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

Endpoint summary

Description of key information

- study conducted according to OECD guideline 429, GLP, LLNA, partially usaturated IQAC, DMS quaternised (no solvent) (CAS-No. 98219-51-3, purity 100%) in methyl ethyl ketone at concentrations of 1.5% (w/v) was applied to 5 mice for sensitisation and challenge (CBA/CaOlaHsd), non-sensitising, read-across, WoE

- study conducted according to OECD guideline 406, GPMT, partially unsaturated IQAC, DMS quaternised (NO SOLVENT, 100 % pure) in PEG 300 applied to 10 Dunkin-Hartley guinea pigs , Induction: intradermal: test: 1. 1:1 (v/v) mixture of FCA and 0.9% NaCl, 2. The test item at 0.5% in PEG 300, 3. The test item at 0.5% in a 1:1 (v/v) FCA and 0.9%NaCl - epicutaneous: 10% (v/v) - challenge: 0.5% intradermal; 10 % epicutaneous, non-sensitising, read-across, WoE

- study conducted according to OECD guideline 406, GPMT, partially unsaturated IQAC, DMS quaternised (NO SOLVENT, 100 % pure) applied to 15 Pirbright-Hartley guinea pigs, 2.5, 5, and 10 % in water, non-sensitising, read-across, WoE

- study conducted according to OECD guideline 429, LLNA, CBA/CaOlaHsd mice were exposed to 2.5%, 5% and 10% partially usaturated IQAC, DMS quaternised (no solvent) (CAS-No. 98219-51-3, purity 100%) in methyl ethyl ketone, sensitising, read-across, supporting study

- study conducted according to the method of Buehler, induction and challenge in guinea pigs with 1% of partially unsaturated IQAC, DMS quarternised, very faint nonconfluent erythema, not enough to consider the substance as sensitising (non-sensitising), read-across, supporting study

- study conducted according to OECD guideline 429, extented with challenge, Mice, CBA/CaOlaHsd were exposed to partially unsaturated IQAC, DMS quarternised at concentrations of 0.25, 1 and 2.5% (w/v) in methyl ethyl ketone for induction and 2.5% for challenge, determination of SI, inconclusive, read-across, RL3

- study conducted according to OECD guideline 406, GPMT, guinea pigs were exposed to partially unsaturated IQAC, DMS quaternised 0.1% in parafin oil (intradermal) and 25% in parafin oil (epicutaneously), challenge with 10% (epicutaneously), occurrence of skin irritation, inconclusive, read-across, RL3

- study conducted according to the method of Buehler, guinea pigs were exposed to 1% partially unsaturated IQAC, DMS quarternised at 1% epicutaneously for induction and challenge, not sensitising, read-across, supporting study

- study conducted according to the method of Buehler, guinea pigs were exposed to 1% partially unsaturated IQAC, DMS quarternised at 1% epicutaneously for induction and challenge, not sensitising, read-across, supporting study

Short description of key information:
No skin sensitisation hazard was identified

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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).

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: relevant methodological deficiencies.

Qualifier:

according to guideline

Guideline:

other: US Interagency Regulatory Liaison Group (IRLG, January 1981)

Qualifier:

according to guideline

Guideline:

other: Magnusson B., Kligman A. M., "The Identification of Contact Allergens by Animal Assay". The Guinea Pig Maximisation Test, Invest. Derm. 1959, 52, 268

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Species:

guinea pig

Strain:

other: Pirbright-White

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: young adult
- Weight at study initiation: 270 - 410 g
- Housing: collective housing up to a maximum of 5 animals per cage (Macrolon type)
- Diet: ad libitum, standard laboratory guinea pig diet Ssniff-G (Ssniff Versuchstier-Diäten GmbH, Soest, Germany)
- Water: ad libitum, drinking water as for human consumption
- Acclimation period: 7 d


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23 °C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 10 x
- Photoperiod (hrs dark / hrs light): 12 / 12

Route:

intradermal and epicutaneous

Vehicle:

paraffin oil

Concentration / amount:

Intradermal induction: 0.1 % v/v in paraffin oil
Epicutaneous induction: 25 % v/v in paraffin oil
Challenge-application: 10 % v/v in paraffin oil
Concentration selection based on a small scale dose range study

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Concentration / amount:

Intradermal induction: 0.1 % v/v in paraffin oil
Epicutaneous induction: 25 % v/v in paraffin oil
Challenge-application: 10 % v/v in paraffin oil
Concentration selection based on a small scale dose range study

No. of animals per dose:

Range finding test: 100 %, 50 %, 25 % 10 % v/v in paraffin oil: 4 animals patch test, 5 %, 1 %, 0.5 %, 0.1 % v/v paraffin oil: 2 animals intradermal injection
treatment group: 10
control group: 5

Details on study design:

RANGE FINDING TESTS:
An area of 4 x 6 cm of the anterior shoulder region was clipped short 24 h before treatment.
- Intradermal Injection:
2 animals were treated at 4 treatment sites each with test item concentrations of 5, 1, 0.5, 0.1 % v/v (solutions in paraffin oil).
Skin reactions were recorded 24 h after treatment.

- Dermal Application:
3 animals were treated at 4 treatment sites each with test item concentrations of 100 % (as delivered by the sponsor) and 50, 25 or 10 V/V (solutions in paraffin oil). A closed patch (saturated filter paper pieces (Whatman No. 3 MM, 1 x 1 cm)) exposure was effected by means of an occlusive bandage. Exposure period was 24 hours. Skin reactions were recorded 3 h after patch removal.

