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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
2008-04-06 to 2008-04-24
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies

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:
yes
Remarks:
extended test procedure with challenge
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
partially unsaturated IQAC, DMS quaternised
IUPAC Name:
partially unsaturated IQAC, DMS quaternised
Constituent 2
Reference substance name:
Imidazolium compounds, 2-(C15-17(odd numbered), C17-unsatd. alkyl)-1-[2-(C16-18(even numbered), C18-unsatd. amido)ethyl]-4,5-dihydro-N-methyl, Me sulfates)
IUPAC Name:
Imidazolium compounds, 2-(C15-17(odd numbered), C17-unsatd. alkyl)-1-[2-(C16-18(even numbered), C18-unsatd. amido)ethyl]-4,5-dihydro-N-methyl, Me sulfates)
Constituent 3
Chemical structure
Reference substance name:
Fatty acids, C16-18 (even numbered) and C18 unsatd., reaction products with diethylene triamine, di-Me sulfate quaternized
EC Number:
937-237-2
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: 8-12 weeks
- Weight at study initiation: 18.0 - 22.9 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: at least 5 days

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 (LLNA)

Vehicle:
methyl ethyl ketone
Concentration:
0.25, 1 and 2.5% (w/v) in methyl ethyl ketone.
Vehicle and dose selection based on the outcome of RCC-CCR Study 1133501 (also cited in this IUCLID)
For group design see table in section "Any other information on materials and methods incl. tables"
No. of animals per dose:
5
Details on study design:
AIMS OF THE STUDY
The purpose of this Local Lymph Node Assay was to differentiate whether a stimulation of lymph node proliferation observed after treatment with PC-2007-140 (W 575 NO SOLVENT) (see RCC Study 1133503 also cited in this IUCLID) is due to a sensitizing effect or due to severe irritation. The study comprises of 7 groups containing 5 female mice each. The study was divided into 2 treatment phases. In the first phase (sensitization phase) two
groups were treated with 2.5 % of the test item by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. Additionally one group each was treated with 0.25 and 1% of the test item to determine the EC3 value. Three control groups were treated similarly with the vehicle only. On day 6 of the experiment one group for each test item concentration and one vehicle group were used for the assessment of the induced lymphocyte proliferation after the sensitization phase. The remaining groups were used for the second phase of the experiment (the challenge phase). In the challenge phase, which was performed on day 16 of the experiment, the remaining test item treated group was challenged with the same dose of the test item (2.5 %). Of the two remaining vehicle treated groups one group was treated with 2.5 % test item and the other with the vehicle. Two days after the challenge phase (on day 18) the lymph proliferation of the individual animals of each group were assessed.
The principle of the assay is based on the immunological memory function of a sensitizing agent, which does not occur in irritation reactions. Thus, the animals are sensitized by a triple application on consecutive days. After a recovery period during which possible irritation effects are subdued the
animals are treated (challenged) with the test item. If the test item has a sensitizing potential the induced reaction (lymph node proliferation) after the challenge phase should be more enhanced as compared to the response obtained directly after the sensitization phase. As a control animals are treated with the vehicle during the sensitization phase and treated with the test item during the challenge phase.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: murine local lymph node assay with challenge
- 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.
-- Third, that the challenge exposure to the test item resulted in an incorporation of 3HTdR that was significantly higher than the primary response.

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 while warming at 50 to 60°C under nitrogen atmosphere. The preparations were made freshly before each dosing.

- Group design: For group design see table in section "Any other information on materials and methods incl. tables"

- Topical Application: Each test group of mice was treated by topical (epidermal) application to the dorsal area of each ear lobe (left and right) with different test item concentrations in methyl ethyl ketone. The application volume, 25 µl, 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).

- Determination of Ear Thickness: Prior to the first application of the test item and prior to the application of 3HTdR the ear thickness was determined using a micrometer (80247 Kroeplin, D-36381 Schlüchtern, Germany).

- 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). On day six all mice of groups 1 and 4 were administered with 20.1 µCi of 3HTdR by intravenous injection via a tail vein with 250 µl of 81.3 µCi/ml 3HTdR. On day eighteen all mice of groups 2, 3, and 5 were administered with 20.4 µCi of 3HTdR by intravenous injection via a tail vein with 250 µl of 81.7 µCi/ml 3HTdR.

