Fatty acids, C16-18 and C18-unsatd., reaction products with diethylenetriamine, di-Me sulfate-quaternized

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2007-10-23 to 2007-11-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2002

Deviations:

no

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:

Fatty acids, C16-18 (even numbered) and C18 unsatd., reaction products with diethylene triamine, di-Me sulfate quaternized

Cas Number:

1370006-50-0

Molecular formula:

Molecular formula cannot be given as substance is a mixture.

IUPAC Name:

Fatty acids, C16-18 (even numbered) and C18 unsatd., reaction products with diethylene triamine, di-Me sulfate quaternized

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Mice, CBA/CaOlaHsd
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 18.6 - 21.6 g
- Housing: single caging in Makrolon Type I cages, with wire mesh top (EHRET GmbH, D-79302 Emmendingen, Germany)
- Diet: ad libitum, pelleted standard diet (Harlan Winkelmann GmbH, D-33178 Borchen, Germany)
- Water: ad libitum, tap water (Gemeindewerke, D-64380 Rossdorf, Germany)
- Acclimation period: animals were acclimated under test conditions after health examination, no data on length of acclimation period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 .± 3°
- Humidity (%): 30-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (non-LLNA)

Positive control substance(s):

yes

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

Low Dose: 2.5 % (w/v) in methyl ethyl ketone
Mid Dose: 5 % (w/v) in methyl ethyl ketone
High Dose: 10 % (w/v) in methyl ethyl ketone
The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used was a 50 % emulsion in methyl ethyl ketone.
- Irritation: To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 5, 10, 25, and 50 % (w/v) on one ear each on three consecutive days. Clinical signs were recorded 24 ± 4 hours after each application.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: murine local lymph node assay
- 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 Preparation: The test item was placed into a volumetric flask on a tared balance and methyl ethyl ketone was quantitatively added. The test item formed a clear solution at the concentrations used for the main experiment. The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied.

- Topical Application: Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 2.5, 5, and 10 % (w/v) in methyl ethyl ketone. The application volume, 25µl, was spread over the entire dorsal surface (diameter approximately 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

- Administration of 3H-Methyl Thymidine: 3H-methyl thymidine (3HTdR) was purchased from GE Healthcare (GE Healthcare product code no. TRA 310; specific activity, 2 Ci/mol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were administered with 250 µl of 80.1µCi/ml 3HTdR (corresponds to 20.0 µCi 3HTdR per mouse) by intravenous injection via a tail vein.

- Determination of Ear Thickness: Prior to the first application of the test item and prior to treatment with 3HTdR the ear thickness was determined using a micrometer (S0247 Kroeplin, D-36381 Schlüchtern, Germany).

- Determination of Incorporated 3HTdR: Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium (Release®, WDT, D-30827 Garbsen, Germany). 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 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, D-63110 Rodgau, Germany) and thoroughly mixed. The level of 3HTdR incorporation was then measured on beta-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, D-63110 Rodgau, Germany). Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (OPM).

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

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

- Observations: In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
-- Mortality / Viability: once daily (week day) from experimental start to necropsy
-- Body weights: prior to the first application and prior to treatment with 3HTdR
-- Ear thickness: prior to the first application and prior to treatment with 3HTdR
-- Ear weights after sacrifice: Biopsy punches were taken from each ear
-- Clinical signs: once daily (week day), 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. A statistical analysis was conducted for assessment of the dose-response relationship and the EC3 value was 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.
The ANOVA (Dunnett-test) was conducted to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group. Statistical significance was 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: 17.20, 13.34 and 11.72 at concentrations of 2.5, 5, 10 % (w/v), respectively. The EC3 value could not be calculated, since all S.I.s are above 3.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

11552.5, 8964.3 and 7876.1 at concentrations of 2.5, 5, 10 % (w/v), respectively. DPM/node was determined by dividing the sum of the measured values from all lymph nodes within a group by the number of lymph nodes taken from that group. Mean DPM value for all groups was according to the ANOVA (Dunnett-test) significantly higher than corresponding control value. The p value for the analysis was 0.005.

Any other information on results incl. tables

RANGE FINDING TESTS:

Redness of the ear skin of both ears as well as signs of systemic toxicity like eyelid closure and ruffled fur were observed in the animal treated with 25 and 50%. After treatment with 5 and 10% slight redness of the ear skin was observed after the second application of the test item but systemic toxicity did not occur.

FURTHER RESULTS:

- Viability / Mortality: No deaths occurred during the study period.

- Clinical Signs: The animals did not show any clinical signs of toxicity during treatment. On the day of preparation oedema formation was recorded for all dose groups. Reddening of the ear skin was not observed during the main experiment

- 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.

- Ear Thickness: The measured ear thickness of all animals treated was recorded prior to the 1st application and prior to treatment with 3HTdR. Ear thickness was dose-dependently increased with statistically significance for all tested concentrations.

- Ear Weight: The measured ear weight of all animals treated was recorded prior to the 1st application and after sacrifice. Ear thickness was dose-dependently increased with statistically significance for all tested concentrations.

DISCUSSION

A test item is regarded as a sensitiser in the LLNA if the 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.

On the basis of the determined stimulation indices (S.I.) of 17.20, 13.34, and 11.72 at concentrations of 2.5, 5, 10 % (w/v), respectively, a sensitising activity of the test item might be supposed. However, the unusual inverse dose-response prompts question whether the increased incorporation of 3HTdR seen is really based on a sensitising activity of the test item.

For further evidence regarding the question whether the obtained effect is truly of a sensitising nature an additional LLNA test with challenge was initiated. In this experiment, a sensitising activity of the test item could not be confirmed (see entry on RCC Study 1180400 also cited in this

IUCLID).

Applicant's summary and conclusion

Interpretation of results:

ambiguous

Remarks:

Migrated information

Conclusions:

A test item is regarded as a sensitizer in the LLNA if the 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.
On the basis of the determined stimulation indices (S.I.) of 17.20, 13.34, and 11.72 at concentrations of 2.5, 5, 10 % (w/v), respectively, a sensitising activity of the test item might be supposed. However, the unusual inverse dose-response prompts question whether the increased incorporation of 3HTdR seen is really based on a sensitising activity of the test item. For further evidence regarding the question whether the obtained effect is truly of a sensitising nature an additional LLNA test with challenge was initiated.

Executive summary:

In a dermal sensitisation study with the partially unsaturated IQAC, DMS quaternised (no solvent) (CAS-No. 98219-51-3, purity 100%) in methyl ethyl ketone, groups of 5 female CBA/CaOlaHsd mice were tested using the LLNA method according to OECD Guideline 429, 2002. Positive control substance was alpha hexyl cinnamic aldehyde, which gave a positive result (STIMULATION INDEX (S.I.) of 3.03) at a concentration of 25 % in acetone:olive oil, 4:1 (w/v).

STIMULATION INDICES of 17.20, 13.34 and 11.72 were determined with the test item at concentrations of 2.5, 5 and 10 % (w/v), respectively.

A test item is regarded as a sensitiser in the LLNA if the 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.

On the basis of the determined stimulation indices (S.I.) of 17.20, 13.34, and 11.72 at concentrations of 2.5, 5, 10 % (w/v), respectively, a sensitising activity of the test item might be supposed. However, the unusual inverse dose-response prompts question whether the increased incorporation of 3HTdR seen is really based on a sensitising activity of the test item.

For further evidence regarding the question whether the obtained effect is truly of a sensitising nature an additional LLNA test with challenge was initiated. In this experiment, a sensitising activity of the test item could not be confirmed (see entry on RCC Study 1180400 also cited in this IUCLID).