MAIN STUDY
A. INDUCTION EXPOSURE
- First stage -an area of 4 x 6 cm over the shoulders was clipped short with electric clippers. Three pairs of intradermal injections were then made symmetrically in two rows on either side of the spine:
-- Test groups:
1. 0.1 ml FCA (50 % v/v in water ad injection)
2. 0.1 ml test article (0.1 % v/v in paraffin oil)
3. 0.1 ml test article (0.1 % v/v in paraffin oil emulsified in FCA (50 %))
-- Control group:
1. 0.1 ml FCA (50 % v/v in water ad injection)
2. 0.1 ml paraffin oil
3. 0.1 ml FCA (50 % v/v in paraffin oil)

- Second stage - One week after intradermal injection the clipped area was reclipped. A filter paper (Whatman No. 3 MM (2 x 4 cm)) was saturated with the test substance (25 % in paraffin oil), applied to clipped area and covered for 48 h.

Control animals received a patch loaded with the vehicle.

B. CHALLENGE EXPOSURE
Both control and test animals were subjected to a challenge exposure two weeks after induction phase. A challenge test was performed on clipped areas (5 x 5 cm) of the flank. The test substance (10 % in paraffin oil) was applied by a 2 x 2 cm filter paper to the left flank of both test and control animals. In each case the duration of exposure was 24 h under an occlusive dressing. 24 and 48 h after patch removal (= 48 and 72 hours after challenge initiation), allergic responses were evaluated on a numerical scale.

C. SCORING SYSTEM:
No skin reaction: score 0
Discrete slight erythema: score 1
Moderate to diffuse erythema: score 2
Intense erythema and swelling: score 3

D. SENSITIZATION CLASSIFICATION SYSTEM used by STUDY AUTHOR:
Sensitisation rate 0%: nonsensitizer
Sensitisation rate 10-20%: weak sensitizer
Sensitisation rate 30-60%: moderate sensitizer
Sensitisation rate 70-100%: strong sensitizer

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10 %

No. with + reactions:

9

Total no. in group:

10

Clinical observations:

discrete slight or moderate to diffuse erythema (score 1 or 2), scaling in all animals

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10 % . No with. + reactions: 9.0. Total no. in groups: 10.0. Clinical observations: discrete slight or moderate to diffuse erythema (score 1 or 2), scaling in all animals.

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

10 %

No. with + reactions:

5

Total no. in group:

5

Clinical observations:

discrete slight or moderate to diffuse erythema (score 1 or 2), scaling in all animals

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 10 % . No with. + reactions: 5.0. Total no. in groups: 5.0. Clinical observations: discrete slight or moderate to diffuse erythema (score 1 or 2), scaling in all animals.

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

10 %

No. with + reactions:

8

Total no. in group:

10

Clinical observations:

discrete slight erythema (score 1), scaling in all animals

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 10 %. No with. + reactions: 8.0. Total no. in groups: 10.0. Clinical observations: discrete slight erythema (score 1), scaling in all animals.

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

10 %

No. with + reactions:

4

Total no. in group:

5

Clinical observations:

discrete slight erythema (score 1), scaling in all animals

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 10 %. No with. + reactions: 4.0. Total no. in groups: 5.0. Clinical observations: discrete slight erythema (score 1), scaling in all animals.

RESULTS

Pilot study (range finding):

- Intradermal:

-- 5% (v/v): necrosis with dark discolouration at the application site

-- 1 and 0.5% (v/v): necrosis with light discolouration at the application site.

-- 0.1 % (v/v): discrete slight erythema (score 1) in 2/4 animals

- Epicutaneous:

- 100% (v/v): no skin reactions were observed

- 50 % (v/v): moderate to diffuse erythema (score 2) in 4/4 animals

- 25 % (v/v): discrete slight erythema (score 1) in 4/4 animals

- 10 % (v/v): moderate to diffuse erythema (score 2) in 4/4 animals

Main study:

- Induction:

-- intradermal: after intradermal induction, test group and negative control animals showed discrete slight (score 1) or moderate to diffuse erythema (score 2)

-- epicutaneous: 48 hours after epicutaneous induction, test group and negative control animals showed moderate to diffuse erythema (score 2)

- Challenge:

-- test group

--- 48 h scoring: discrete slight erythema (score 1) in 5/10 animals, moderate to diffuse erythema (score 2) in 4/10 animals, all animals showed scaling

--- 72 h scoring: discrete slight erythema (score 1) in 8/10 animals, all animals showed scaling

-- negative control group:

--- 48 h scoring: discrete slight erythema (score 1) in 4/5 animals, moderate to diffuse erythema (score 2) in 1/5 animals, all animals showed scaling

--- 72 h scoring: discrete slight erythema (score 1) in 4/5 animals, all animals showed scaling

In total 90 % of the animals treated with the test item and 100 % of the negative control animals showed erythema after the challenge treatment. In addition scaling occurred in all animals of both test group and negative control group.

Discussion:

The test item has been judged by the study author as non sensitising. However, the test has been insufficiently performed and thus the results are inconclusive. After intradermal and dermal induction, the control animals treated with the solvent only showed clear skin reactions in the form of discrete slight (score 1) or moderate to diffuse erythema (score 2). These anomalous unexpected findings suggest the assumption that the animals were inaccurately treated or contaminated means were used. The same reactions were also seen in the test group during induction. After challenge application, the test group and the control group showed discrete slight (score 1) or moderate to diffuse erythema (score 2) and squamation. In both groups, the reactions were almost similar in incidence and intensity. It is impossible to distinguish between allergic and irritative reactions and therefore one cannot rule out the possibility that allergic sensitisation might be mask by irritative reactions. Therefore, the findings in the test group cannot be evaluated regarding sensitisation. Consequently the test is judged as invalid.

