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

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physicochemical, ecotoxicological and toxicological properties because
• they are manufactured from similar or identical precursors under similar conditions
• they share structural similarities with common functional groups: quaternary amines, amide bonds, and fatty acid chains with small differences in their chain length distribution and differences in the amount of unsaturated carbon chains
• the metabolism pathway may lead to comparable products: Imidazolium and free fatty acid chains
Therefore, read-across from the existing physicochemical, ecotoxicity and toxicity studies on the source substances is considered as an appropriate adaptation to the standard information requirements of REACH regulation

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see “Justification for read-across” attached to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
see “Justification for read-across” attached to IUCLID section 13

4. DATA MATRIX
see “Justification for read-across” attached to IUCLID section 13

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2008-05-18 to 2008-06-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study

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)

Type of study:

mouse local lymph node assay (LLNA)

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: 17.5 - 22.4 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.

Vehicle:

methyl ethyl ketone

Concentration:

1.5 %
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 test substance (see RCC Study 1133503 also cited in this IUCLID) is due to a sensitising effect or due to severe irritation. The study comprises of 5 groups containing 5 female mice each. The study was divided into 2 treatment phases. In the first phase (sensitisation phase) two groups were treated with 1.5 % of the test item by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. Three control groups were treated similarly with the vehicle methyl ethyl ketone only. On day 6 of the experiment one test item and one vehicle group were used for the assessment of the induced lymphocyte proliferation after the sensitisation phase. The remaining group was used for the second phase of the experiment (the challenge phase). In the challenge phase, which was performed on day 21 of the experiment, the remaining test item treated group was challenged with the same dose of the test item (1.5 %). Of the two remaining vehicle treated groups one group was treated with 1.5 % test item and the other with the vehicle. Two days after the challenge phase (on day 23) the lymph proliferation of the individual animals of each group were assessed.
The principle of this study design is based on the immunological memory function of a sensitising agent, which does not occur in irritation reactions. Thus, the animals are sensitised 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 sensitising potential the induced reaction (lymph node proliferation) after the challenge phase should be more enhanced as compared to the response obtained directly after the sensitisation phase. As a control animals are treated with the vehicle during the sensitisation 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 challenge exposure to the test item
results in an incorporation of 3HTdR that is relevantly higher than the primary response.

TREATMENT PREPARATION AND ADMINISTRATION:
- Test Item Preparation: Based on the outcome of previous studies, the test item in the main study was formulated in methyl ethyl ketone and assayed at 1.5 % % (w/v).

- 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 surface of each ear lobe (left and right) with a test item concentrations of 1.5 %(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).

- Determination of Ear Thickness: Prior to the first application of the test item (day 1) and prior to the application of 3HTdR on day 6, additionally on day 16, 18, and 21, as well as prior to the application of 3HTdR on day 23 the ear thickness was determined using a micrometer (S0247 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 20.25 µCi of 3HTdR by intravenous injection via a tail vein with 250 µl of 81.0 µCi/ml 3HTdR. On day eighteen all mice of groups 2, 3, and 5 were administered with 19.7 µCi of 3HTdR by intravenous injection via a tail vein with 250 µl of 78.9 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 challenge exposure to the test item results in an incorporation of 3HTdR that is 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: day 1,16,18,21, and 23
-- Ear weights: after sacrifice. Biopsy punches were taken from each ear
-- Clinical signs: once daily (week day), especially the treatment sites were observed carefully

Statistics:

The mean values and standard deviations were calculated in the body weight and ear thickness tables.
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.

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"

Key result

Parameter:

SI

Value:

1

Variability:

n.a.

Test group / Remarks:

vehicle control day 1-3

Key result

Parameter:

SI

Value:

11.35

Variability:

n.a.

Test group / Remarks:

1.5% test item day 1-3

Key result

Parameter:

SI

Value:

1

Variability:

n.a.

Test group / Remarks:

vehicle control trestment day 21

Key result

Parameter:

SI

Value:

6.3

Variability:

n.a.

Test group / Remarks:

Vehicle/ 1.5% test item day 21

Key result

Parameter:

SI

Value:

8.16

Variability:

n.a.

