be detected in any animal.
- The animal treated with 100% AOO (negative control) showed sticky fur at the application sites on day 4.
- All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the duration of the prescreen test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: The animals were randomly selected using the validated departmental computerised system E-WorkBook (version 10.1.2, ID Business Solutions Ltd.). Identification was ensured by cage number and individual marking (tail, no ear marking).

- Criteria used to consider a positive response: A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the
test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0). The strength of the dose response, the statistical significance and the consistency of the vehicle and positive control responses may also be used when determining whether a borderline result is declared positive.

TREATMENT PREPARATION AND ADMINISTRATION:
Topical Application:
- Before the first application the thickness of both ears of all animals was measured using a digital caliper.
- Each mouse was treated by topical application of 25 μL of the selected solution to the entire dorsal surface of each ear.
- A second measurement of the ear thickness of all animals was carried out approximately 48 hours after the first application.
- Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1.

Administration of 3H-Methyl Thymidine:
- Five days after the first topical application (study day 6) all mice were dosed with 20 μCi 3H-methyl thymidine by intravenous injection (tail vein) of 250 μL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80 μCi/mL.

Preparation of Cell Suspension:
- Approximately 5 hours after injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation.
- Shortly before sacrificing, scoring of ear erythema and the thickness of ears of all animals was measured for a third time.
- The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS).
- A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size)
- After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
- Each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid (equivalent to OECD 429, in order to achieve a more efficient counting by reducing the background noise) was added. This solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of Incorporated 3H -Methyl Thymidine:
- The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM).
- Similarly, background 3H-methyl thymidine levels were also measured (5% TCA) in order to determine a baseline. Determination of radioactivity was performed individually for each animal.

OBSERVATIONS:
- Prior to the start of treatment all animals were examined to ensure that they had no observable skin lesions.
- Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application.
- The following clinical observations were observed and recorded: changes in nervous system function (e.g. pilo-erection, ataxia, tremors, and convulsions); changes in behaviour (e.g. aggressiveness, change in grooming activity, marked change in activity level); changes in respiratory patterns (i.e. changes in frequency and intensity of breathing such as dyspnoea, gasping, and rales), and changes in food and water consumption.
- Signs of lethargy and/or unresponsiveness and any clinical signs of more than slight or momentary pain and distress, or a >10% reduction in body weight from day 1 to day 6, and mortality were considered in the evaluation.
- The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).

EVALUATION OF RESULTS:
- The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX).
- Before DPM/NODE values were determined, background values were subtracted.
- EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3=c+[(3-d)/(b-d)]x(a-c), between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three.
- If all measured points are above or below the stimulation index of three, no EC3 value can be stated.

Positive control substance(s):
other: 1 % phenylenediamine in acetone:olive oil (4/1 v/v)
Statistics:
According to OECD 429 the SI calculation may need to be carried out without outliers. Therefore, DPM data of all dose and control groups were screened for outliers using Grubbs’, Nalimov’s and Dixon’s test, respectively. Values that failed at least one of the tests were labelled as outliers and excluded for SI calculation.

Results and discussion

Positive control results:
The positive-control substance, 1 % phenylenediamine in acetone:olive oil (4/1 v/v), exceeded the stimulation index of 3 (mean SI 8.6) confirming the reliability of the test system.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.6
Variability:
0.4 (SD)
Test group / Remarks:
12.5 % group
Parameter:
SI
Value:
1.4
Variability:
0.2 (SD)
Test group / Remarks:
25 % group
Parameter:
SI
Value:
1.3
Variability:
0.7
Test group / Remarks:
50 % group
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
- The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX).
- Before DPM/NODE values were determined, background values were subtracted.
- One measured DPM value (25% test item concentration) failed Grubbs’, Nalimov’s and Dixon’s outlier tests and was excluded for SI calculation.

EC3 CALCULATION
- Since all three stimulation indices were below the stimulation index of three, no EC3 value can be calculated.
- A dose-response relationship was not observed.

CLINICAL OBSERVATIONS:
- All animals survived throughout the test period without showing any clinical signs.

BODY WEIGHTS
- All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.

EAR THICKNESS:
- The means of the ear thickness per group showed no relevant difference compared to the negative control Please refer to the field "Any other information on results incl. tables" below.

Any other information on results incl. tables

Table 1: Mean ear thickness

Mean ear thickness

day 1

day 3

day 6

12.5% test group

18.4 mm

19.1 mm

19.0 mm

25% test group

18.2 mm

19.0 mm

18.3 mm

50% test group

18.1 mm

19.4 mm

18.5 mm

negative control group

18.3 mm

19.4 mm

19.2 mm

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the present test conditions, zinc 5-nitroisophthalate at concentrations of 12.5%, 25% or 50% (w/w) in acetone/olive oil (4/1 v/v) did not reveal any sensitising properties in the local lymph node assay and therefore should not be classified and labelled according to Regulation (EC) No.: 1272/2008 and its subsequent regulations.