Interpretation of results:

ambiguous

Remarks:

Migrated information

Conclusions:

The test item has been judged by the study author as non sensitising. However, the test has been insufficiently performed and thus the results are inconclusive. After intradermal and dermal induction, the control animals treated with the solvent only showed clear skin reactions in the form of discrete slight (score 1) or moderate to diffuse erythema (score 2). These anomalous unexpected findings suggest the assumption that the animals were inaccurately treated or contaminated means were used. The same reactions were also seen in the test group during induction. After challenge application, the test group and the control group showed discrete slight (score 1) or moderate to diffuse erythema (score 2) and squamation. In both groups, the reactions were almost similar in incidence and intensity. It is impossible to distinguish between allergic and irritative reactions and therefore one cannot rule out the possibility that allergic sensitisation might be mask by irritative reactions. Therefore, the findings in the test group cannot be evaluated regarding sensitisation. Consequently the test is judged as invalid.

Executive summary:

In a dermal sensitisation study with partially unsaturated IQAC, DMS quaternised /PG, young adult Pirbright white guinea pig (10 female) were tested using the MAXIMISATION TEST method described by the US Interagency Regulatory Liaison Group (IRLG, January 1981) and Magnusson B., Kligman A. M., "The Identification of Contact Allergens by Animal Assay". The Guinea Pig Maximisation Test, Invest. Derm. 1959, 52, 268.The negative control group comprised 5 animals.

The test item has been judged by the study author as non sensitising. However, the test has been insufficiently performed and thus the results are inconclusive. After intradermal and dermal induction, the control animals treated with the solvent only showed clear skin reactions in the form of discrete slight (score 1) or moderate to diffuse erythema (score 2). These anomalous unexpected findings suggest the assumption that the animals were inaccurately treated or contaminated means were used. The same reactions were also seen in the test group during induction. After challenge application, the test group and the control group showed discrete slight (score 1) or moderate to diffuse erythema (score 2) and squamation. In both groups, the reactions were almost similar in incidence and intensity. It is impossible to distinguish between allergic and irritative reactions and therefore one cannot rule out the possibility that allergic sensitisation might be mask by irritative reactions. Therefore, the findings in the test group cannot be evaluated regarding sensitisation. Consequently the test is judged as invalid.

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1981-07-22 to 1981-08-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: basic data given: comparable to guidelines/standards

Qualifier:

equivalent or similar to guideline

Guideline:

other: Buehler method

GLP compliance:

not specified

Type of study:

Buehler test

Species:

guinea pig

Strain:

not specified

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: 333 to 426 g
- Housing: individually housed in screen-bottom cages
- Diet: ad libitum, Purina guinea pig chow
- Water: ad libitum
- Acclimation period: the animals were acclimated, no further data given

ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled, no further data given
- Humidity (%): controlled, no further data given
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

Induction: 1%
Challenge: 1%
Concentration selection based on a small scale dose range study

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

Induction: 1%
Challenge: 1%
Concentration selection based on a small scale dose range study

No. of animals per dose:

dose group: 20 (10 males, 10 females)
naive control group: 4 (2 males, 2 females)

Details on study design:

RANGE FINDING TESTS:
- in a first experiment, 4 animals were treated at 4 treatment sites each with test item concentrations of 100, 75, 50 or 25 %, one further animal was treated at two treatment sites with a test item concentration of 10 %
- in a second experiment, 2 animals were treated at 2 treatment sites each with test item concentrations of 5 or 1 %
Skin reactions were scored 24 and 48 hours after initiation of treatment.

MAIN STUDY
a: PREPARATION OF ANIMAL
- The day before each application the hair was removed from the left shoulder of each animal with electric clippers.

b: PREAPARTION OF TEST MATERIAL
To prepare a 1.0% weight/volume mixture, 1.0 g of Varisoft Lot 1138K was weighed into an Erlenmeyer flask. Sterile 0.9% saline was added to make a total volume of 100 ml. The mixture was then stirred using a stir plate and a magnetic stir bar to a uniform suspension. The test material was prepared fresh prior to each application.

A. INDUCTION EXPOSURE
The test material was applied to one area on each animal by placing 0.4 ml of the freshly prepared test substance on a Webril pad (7/8 inch x 1 inch) and placing the pad on the test site along the midline of the back. The patch was covered with rubber dam and secured with an overwrap of elastoplast tape. The dressing remained in place for a period of six hours at which time it was removed. The test material was removed by a gentle rinse with warm water before returning the animals to their cages.
The animals received one application per week for three weeks for a total of three applications.

B. CHALLENGE EXPOSURE

Two weeks following the administration of the third induction (sensitising) dose, a challenge dose at a volume of 0.4 ml was administered to the test group in the same manner as during the induction (sensitising) phase of the study. At this time, the four naive (previously untreated) control animals were also treated with a challenge application. The challenge applications were made to a freshly clipped skin site that had not been previously treated.
Twenty-four hours after primary challenge, the animals were depilated with NEET Cream hair removal (Whitehall Laboratories Inc., New York, USA). The depilatory was applied on the test sites and surrounding areas for thirty minutes. Then the depilatory was thoroughly washed off with warm water, the animals dried with a towel and returned to their cages.

C: OBSERVATIONS
The application sites were read and scored for erythema and edema at 24 and 48 hours following each application during the induction (sensitising) phase of the study. Reactions to the challenge dose were read and scored two hours after depilation and 24 hours later (48 hour scores). Scoring system not explicitly mentioned, but very likely a system in the style of the Draize system was used.

OTHER:
The animals were observed for general behavior and appearance once daily during the entire study period. Body weights were taken at study initiation and at weekly intervals during the study.

Positive control substance(s):

no

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

1%

No. with + reactions:

4

Total no. in group:

20

Clinical observations:

very faint nonconfluent erythema (score 0.5)

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 1%. No with. + reactions: 4.0. Total no. in groups: 20.0. Clinical observations: very faint nonconfluent erythema (score 0.5).

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

1%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 20.0.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

1%

No. with + reactions:

1

Total no. in group:

4

Clinical observations:

very faint nonconfluent erythema (score 0.5)

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 1%. No with. + reactions: 1.0. Total no. in groups: 4.0. Clinical observations: very faint nonconfluent erythema (score 0.5).