Test group / Remarks:

1.5% test item/1.5% test item day 21

Table 2 Calculation and results of individual data

	Group	Treatment day 1-3 with	Treatment day 21 with	Day of Preparation	Number of LN	DPM/LN*	S.I.**
Sensitisation	1 (Control Group)	vehicle	-	6	10	564.0	1.00
	4 (Test group)	1.5 % test item	-	6	10	6403.6	11.35
Challenge	2 (Control Group)	vehicle	vehicle	23	10	321.5	1.00
	3 (Control Group)	vehicle	1.5 % test item	23	10	2026.5	6.30
	5 (Test group)	1.5 % test item	1.5 % test item	23	10	2622.1	8.16

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

FURTHER RESULTS:

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

- Clinical Signs: The animals did not show signs of systemic toxicity during the course of the study and no

cases of mortality were observed.

- Ear Thickness: Measurement of ear thickness revealed a strongly increased ear thickness gain of the test item treated group on day 6. The increase persisted until day 16. Therefore, the challenge application was delayed. After day 18 ear thickness of the challenge group started to decrease. After the challenge application as well as in the challenge control the ear thickness was no longer increased.

-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 a Stimulation Index (S.I.) of 11.35 was determined with the test item at a concentration of 1.5 % in methyl ethyl ketone. After a single challenge application on day 21 an S.I. of 8.16 was yielded. As a control (challenge control) animals treated with the vehicle during the sensitisation phase were treated with 1.5 % test item on day 21. The S.I. of this group was 6.30.

These data indicate that a single application of the test item at 1.5% is sufficient to induce Iymphoproliferation in the draining lymph nodes. This proliferation is, however, not due to sensitising effects but result from strong irritation, since the magnitude of the response of challenged animals (S.I. = 8.16) did not differ greatly from the challenge control group (S.I. = 6.30). Thus, showing that the induced response does not bear any

immunological memory.

The test item was not a skin sensitiser under the described conditions.

 

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The purpose of this Local Lymph Node Assay was to differentiate whether a stimulation of lymph node proliferation induced by the test item is due to a sensitizing effect or due to severe irritation. In the sensitisation phase of this study a Stimulation Index (S.I.) of 11.35 was determined with the test item at a concentration of 1.5 % in methyl ethyl ketone. After a single challenge application on day 21 an S.I. of 8.16 was yielded. As a control (challenge control) animals treated with the vehicle during the sensitisation phase were treated with 1.5 % test item on day 21. The S.I. of this group was 6.30. These data indicate that a single application of the test item at 1.5% is sufficient to induce Iymphoproliferation in the draining lymph nodes. This proliferation is, however, not due to sensitising effects but result from strong irritation, since the magnitude of the response of challenged animals (S.I. = 8.16) did not differ greatly from the challenge control group (S.I. = 6.30). Thus, showing that the induced response does not bear any immunological memory.

Executive summary:

In a dermal sensitisation study with the partially usaturated 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 test substance (see RCC Study 1133503 also cited in this IUCLID) is due to a sensitising effect or due to severe irritation. The study comprises of 5 groups containing 5 female mice each. The study was divided into 2 treatment phases. In the first phase (sensitisation phase) two groups were treated with 1.5 % of the test item by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. Three control groups were treated similarly with the vehicle methyl ethyl ketone only. On day 6 of the experiment one test item and one vehicle group were used for the assessment of the induced lymphocyte proliferation after the sensitisation phase. The remaining group was used for the second phase of the experiment (the challenge phase). In the challenge phase, which was performed on day 21 of the experiment, the remaining test item treated group was challenged with the same dose of the test item (1.5 %). Of the two remaining vehicle treated groups one group was treated with 1.5 % test item and the other with the vehicle. Two days after the challenge phase (on day 23) the lymph proliferation of the individual animals of each group were assessed.

The principle of this study design is based on the immunological memory function of a sensitising agent, which does not occur in irritation reactions. Thus, the animals are sensitised 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 sensitising potential the induced reaction (lymph node proliferation) after the challenge phase should be more enhanced as compared to the response obtained directly after the sensitisation phase. As a control animals are treated with the vehicle during the sensitisation phase and treated with the test item during the challenge phase.