- 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).

- Interpretation of Raw Data: The proliferative responses of lymph node cell 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 are determined, mean scintillation-background DPM is 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.
-- Third, that the challenge exposure to the test item resulted in an incorporation of 3HTdR that was relevantly higher than the primary response.

- 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
-- Clinical signs (local/systemic): once daily (week day), especially the treatment sites were observed carefully
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
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Table 2 Calculation and results of individual data in section "Remarks on results including tables and figures"

Any other information on results incl. tables

Table 2 Calculation and results of individual data

 

Group

Treatment day 1-3 with

Treatment day 16 with

Day of Preparation

Number of LN

DPM/LN*

S.I.**

Sensitisation

1 (Control Group)

vehicle

-

6

10

532.5

1.00

4 (Test group)

0.25 % test item

-

6

10

1206.6

2.27

5 (Test group)

1 % test item

-

6

10

3353.7

6.30

6 (Test group)

2.5 % test item

-

6

10

8179.6

15.36

Challenge

2 (Control Group)

vehicle

vehicle

18

10

545.0

1.00

3 (Control Group)

vehicle

2.5 % test item

18

10

2129.9

3.91

7 (Test group)

2.5 % test item

2.5 % test item

18

10

3197.1

5.87

 

Vehicle: methyl ethyl ketone

- no treatment performed

LN: lymph node

* DPM values per group divided by the number of lymph nodes per group

** S.I. values calculated by dividing the mean DPM per group by the mean DPM per group obtained for the respective control group

Table 3 EC3 Calculation

 

Test item concentration %

S.I.

Test Group 3

0.25 (a)

2.27 (b)

Test Group 4

1 (c)

6.30 (d)

EC3= (a-c) [(3-d)/(b-d)]+c = 0.4% (w/v)

 

EC3 =Estimated concentration for a S.1. of 3.

a,b,c,d = Co-ordinates of the two pairs of data lying immediately above and below the S.1.

value of 3 on the LLNA dose response plot.

FURTHER RESULTS:

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

- Clinical Signs: Visible oedema formation of the ear skin occurred for the animals treated with 2.5% of the test item on day 6. Measurement of ear thickness revealed an increased ear thickness gain of the test item treated groups during both the sensitisation and the challenge phase

as determined on day 6 or 18, respectively. In contrast, the challenge control was in the range of the vehicle controls.

- Ear Thickness: Measurement of ear thickness revealed an increased ear thickness gain of the test item treated groups during both the sensitisation and the challenge phase as determined on day 6 or 18, respectively. In contrast, the challenge control was in the range of the vehicle

controls.

-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.

DISCUSSION

In the sensitisation phase of this study Stimulation Indices (S.I.) of 2.27, 6.30, and 15.36 were determined with the test item at concentrations of 0.25, 1, and 2.5% in methyl ethyl ketone, respectively. The EC3 value was 0.4 %. After a single challenge application on day 16 an S.I. of 5.87 was yielded for the test item concentration of 2.5%. As a control (challenge control) animals treated with the vehicle during the sensitisation phase were

treated with 2.5 % of the test item on day 16. The S.I. of this group was 3.91. Thus in both primed and naive animals S.I. values above 3 were induced after a single application of the test item (at 2.5%). The magnitude of the response obtained in the challenge group (S.I. = 5.87) does not clearly indicate a secondary response. Therefore, the observed data indicate that the effects induced are solely due to irritative effects. However, the analysis of ear thickness indicates other effects. A dose-dependent increase in ear thickness gain was observed on day 6 for 1 % as well as for 2.5 % of the test item. On day 18 for the test item group treated with 2.5 % ear thickness was still higher than the control or challenge control animals. In contrast, the animals of the challenge control did not show values above the vehicle control range. Unfortunately, it has not been evaluated if ear thickness was still increased on the day the challenge application was performed (day 16). Therefore, it cannot be clearly distinguished, if the observed lymphocyte proliferation is due to irritation alone. The reason for the observed inconsistency in the results is the high

levels of irritation induced which clearly mask the possibility to distinguish between a true memory response and an irritation effect.

The test was found to be inconclusive, since the concentrations were to high and the missing measurement of ear thickness on the day of

the challenge application.