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

1%

No. with + reactions:

0

Total no. in group:

4

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 4.0.

GENERAL BEHAVIOR and APPEARANCE:

All the guinea pigs used in this study appeared normal throughout the study period. Normal body weight gains were recorded for all animals during the course of the study.

SKIN REACTIONS

Range finding tests:

Concentrations of 5, 25, 50, 75 and 100 % induced skin reactions, no skin reactions were seen after treatment with concentrations of 1 or 10 %:

- 100% (4 animals at one application site each treated): 24 h and 48 h after treatment erythema scores of 3, 3, 3, 3 and edema scores of 3, 3, 3, 3

- 75 % (4 animals at one application site each treated): 24 h and 48 h after treatment erythema scores of 3, 3, 3, 3 and edema scores of 3, 3, 3, 3

- 50% (4 animals at one application site each treated): 24 h after treatment erythema scores of 3, 3, 2, 2 and edema scores of 3, 3, 2, 3; 48 h after treatment eythema scores of 3, 3, 2, 2 and edema scores of 3, 3, 2, 2

- 25% (4 animals at one application site treated): 24 h after treatment erythema scores of 1, 2, 2, 2 and edema scores of 0, 1, 2, 2; 48 h after treatment erythema scores of 1, 1, 2, 2 and edema scores of 1, 0, 2, 1

- 10% (one animal at two application sites treated): no erythema, no edema

- 5% (two animals at one application site each treated): 24 h after treatment erythema scores of 1 and 0; edema scores of 1 and 0; 48 h after treatment no erythema and no edema

- 1% (two animals at one application site each treated): no erythema, no edema

Main Study:

Induction: during the induction phase, animals were free of erythema and edema

Challenge:

- test animal:

-- 24 hour scoring: one male and three females reacted to the challenge application with a very faint nonconfluent erythema (score 0.5)

-- 48 hour scoring: all animals were free of erythema and edema

- naive controls:

-- 24 hour scoring: one of the two males reacted with a very faint nonconfluent erythema (score 0.5), the two female naive control animals were free of erythema and edema

-- 48 hour scoring: all animals were free of erythema and edema

Because the skin reaction in the treated animals did not exceed the most severe control reaction, the responses were not substantial enough to be considered positive for sensitisation.

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

1/10 male and 3/10 females reacted to the challenge application with a very faint nonconfluent erythema (score 0.5). In the control group 1/2 male and none of the two females reacted to the challenge application with a very faint nonconfluent erythema (score 0.5). Because the skin reaction in the treated animals did not exceed the most severe control reaction, the responses were not substantial enough to be considered positive for sensitisation.

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1981-07-22 to 1981-08-28

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: basic data given: comparable to guidelines/standards

Qualifier:

equivalent or similar to guideline

Guideline:

other: Buehler method

GLP compliance:

not specified

Type of study:

Buehler test

Species:

guinea pig

Strain:

not specified

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: 324 to 416 g
- Housing: individually housed in screen-bottom cages
- Diet: ad libitum, Purina guinea pig chow
- Water: ad libitum
- Acclimation period: the animals were acclimated, no further data given

ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled, no further data given
- Humidity (%): controlled, no further data given
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

Induction: 1%
Challenge: 1%
Concentration selection based on a small scale dose range study

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

Induction: 1%
Challenge: 1%
Concentration selection based on a small scale dose range study

No. of animals per dose:

dose group: 20 (10 males, 10 females)
naive control group: 4 (2 males, 2 females)

Details on study design:

RANGE FINDING TESTS:
- in a first experiment, 2 animals were treated at 2 treatment sites each with test item concentrations of 5 and 1 %
- in a second experiment, 2 animals were treated at 2 treatment sites each with test item concentrations of 0.1 %
Skin reactions were scored 24 and 48 hours after initiation of treatment.

MAIN STUDY
a: PREPARATION OF ANIMAL
- The day before each application the hair was removed from the left shoulder of each animal with electric clippers.

b: PREAPARTION OF TEST MATERIAL
To prepare a 1.0% weight/volume mixture, 1.0 g of Varisoft Lot 1104K was weighed into an Erlenmeyer flask. Sterile 0.9% saline was added to make a total volume of 100 ml. The mixture was then stirred using a stir plate and a magnetic stir bar to a uniform suspension. The test material was prepared fresh prior to each application.

A. INDUCTION EXPOSURE
The test material was applied to one area on each animal by placing 0.4 ml of the freshly prepared test substance on a Webril pad (7/8 inch x 1 inch) and placing the pad on the test site along the midline of the back. The patch was covered with rubber dam and secured with an overwrap of elastoplast tape. The dressing remained in place for a period of six hours at which time it was removed. The test material was removed by a gentle rinse with warm water before returning the animals to their cages.
The animals received one application per week for three weeks for a total of three applications.

B. CHALLENGE EXPOSURE

Two weeks following the administration of the third induction (sensitising) dose, a challenge dose at a volume of 0.4 ml was administered to the test group in the same manner as during the induction (sensitising) phase of the study. At this time, the four naive (previously untreated) control animals were also treated with a challenge application. The challenge applications were made to a freshly clipped skin site that had not been previously treated.
Twenty-four hours after primary challenge, the animals were depilated with NEET Cream hair removal (Whitehall Laboratories Inc., New York, USA). The depilatory was applied on the test sites and surrounding areas for thirty minutes. Then the depilatory was thoroughly washed off with warm water, the animals dried with a towel and returned to their cages.

