Reaction mass of 1,2-Benzenedicarboxylic acid, 4-sulfo-, trisodium salt, 1,2-Benzenedicarboxylic acid, 3-sulfo-, sodium salt (1:3), Sodium sulphate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

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:

Produkt SPS

IUPAC Name:

Produkt SPS

Details on test material:

- Name of test material (as cited in study report): Produkt SPS
- Test-substance No.: 11/0629-1
- Lot/batch No.: 78522224U0
- Purity: Trisodium 4-sulphonophthalate: 25.5 g/100 g; Trisodium 3-sulphonatophthalate: 7.6 g/100 g; Disodium phthalate: 1 g/100 g; Sulphate: 60.6 g/100 g; Water: 4.8 g/100 g; Sum: 99.5 g/100 g; Determined by 1H-NMR-analysis
- Physical state/ colour: Solid/ white
- Homogeneity: The test substance was homogeneous by visual inspection
- Storage conditions: Room temperature
- Expiration date: December 10, 2012

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 11 weeks (pretest), 9 weeks (main test)
- Weight at study initiation: 18.5-22.7 g
- Housing: Single housing in Makrolon cages, type II with bedding (H 15005-29; Ssniff. Spezialitäten GmbH (Experimental Animal Diets Inc., 59494 Soest, Germany))
- Diet: STANRAB (P) SQC; SDS Special Diets Services. 67122 Altrip, Germany ad libitum
- Water: Tap water ad libitum
- Acclimation period: at least 5 days prior to the start of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 2.5, 5, 10% (w/w)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which could be technically used, was 25% (w/w) dilution in N,N- dimethylformamide.
- Irritation: At the test item concentration of 25 % (w/w) signs of local skin irritation such as ear erythema (score 1), incrustations, scaling and test item residues and an increase in ear weight (> 25 % in one animal) and ear thickness (>25 % in one animal) were observed. At the test item concentration of 10% (w/w) signs of local skin irritation such as ear erythema (score 1 and 2), incrustations, scaling and test item residues were also observed. Increases in ear weight and ear thickness were not observed.
- Lymph node proliferation response: Not determined.

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
Test Item Preparation
The test item preparations were produced individually on a weight per weight (w/w) basis shortly before application by stirring with a magnetic stirrer. The homogeneity of the test item preparation during application was ensured by stirring with a magnetic stirrer.

Topical Application
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 N,N-dimethylformamide. The application volume 25 μL/ear/day was spread over the entire dorsal surface of each ear once daily for three consecutive days. The control test group of mice was treated with an equivalent volume of the relevant vehicle alone. The positive control test group of mice was treated with 25 % α-hexyl cinnamaldehyde dissolved in N,N- dimethylformamide.

Administration of BrdU
BrdU was purchased from Roche Diagnostics GmbH. For injection it was dissolved in DPBS (10 mg/mL) before administration and stored in a refrigerator until use. Four days after the first topical application (day 5) 0.5 mL of BrdU solution (5mg/per mouse/injection) were intraperitoneally injected.

Determination of Incorporated BrdU
Approximately 24 hours after treatment with BrdU the mice were sacrificed and the draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through a 70 μm nylon mesh. The lymph node cells were resuspended in 6 mL DPBS. After cell count with a cell counter (Casy Cell Counter. Innovatis), cell suspensions of 100 000 cells / mL were adjusted.
The incorporation of BrdU into lymph node cells was determined using a commercial cell proliferation assay kit (Roche Applied Science. Mannheim):
100 μL of the lymph node cell suspension (100 000 cells /mL) were added to the wells of a flat-bottom microplate in triplicate. After fixation and denaturation of the lymph node cells with FixDenat (included in Cell Proliferation Elisa Kit), anti BrdU antibody was added to each well and allowed to react. Subsequently the anti-BrdU antibody was removed by washing and the substrate solution TMB (included in Cell Proliferation Elisa Kit) was then added and allowed to produce chromogen. After stopping the substrate reaction the absorbance was measured at 450 nm with a reference wavelength of 690 nm.

Determination of Lymph Node Weight and Cell Count
After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions mentioned in section 8.3.3 was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter (Casy Cell Counter, Innovatis). The values obtained were taken down manually.

Determination of Ear Weights
After the lymph nodes were excised, both ears of mice were punched at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.

Interpretation of Raw Data
The proliferative response of the lymph node cells is expressed as the mean SI. The SI is derived by dividing the mean BrdU labeling index/mouse within each test substance group as well as for the positive control group by the mean BrdU labeling index for the vehicle group.
A test item is regarded as a sensitizer 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 BrdU at least 1.6-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.
Furthermore, an index was calculated for the lymph node weight and -cell count as well as for the ear weight by dividing the mean values of the test item treated groups by the mean of the vehicle control group. For BALB/c mice, a cutoff-value for the lymph node cell count index of 1.55 was reported for a positive response.Furthermore, according to OECD guideline 442B, an increase in ear weight exceeding the threshold value of 25% was considered to be indicative for excessive local skin irritation.

Observations
In addition to the sensitizing 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: Prior to the first application and prior to sacrifice (pretest and main experiment)
- Ear thickness: In the pre-test prior to the first application of the test item (day 0), on day 2 and before sacrifice (day 5) the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany)
- Ear weights: In the pre-test and main experiment after sacrifice; biopsy punches were taken from each ear
- Lymph node weights: After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance
- Lymph node cell count: The lymph node cell count was determined in aliquots taken from the prepared single cell suspensions of the lymph nodes using a cell counter
- Clinical signs (local / systemic): Clinical signs (local irritation at the application site or systemic toxicity) 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)

Statistics:

The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the BrdU values. A statistical analysis was conducted on the BrdU values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between test item groups and negative control group. For all statistical calculations Statistica (Version 10) was used. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test or the Student Newman Keuls test. Statistical significance was set at the five per cent level (p < 0.05). However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Concentration (% w/w) Stimulation index Result 0 1.0 - 2.5 0.9 negative 5 0.5 negative 10 1.1 negative

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Concentration (% w/v) Mean BrdU labeling Index 0 0.071 2.5 0.066 5 0.039 10 0.060

Any other information on results incl. tables

Viability / Mortality

No deaths occurred during the study period.

 

Clinical Signs

No symptoms of local toxicity at the ears of the animals, except scaling, incrustation and test item residues in the mean and high dose group and no systemic findings were observed during the study period.

 

Body Weights

The body weight of the animals recorded prior to the first application and prior to sacrifice were within the range commonly recorded for animals of this strain and age.

 

Lymph Node Weights and Cell Counts

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in lymph node weights or cell counts was not observed in any of the test item treated groups in comparison to the vehicle control group.

 

Ear Weights

The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. According to OECD guideline 442B, an increase in ear weight exceeding the threshold value of 25% was considered to be indicative for excessive local skin irritation. This threshold was not exceeded in any test item treated group.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information