To further investigate the true nature of the response an additional LLNA with challenge has been performed in the meantime using a lower concentration of the test item (i.e. 1.5 %) that is unlikely to induce an irritation reaction that will persist until day 16. Additionally, the challenge application has been performed after ear thickness measurements indicated values for

the test group that are clearly within the range of the vehicle control.

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:
study cannot be used for classification
Conclusions:
In the sensitisation phase of this study Stimulation Indices (S.I.) of 2.27, 6.30, and 15.36 were determined with the test item at concentrations of 0.25, 1, and 2.5% in methyl ethyl ketone, respectively. The EC3 value was 0.4 %. However, the test was found to be inconclusive, since the concentrations were to high and the missing measurement of ear thickness on the day of the challenge application.
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 including challenge.

The purpose of this Local Lymph Node Assay was to differentiate whether a stimulation of lymph node proliferation observed after treatment with the partially unsaturated IQAC, DMS quaternised (no solvent)

(see RCC Study 1133503 also cited in this IUCLID) is due to a sensitizing effect or due to severe irritation. The study comprises of 7 groups containing 5 female mice each. The study was divided into 2 treatment phases. In the first phase (sensitization phase) two groups were treated with 2.5 % of the test item by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. Additionally one group each was treated with 0.25 and 1% of the test item to determine the EC3 value. Three control groups were treated similarly with the vehicle only. On day 6 of the experiment one group for each test item concentration and one vehicle group were used for the assessment of the induced lymphocyte proliferation after the sensitization phase. The remaining groups were used for the second phase of the experiment (the challenge phase). In the challenge phase, which was performed on day 16 of the experiment, the remaining test item treated group was challenged with the same dose of the test item (2.5 %). Of the two remaining vehicle treated groups one group was treated with 2.5 % test item and the other with the vehicle. Two days after the challenge phase (on day 18) the lymph proliferation of the individual animals of each group were assessed.

The principle of the assay is based on the immunological memory function of a sensitizing agent, which does not occur in irritation reactions. Thus, the animals are sensitized by a triple application on consecutive days. After a recovery period during which possible irritation effects are subdued the animals are treated (challenged) with the test item. If the test item has a sensitizing potential the induced reaction (lymph node proliferation) after the challenge phase should be more enhanced as compared to the response obtained directly after the sensitization phase. As a control animals are treated with the vehicle during the sensitization phase and treated with the test item during the challenge phase.

In the sensitisation phase of this study Stimulation Indices (S.I.) of 2.27, 6.30, and 15.36 were determined with the test item at concentrations of 0.25, 1, and 2.5% in methyl ethyl ketone, respectively. The EC3 value was 0.4 %. After a single challenge application on day 16 an S.I. of 5.87 was yielded for the test item concentration of 2.5%. As a control (challenge control) animals treated with the vehicle during the sensitisation phase were treated with 2.5 % of the test item on day 16. The S.I. of this group was 3.91. Thus in both primed and naive animals S.I. values above 3 were induced after a single application of the test item (at 2.5%). The magnitude of the response obtained in the challenge group (S.I. = 5.87) does not clearly indicate a secondary response. Therefore, the observed data indicate that the effects induced are solely due to irritative effects. However, the analysis of ear thickness indicates other effects. A dose-dependent increase in ear thickness gain was observed on day 6 for 1 % as well as for 2.5 % of the test item. On day 18 for the test item group treated with 2.5 % ear thickness was still higher than the control or challenge control animals. In contrast, the animals of the challenge control did not show values above the vehicle control range. Unfortunately, it has not been evaluated if ear thickness was still increased on the day the challenge application was performed (day 16). Therefore, it cannot be clearly distinguished, if the observed lymphocyte proliferation is due to irritation alone. The reason for the observed inconsistency in the results is the high

levels of irritation induced which clearly mask the possibility to distinguish between a true memory response and an irritation effect.

The test was found to be inconclusive, since the concentrations were to high and the missing measurement of ear thickness on the day of the challenge application.

To further investigate the true nature of the response an additional LLNA with challenge has been performed in the meantime using a lower concentration of the test item (i.e. 1.5 %) that is unlikely to induce an irritation reaction that will persist until day 16. Additionally, the challenge application has been performed after ear thickness measurements indicated values for

the test group that are clearly within the range of the vehicle control.

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).

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