B.1 RECHALLENGE

Because one animal exhibited a possible sensitising response to the challenge dose, a rechallenge was conducted on this animal 7 days after the primary challenge. The sensitised animal along with 4 naive control (once challenged) animals, were treated with 0.4 ml of 1% preparation (weight/volume of VARISOFT 475, LOT 1138K) and 0.4 ml of 1% preparation (weight/volume) of VARISOFT 475, LOT 195-136 (tested in Raltech Buehler test study NO. 871607, also cited in this IUCLID) in the same manner as for the primary challenge procedure, including depilation and 24 and 48 hour scores

C: OBSERVATIONS
The application sites were read and scored for erythema and edema at 24 and 48 hours following each application during the induction (sensitising) phase of the study. Reactions to the challenge dose were read and scored two hours after depilation and 24 hours later (48 hour scores). Scoring system not explicitly mentioned, but very likely a system in the style of the Draize system was used.

OTHER:
The animals were observed for general behavior and appearance once daily during the entire study period. Body weights were taken at study initiation and at weekly intervals during the study.

Positive control substance(s):

no

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

1%

No. with + reactions:

3

Total no. in group:

20

Clinical observations:

very faint nonconfluent erythema (score 0.5) in two females, slight edema (score 1) in one male

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 1%. No with. + reactions: 3.0. Total no. in groups: 20.0. Clinical observations: very faint nonconfluent erythema (score 0.5) in two females, slight edema (score 1) in one male.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

1%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 20.0.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

1%

No. with + reactions:

1

Total no. in group:

4

Clinical observations:

very faint nonconfluent erythema (score 0.5)

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 1%. No with. + reactions: 1.0. Total no. in groups: 4.0. Clinical observations: very faint nonconfluent erythema (score 0.5).

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

1%

No. with + reactions:

0

Total no. in group:

4

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 4.0.

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

1%

No. with + reactions:

0

Total no. in group:

1

Clinical observations:

one animal with greater responses after primary challenge than seen in control animals tested

Remarks on result:

other: see Remark

Remarks:

Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 1.0. Clinical observations: one animal with greater responses after primary challenge than seen in control animals tested.

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

1%

No. with + reactions:

0

Total no. in group:

4

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 4.0.

GENERAL BEHAVIOR and APPEARANCE:

All the guinea pigs used in this study appeared normal throughout the study period. Normal body weight gains were recorded for all animals during he course of the study.

SKIN REACTIONS

Range finding tests:

Concentrations of 1 and 5% induced skin reactions, no skin reactions were seen after treatment with concentrations of 0.1 %; 48 h after treatment no erythema and no edema were seen on either concentration:

- 5% (2 animals at one application site each treated): 24 h after treatment erythema and edema scores of 1 in one of the two animals whereas the second animal was free of erythema and edema; 48 h after treatment no erythema and no edema

- 1% (2 animals at one application site each treated): 24 h after treatment erythema and edema scores of 1 in one of the two animals whereas the second animal was free of erythema and edema; 48 h after treatment no erythema and no edema

- 0.1% (two animals at two application sites each treated): no erythema, no edema

Main Study:

Induction: during the induction phase, animals were free of erythema and edema

Challenge

- test animal:

-- 24 hour scoring: one male animal responded to the challenge application with a slight edema reaction (score 1), two females reacted to the challenge application with a very faint nonconfluent erythema (score 0.5)

-- 48 hour scoring: all animals were free of erythema and edema

- naive controls:

-- 24 hour scoring: one of the two males reacted with a very faint nonconfluent erythema (score 0.5), the two female naive control animals were free of erythema and edema

-- 48 hour scoring: all animals were free of erythema and edema

The skin reactions in the two treated females were not greater than the reaction seen in the naive control animals and are therefore not considered sensitisation responses.

Rechallenge

The reaction of the one treated male was greater than the reaction seen in the control animals and indicates sensitisation. Because of this reaction to the challenge dose, a rechallenge was conducted on this animal and four naive control (once challenged) animals seven days following. None of the animals responded to the rechallenge dose with any sensitisation reaction at either of the observation periods.

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

1/10 male animal responded to the challenge application with a slight edema reaction (score 1), 2/10 females reacted with a very faint nonconfluent erythema (score 0.5). In the control group 1/2 male and none of the two females reacted to the challenge application with a very faint nonconfluent erythema (score 0.5). The reaction of the one treated male was greater than the reaction seen in the control animals and indicates sensitisation. Because of this, a rechallenge was conducted on this animal and four naive control (once challenged) animals seven days following. None of the animals responded to the rechallenge dose with any sensitisation reaction at either of the observation periods.

Reference 5

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1981-07-22 to 1981-08-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: basic data given: comparable to guidelines/standards

Qualifier:

equivalent or similar to guideline

Guideline:

other: Buehler method

GLP compliance:

not specified

Type of study:

Buehler test

Species:

guinea pig

Strain:

not specified

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: 322 to 402 g
- Housing: individually housed in screen-bottom cages
- Diet: ad libitum, Purina guinea pig chow
- Water: ad libitum
- Acclimation period: the animals were acclimated, no further data given

ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled, no further data given
- Humidity (%): controlled, no further data given
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

Induction: 1%
Challenge: 1%
Concentration selection based on a small scale dose range study

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

Induction: 1%
Challenge: 1%
Concentration selection based on a small scale dose range study

No. of animals per dose:

dose group: 20 (10 males, 10 females)
naive control group: 4 (2 males, 2 females)

Details on study design:

RANGE FINDING TESTS:
- in a first experiment, 4 animals were treated at 4 treatment sites each with test item concentrations of 100, 75, 50 or 25 %
- in a second experiment one further animal was treated at two treatment site with a test item concentration of 10 %
- in third experiment, 2 animals were treated at 2 treatment sites each with test item concentrations of 5 or 1 %
Skin reactions were scored 24 and 48 hours after initiation of treatment.

MAIN STUDY
a: PREPARATION OF ANIMAL
- The day before each application the hair was removed from the left shoulder of each animal with electric clippers.

b: PREAPARTION OF TEST MATERIAL
To prepare a 1.0% weight/volume mixture, 1.0 g of Varisoft Lot 195-136 was weighed into an Erlenmeyer flask. Sterile 0.9% saline was added to make a total volume of 100 ml. The mixture was then stired using a stir plate and a magnetic stir bar to a uniform suspension. The test material was prepared freshly prior to each application.