In the sensitisation phase of this study a Stimulation Index (S.I.) of 11.35 was determined with the test item at a concentration of 1.5 % in methyl ethyl ketone. After a single challenge application on day 21 an S.I. of 8.16 was yielded. As a control (challenge control) animals treated with the vehicle during the sensitisation phase were treated with 1.5 % test item on day 21. The S.I. of this group was 6.30.

These data indicate that a single application of the test item at 1.5% is sufficient to induce Iymphoproliferation in the draining lymph nodes. This proliferation is, however, not due to sensitising effects but result from strong irritation, since the magnitude of the response of challenged animals (S.I. = 8.16) did not differ greatly from the challenge control group (S.I. = 6.30). This result shows that the induced response does not bear any immunological memory.

The test item partially usaturated IQAC, DMS quaternised (no solvent) was not a skin sensitiser under the described conditions.

Cross-reference 2

Reason / purpose for cross-reference:

read-across: supporting information

Reference

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

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)

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.

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.

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.

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.

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).

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).

Cross-reference 3

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2008-04-06 to 2008-04-24

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Due to error in conduct of challenge application, study had to be repeated

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)

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.

Positive control substance(s):

yes

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 (see RCC Study 1133503 also cited in this IUCLID) is due to a sensitising 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 (sensitisation 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 sensitisation 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 sensitising agent, which does not occur in irritation reactions. Thus, the animals are sensitised 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 sensitising potential the induced reaction (lymph node proliferation) after the challenge phase should be more enhanced as compared to the response obtained directly after the sensitisation phase. As a control animals are treated with the vehicle during the sensitisation 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.

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"

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).

 

Interpretation of results:

ambiguous

Remarks:

Migrated information

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 sensitising 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 (sensitisation 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 sensitisation 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 sensitising agent, which does not occur in irritation reactions. Thus, the animals are sensitised 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 sensitising potential the induced reaction (lymph node proliferation) after the challenge phase should be more enhanced as compared to the response obtained directly after the sensitisation phase. As a control animals are treated with the vehicle during the sensitisation 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).

Cross-reference 4

Reason / purpose for cross-reference:

read-across: supporting information

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test material

Test material information

Constituent 1

Reference substance name:

Imidazolium compounds, 2-(C15 and C17 -alkyl)-1-[2-(C16 and C18) amidoethyl]-4,5-dihydro-(1 or 3)-methyl, Me sulfates

Molecular formula:

Molecular formula cannot be given as substance is a mixture.

IUPAC Name:

Imidazolium compounds, 2-(C15 and C17 -alkyl)-1-[2-(C16 and C18) amidoethyl]-4,5-dihydro-(1 or 3)-methyl, Me sulfates

Constituent 2

Reference substance name:

Imidazolium compounds, 2-(C15 and C17)-1-[2-(C16 and C18) amidoethyl]-4,5-dihydro, Me sulfates

Molecular formula:

Molecular formula cannot be given as substance is a mixture.

IUPAC Name:

Imidazolium compounds, 2-(C15 and C17)-1-[2-(C16 and C18) amidoethyl]-4,5-dihydro, Me sulfates

Constituent 3

Reference substance name:

N,N,N-trimethyl-2-[2-(C15 and C17)]-4,5-dihydro-1H-imidazol-1-yl)ethanaminium, Me sulfates

Molecular formula:

Molecular formula cannot be given as substance is a mixture.

IUPAC Name:

N,N,N-trimethyl-2-[2-(C15 and C17)]-4,5-dihydro-1H-imidazol-1-yl)ethanaminium, Me sulfates

1

Reference substance name:

Glycerol

EC Number:

200-289-5

EC Name:

Glycerol

Cas Number:

56-81-5

Molecular formula:

C3H8O3

IUPAC Name:

glycerol

2

Reference substance name:

Glycerides, C16-18 mono-

EC Number:

293-208-8

EC Name:

Glycerides, C16-18 mono-

Cas Number:

91052-47-0

Molecular formula:

C19H38O4 to C21H42O4

IUPAC Name:

Glycerides, C16-18 mono-

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Variability:

n.a.

Test group / Remarks:

vehicle control day 1-3

Remarks on result:

other:

Remarks:

test item 98219-51-3

Results 2

Key result

Parameter:

SI

Value:

11.35

Variability:

n.a.