A. INDUCTION EXPOSURE
The test material was applied to one area on each animal by placing 0.4 ml of the freshly prepared test substance on a Webril pad (7/8 inch x 1 inch) and placing the pad on the test site along the midline of the back. The patch was covered with rubber dam and secured with an overwrap of elastoplast tape. The dressing remained in place for a period of six hours at which time it was removed. The test material was removed by a gentle rinse with warm water before returning the animals to their cages.
The animals received one application per week for three weeks for a total of three applications.

B. CHALLENGE EXPOSURE

Two weeks following the administration of the third induction (sensitising) dose, a challenge dose at a volume of 0.4 ml was administered to the test group in the same manner as during the induction (sensitising) phase of the study. At this time, the four naive (previously untreated) control animals were also treated with a challenge application. The challenge applications were made to a freshly clipped skin site that had not been previously treated.
Twenty-four hours after primary challenge, the animals were depilated with NEET Cream hair removal (Whitehall Laboratories Inc., New York, USA). The depilatory was applied on the test sites and surrounding areas for thirty minutes. Then the depilatory was thoroughly washed off with warm water, the animals dried with a towel and returned to their cages.

C: OBSERVATIONS
The application sites were read and scored for erythema and edema at 24 and 48 hours following each application during the induction (sensitising) phase of the study. Reactions to the challenge dose were read and scored two hours after depilation and 24 hours later (48 hour scores). Scoring system not explicitly mentioned, but very likely a system in the style of the Draize system was used.

OTHER:
The animals were observed for general behavior and appearance once daily during the entire study period. Body weights were taken at study initiation and at weekly intervals during the study.

Positive control substance(s):

no

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

1%

No. with + reactions:

2

Total no. in group:

20

Clinical observations:

very faint nonconfluent erythema (score 0.5), both of the responses were not substantial enough to be considered positive for sensitisation.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 1%. No with. + reactions: 2.0. Total no. in groups: 20.0. Clinical observations: very faint nonconfluent erythema (score 0.5), both of the responses were not substantial enough to be considered positive for sensitisation..

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

1%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 20.0.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

1%

No. with + reactions:

0

Total no. in group:

4

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 4.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

1%

No. with + reactions:

0

Total no. in group:

4

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 4.0.

GENERAL BEHAVIOR and APPEARANCE:

All the guinea pigs used in this study appeared normal throughout the study period. Normal body weight gains were recorded for all animals during he course of the study.

SKIN REACTIONS

Range finding tests:

Concentrations of 10, 25, 50, 75 and 100 % induced skin reactions, no skin reactions were seen after treatment with concentrations of 5 and 1 %:

- 100% (4 animals at one application site each treated): 24 h after treatment erythema scores of 2, 2, 2, 2 and edema scores of 1, 1, 2, 2; 48 h after treatment erythema scores of 2, 2, 2, 2 and edema scores of 2, 2, 2, 2

- 75% (4 animals at one application site each treated): 24 h after treatment erythema scores of 2, 2, 2, 2 and edema scores of 1, 1, 2, 2; 48 h after treatment erythema scores of 2, 2, 2, 2 and edema scores of 2, 2, 2, 2

- 50% (4 animals at one application site each treated): 24 h after treatment erythema scores of 1, 2, 2, 2 and edema scores of 1, 1, 2, 2; 48 h after treatment erythema scores of 2, 2, 2, 2 and edema scores of 2, 2, 2, 2

- 25% (4 animals at one application site treated): 24 h after treatment erythema scores of 1, 2, 1, 1 and edema scores of 1, 1, 0, 0; 48 h after treatment erythema scores of 2, 2, 2, 2 and edema scores of 2, 2, 2, 1

- 10% (one animal at two application sites treated): 24 h after treatment erythema and edema score 1 at both test sites, 48 h after treatment no erythema and no edema

- 5% (two animals at one application site each treated): no erythema, no edema

- 1% (two animals at one application site each treated): no erythema, no edema

Main Study:

Induction: during the induction phase, animals were free of erythema and edema

Challenge

-test animal:

-- 24 hour scoring: One male and one females reacted to the challenge application with a very faint nonconfluent erythema (score 0.5). Both of the responses were not substantial enough to be considered positive for sensitisation.

-- 48 hour scoring: all animals were free of erythema and edema

-- naive controls:

-- 24 and 48 hour scoring: all animals were free of erythema and edema at both scorings

The responses were not substantial enough to be considered positive for sensitisation.

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

One male and one females reacted to the challenge application with a very faint nonconfluent erythema (score 0.5). Both of the responses were not substantial enough to be considered positive for sensitisation.

Reference 6

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).

Reference 7

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2009-04-01 to 2009-06-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

as layed down in Commission Regulation (EC) No 440/2008 of 30 May 2008

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A valid GPMT conducted according to guideline is available, which is reliable without restrictions and adequate for classification and labelling purposes. Potency estimation is not mandatory when existing guideline and GLP conforming data are available, which were conducted before the new annex of the REACH Regulation entered into force. Moreover, no indication for skin sensitisation was observed in this study, thus, no dose response information is needed. For this reason and for reasons of animal welfare no additional LLNA was conducted.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
Albino Dunkin Hartley Guinea Pig, CRL:(HA)BR, SPF
- Age at study initiation: 4-6 weeks
- Weight at study initiation: pretest groups: 332 - 349 g, test and control group: 330 - 366 g
- Housing: individually in Makrolon type-4 cages with standard softwood bedding ("Lignocel", Schill AG, 4132 Muttenz, Switzerland).
- Diet: ad libitum, pelleted standard Provimi Kliba 3418 guinea pig breeding / maintenance diet batch no. 82/08, Provimi Kliba AG, 4303 Kaiseraugst, Switzerland
- Water: ad libitum, tap water as for human consumption
- Acclimation period: animals of test and control group were acclimatised, no data on acclimation length


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): automatically controlled light cycle of 12 hours light and 12 hours dark

Route:

intradermal and epicutaneous

Vehicle:

polyethylene glycol

Concentration / amount:

induction: 0.5 % (V/V) in intradermal, 10 % (V/V) epicutaneous
challenge: 1 % (V/V)
Concentration selection based on a small scale dose range studies

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Concentration / amount:

induction: 0.5 % (V/V) in intradermal, 10 % (V/V) epicutaneous
challenge: 1 % (V/V)
Concentration selection based on a small scale dose range studies

No. of animals per dose:

treatment group: 10
control group: 5

Details on study design:

RANGE FINDING STUDIES
Intradermal injection:
For intradermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of three guinea pig. Six days later intradermal injections (0.1 mL/site) were made into the clipped flank . One animal received spatially divided injections of 25, 15, and 10 % , one of 5, 2.5, and 1 % and one of 0.5 and 0.1 %. Concentrations were formulated in polyethylene glycol 300 (PEG 300). Dermal reactions were assessed 24 hours later.

Dermal application:
Four intradermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of two guinea pigs. Six days later, four patches of filter paper (3 x 3 cm) were saturated with the test item at 100% (technically the highest possible concentration to be applied sufficiently), 75%, 50% and 25% in PEG 300 and applied to the clipped and shaved flanks of the same guinea pigs. The amount or volume of test item preparation applied was approximately 0.2 g at 100%, 75% and 50% or 0.2 mL at 25%. The concentration causing mild (to moderate) local skin reaction and the concentration being the highest tested non-irritating concentration could not be determined after the epidermal pretest described above. Therefore, a second pretest was performed with two additional naive guinea pigs, treated in the same way as those described previously, with the concentrations of 15%, 10%, 5% and 1% in PEG 300, using an application volume of approximately 0.2 mL.

MAIN STUDY
A. INDUCTION EXPOSURE
- First stage (test day 1)
An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 mL/site) were made symmetrically in two rows on either side of the sine just within the boundaries of a 4 x 6 cm area in the clipped region.
-- Test groups:
1. 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline
2. The test item at 0.5% in PEG 300
3. The test item at 0.5% in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline
- Control group:
1. 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline
2. PEG 300
3. 1:1 (w/w) mixture of PEG 300 in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline

- Second stage - One week after the intradermal injections (test day 8), the scapular area (approximately 6 x 8 cm) was again clipped and shaved free of hair prior to the application. A 2 x 4 cm patch of filter paper was saturated with the test item at 10% in PEG 300 and placed over the injection sites of the test animals. The volume of test item preparation applied was approximately 0.3 mL. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressings were left in place
for 48 hours. The epidermal application procedure described ensured intensive contact of the test item.

The guinea pigs of the control group were treated as described above with PEG 300 only, applied at a volume of approximately 0.3 mL.

The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.

B. CHALLENGE EXPOSURE (test day 22)
The test and control guinea pigs were challenged two weeks after the epidermal induction application and were treated in the same way.

Two patches (3 x 3 cm) of filter paper were saturated with the test item at the highest tested nonirritating concentration of 1% (applied to the left flank) and the vehicle only (PEG 300 applied to the right flank) using the same method as for the epidermal application. The volume of test item preparation and vehicle alone applied was approximately 0.2 mL. The dressings were left in place for 24 hours.

C. OBSERVATION and SCORING
The test item skin area of the animals used for the epidermal pretest and challenge was depilated approximately 21 hours after the patches have been removed, using an approved depilatory cream (VEET Cream, Reckitt & Colman AG, 4123 Allschwil, Switzerland). The depilation was performed to clean the stratum corneum from the possible staining produced by the test item and to facilitate the reading of the possible skin reaction. The depilatory cream was placed on the patch sites and surrounding areas, and left on for up to 3-5 minutes. It was then thoroughly washed off with a stream of warm, running water. The animals were then dried with a disposable towel, and returned to their cages.

The scoring system was performed by visual assessment of erythema and oedema.
0 = no visible change
1 = discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling

Any other gross lesions not covered by this scoring system were described.

The grading method used for the pretest, induction and challenge was identical. It was performed 24 ± 2 hours after removal of the patches for the intradermal and epidermal pretest and induction and for challenge and repeated 24 ± 2 hours later (48-hour grades) for the epidermal pretest,
epidermal induction and challenge.

OTHER OBSERVATIONS
- Viability / Mortality: Daily from delivery of the animals to the termination of the test.
- Clinical Signs / Grading of Skin Response: Daily from delivery of the animals to the termination of test. Skin responses were graded during the pretest, induction and challenge periods.
- Body Weights: At delivery/acclimatization start, at the end of the pretest, at test day 1 (day of treatment) and at the termination of the study.

POSITIVE CONTROL (RELIABILITY CHECK):
- The sensitivity and reliability of the experimental technique employed was assessed by use of ALPHA-HEXYLCINNAMALDEHYDE which is recommended by the OECD 406 Guidelines and is known to have moderate skin sensitisation properties in the guinea pig strain. The results from the most recent test run (Harlan Laboratories Study C16261, performed from 08-Oct-2008 to 14-Nov-2008) were included in the study report.

STATISTICAL ANALYSIS
Descriptive statistics (means and standard deviations) were calculated for body weights. No inferential statistics were used.

Positive control substance(s):

yes

Remarks:

ALPHA-HEXYLCINNAMALDEHYDE, periodically tested, most recent test run performed from 08-Oct-2008 to 14-Nov-2008

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

1 %

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

discrete or patchy erythema (score 1)

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 1 %. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: discrete or patchy erythema (score 1).