Test group / Remarks:

1.5% test item day 1-3

Remarks on result:

other:

Remarks:

test item 98219-51-3

Results 3

Key result

Parameter:

SI

Value:

1

Variability:

n.a.

Test group / Remarks:

vehicle control treatment day 21

Remarks on result:

other:

Remarks:

test item 98219-51-3

Results 4

Key result

Parameter:

SI

Value:

6.3

Variability:

n.a.

Test group / Remarks:

vehicle/1.5% test item day 21

Remarks on result:

other:

Remarks:

test item 98219-51-3

Results 5

Key result

Parameter:

SI

Value:

8.16

Variability:

n.a.

Test group / Remarks:

1.5 % test item/1.5% test item day 21

Remarks on result:

other:

Remarks:

test item 98219-51-3

Results 6

Key result

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.

Remarks:

test substance 98219-51-3 (supporting study)

Results 7

Key result

Parameter:

SI

Value:

1

Variability:

n.a.

Test group / Remarks:

vehicel control day 1-3

Remarks on result:

other:

Remarks:

test item 98219-51-3 (RL3 study; 429 error challenge)

Results 8

Key result

Parameter:

SI

Value:

2.27

Variability:

n.a.

Test group / Remarks:

test group (0.25% test item) day 1-3

Remarks on result:

other:

Remarks:

test item 98219-51-3 (RL3 study; 429 error challenge)

Results 9

Key result

Parameter:

SI

Value:

6.3

Variability:

n.a.

Test group / Remarks:

test group (1% test item) day 1-3

Remarks on result:

other:

Remarks:

test item 98219-51-3 (RL3 study; 429 error challenge)

Results 10

Key result

Parameter:

SI

Value:

15.36

Variability:

n.a.

Test group / Remarks:

test group (2.5% test item) day 1-3

Remarks on result:

other:

Remarks:

test item 98219-51-3 (RL3 study; 429 error challenge)

Results 11

Key result

Parameter:

SI

Value:

1

Variability:

n.a.

Test group / Remarks:

vehicle control day 16

Remarks on result:

other:

Remarks:

test item 98219-51-3 (RL3 study; 429 error challenge)

Results 12

Key result

Parameter:

SI

Value:

3.91

Variability:

n.a.

Test group / Remarks:

vehicle/2.5% test item day 16

Remarks on result:

other:

Remarks:

test item 98219-51-3 (RL3 study; 429 error challenge)

Results 13

Key result

Parameter:

SI

Value:

5.87

Variability:

n.a.

Test group / Remarks:

test item 2.5%/test item 2.5% day 16

Remarks on result:

other:

Remarks:

test item 98219-51-3 (RL3 study; 429 error challenge)

Any other information on results incl. tables

Test item 98219 -51 -3:

Table 2 Calculation and results of individual data

	Group	Treatment day 1-3 with	Treatment day 21 with	Day of Preparation	Number of LN	DPM/LN*	S.I.**
Sensitisation	1 (Control Group)	vehicle	-	6	10	564.0	1.00
	4 (Test group)	1.5 % test item	-	6	10	6403.6	11.35
Challenge	2 (Control Group)	vehicle	vehicle	23	10	321.5	1.00
	3 (Control Group)	vehicle	1.5 % test item	23	10	2026.5	6.30
	5 (Test group)	1.5 % test item	1.5 % test item	23	10	2622.1	8.16

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

FURTHER RESULTS:

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

-Clinical Signs: The animals did not show signs of systemic toxicity during the course of the study and no

cases of mortality were observed.

- Ear Thickness: Measurement of ear thickness revealed a strongly increased ear thickness gain of the test item treated group on day 6. The increase persisted until day 16. Therefore, the challenge application was delayed. After day 18 ear thickness of the challenge group started to decrease. After the challenge application as well as in the challenge control the ear thickness was no longer increased.

-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 a Stimulation Index (S.I.) of 11.35 was determined with the test item at a concentration of 1.5 % in methyl ethyl ketone. After a single challenge application on day 21 an S.I. of 8.16 was yielded. As a control (challenge control) animals treated with the vehicle during the sensitisation phase were treated with 1.5 % test item on day 21. The S.I. of this group was 6.30.

These data indicate that a single application of the test item at 1.5% is sufficient to induce Iymphoproliferation in the draining lymph nodes. This proliferation is, however, not due to sensitising effects but result from strong irritation, since the magnitude of the response of challenged animals (S.I. = 8.16) did not differ greatly from the challenge control group (S.I. = 6.30). Thus, showing that the induced response does not bear any

immunological memory.

The test item was not a skin sensitiser under the described conditions.

Test item 98219 -51 -3 (Supporting study):

-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.

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Test item 98219-51-3:
The purpose of this Local Lymph Node Assay was to differentiate whether a stimulation of lymph node proliferation induced by the test item is due to a sensitizing effect or due to severe irritation. In the sensitisation phase of this study a Stimulation Index (S.I.) of 11.35 was determined with the test item at a concentration of 1.5 % in methyl ethyl ketone. After a single challenge application on day 21 an S.I. of 8.16 was yielded. As a control (challenge control) animals treated with the vehicle during the sensitisation phase were treated with 1.5 % test item on day 21. The S.I. of this group was 6.30. These data indicate that a single application of the test item at 1.5% is sufficient to induce Iymphoproliferation in the draining lymph nodes. This proliferation is, however, not due to sensitising effects but result from strong irritation, since the magnitude of the response of challenged animals (S.I. = 8.16) did not differ greatly from the challenge control group (S.I. = 6.30). Thus, showing that the induced response does not bear any immunological memory.

Test item 98219-51-3 (supporting study): 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.

Test item 98219-51-3 (RL3 study, error challenge):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:

Test item 98219 -51 -3:

In a dermal sensitisation study with the partially usaturated 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 test substance (see RCC Study 1133503 also cited in this IUCLID) is due to a sensitising effect or due to severe irritation. The study comprises of 5 groups containing 5 female mice each. The study was divided into 2 treatment phases. In the first phase (sensitisation phase) two groups were treated with 1.5 % of the test item by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. Three control groups were treated similarly with the vehicle methyl ethyl ketone only. On day 6 of the experiment one test item and one vehicle group were used for the assessment of the induced lymphocyte proliferation after the sensitisation phase. The remaining group was used for the second phase of the experiment (the challenge phase). In the challenge phase, which was performed on day 21 of the experiment, the remaining test item treated group was challenged with the same dose of the test item (1.5 %). Of the two remaining vehicle treated groups one group was treated with 1.5 % test item and the other with the vehicle. Two days after the challenge phase (on day 23) the lymph proliferation of the individual animals of each group were assessed.

The principle of this study design is based on the immunological memory function of a sensitising agent, which does not occur in irritation reactions. Thus, the animals are sensitised 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 asensitising potential the induced reaction (lymph node proliferation) after the challenge phase should be more enhanced as compared to the response obtained directly after the sensitisation phase. As a control animals are treated with the vehicle during the sensitisation phase and treated with the test item during the challenge phase.

In the sensitisation phase of this study a Stimulation Index (S.I.) of 11.35 was determined with the test item at a concentration of 1.5 % in methyl ethyl ketone. After a single challenge application on day 21 an S.I. of 8.16 was yielded. As a control (challenge control) animals treated with the vehicle during the sensitisation phase were treated with 1.5 % test item on day 21. The S.I. of this group was 6.30.

These data indicate that a single application of the test item at 1.5% is sufficient to induce Iymphoproliferation in the draining lymph nodes. This proliferation is, however, not due to sensitising effects but result from strong irritation, since the magnitude of the response of challenged animals (S.I. = 8.16) did not differ greatly from the challenge control group (S.I. = 6.30). This result shows that the induced response does not bear any immunological memory.

The test itempartially usaturated IQAC, DMS quaternised (no solvent)was not a skin sensitiser under the described conditions.

Test item 98219 -51 -3:

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.

Test item 98219 -51 -3 (RL3 study, error challenge):

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 withthe partially unsaturated IQAC, DMS quaternised(no solvent) is due to a sensitising 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 (sensitisation 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 sensitisation 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 sensitising agent, which does not occur in irritation reactions. Thus, the animals are sensitised 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 sensitising potential the induced reaction (lymph node proliferation) after the challenge phase should be more enhanced as compared to the response obtained directly after the sensitisation phase. As a control animals are treated with the vehicle during the sensitisation 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.