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

1 %

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1 %. No with. + reactions: 0.0. Total no. in groups: 10.0.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

1 %

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 1 %. No with. + reactions: 0.0. Total no. in groups: 5.0.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

1 %

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 1 %. No with. + reactions: 0.0. Total no. in groups: 5.0.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

other: vehicle (PEG) control in initiated animals

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: vehicle (PEG) control in initiated animals. Dose level: 0 %. No with. + reactions: 0.0. Total no. in groups: 10.0.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

other: vehicle (PEG) control in initiated animals

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: vehicle (PEG) control in initiated animals. Dose level: 0 %. No with. + reactions: 0.0. Total no. in groups: 10.0.

Key result

Reading:

other: 1st and 2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

3%

Remarks on result:

positive indication of skin sensitisation

Remarks:

total number of animals not reported, all animals were reported to show positive reactions on challenge

RESULTS

Range finding studies:

Intradermal injection: All tested concentrations caused skin reactions:

- 25 and 10 %: moderate and confluent erythema (score 2) with swelling

- 10, 5, 2.5 and 1 %: moderate and confluent erythema (score 2)

- 0.5 %: discrete or patchy erythema (score 1)

- 0.1 %: discrete or patchy erythema (score 1) with blanching

Dermal application: The test item at a concentration of 1 % caused no skin reactions. Concentration depended skin reactions after application of 5 to 100 %:

- 100 and 75 %: at 24 and 48 h scoring intense erythema and swelling (score 3), at 48 h with scaling and necrosis

- 50 %: at 24 and 48 h scoring moderate and confluent erythema (score 2), at 24 with swelling, at 48 h with scaling and necrosis

- 25 %: at 24 h scoring in both animals moderate and confluent erythema (score 2), at 48 h scoring one animals with discrete or patchy erythema (score 1) with scaling and one animals with moderate and confluent erythema (score 2) with scaling and necrosis

- 15, 10 and 5 %: at 24 and 48 h scoring discrete or patchy erythema (score 1), at 48 h with scaling

- 1 %: at 24 and 48 h scoring no visible changes

Main study:

- Induction:

-- intradermal: The expected and common findings were observed in the control and test group after the different applications using FCA intradermally. These findings consisted of erythema, oedema, necrotizing dermatitis, encrustation and exfoliation of encrustation.

-- epicutaneous: No erythematous or oedematous reaction was observed in the animals treated with PEG 300 only. Discrete or patchy (score 1) to moderate and confluent (score 2) erythema was observed in all animals at the 24- and 48 -hour scorings after treatment with the test item at 10% in PEG 300; in detail at 24 h scoring 8/10 animals with discrete or patchy erythema (score 1), 2/10 animals with moderate and confluent erythema (score 2), at 48 h scoring 9/10 animals with discrete or patchy erythema (score 1), 1/10 animals with moderate and confluent erythema (score 2). Scaling was observed in 70% of the test animals at the 48-hour reading.

- Challenge

-- test group: Discrete or patchy erythema (score 1) was observed in 1/10 of the test animals at the 24-hour scoring after treated with the test item at 1% in PEG 300. No local skin reaction was observed at the 48-hour scoring.

No skin reactions were observed in the test animals when treated with PEG 300 only.

-- control group: No skin reactions were observed in the animals when treated with PEG 300 alone or when treated with the test item at 1% in PEG 300.

OTHER OBSERVATIONS

- Viability / Mortality: There were no deaths during the course of the study, hence no necropsies were performed.

- Clinical Signs: No signs of systemic toxicity were observed in the animals.

- Body Weights: The body weight of the animals was within the range commonly recorded for animals of this strain and age.

Reliability check (positive control):

The test was performed from 08-Oct-2008 to 14-Nov-2008. All test animals showed skin reactions after the challenge treatment with ALPHAHEXYLCINNAMALDEHYDE at 3% in PEG 300. No skin effect was observed in the animals treated with ALPHA-HEXYLCINNAMALDEHYDE at 1% in PEG 300. No skin effect was observed in the control group.

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Ten percent of the test animals showed discrete/patchy erythema at the 24-hour scoring after the challenge treatment with the partially unsaturated IQAC, DMS quaternised (NO SOLVENT) at 1% (w/v) in PEG 300; at the 48 h scoring the 10 animals were free of skin reactions. None of the 5 control animals showed skin reactions at challenge. Based on these findings in an adjuvant sensitization test (Magnusson Kligman maximisation test) in guinea pigs, the partially unsaturated IQAC, DMS quaternised does not have to be classified and labelled as a skin sensitizer.

Executive summary:

In a dermal sensitisation study with the partially unsaturated IQAC, DMS quaternised (NO SOLVENT, 100 % pure) in PEG 300, 15 Dunkin Hartley guinea pigs (10 in treatment group, 5 in control group), were tested using the MAXIMISATION METHOD described by OECD Guideline No. 406 and the Commission Regulation (EC) No 440/2008, B.6. The sensitivity of the test system was assessed by use of the positive control substance ALPHA-HEXYLCINNAMALDEHYDE.

The intradermal induction of sensitisation in the test group was performed in the nuchal region with a 0.5% dilution of the test item in PEG 300 and in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline. The epidermal induction of sensitisation was conducted for 48 hours under occlusion with the test item at 10% in PEG 300 one week after the intradermal induction. The animals of the control group were intradermally induced with PEG 300 and FCA/physiological saline and epidermally induced with PEG 300 under occlusion. Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test item at 1% in PEG 300 and PEG 300 alone under occlusive dressing.

Ten percent of the test animals showed discrete or patchy erythema at the 24-hour scoring after the challenge treatment with test substance at 1% (w/v) in PEG 300; at the 48 h scoring the 10 animals were free of skin reactions. None of the 5 control animals showed skin reactions at challenge.

Based on the above mentioned findings in an adjuvant sensitisation test (Magnusson Kligman maximisation test) in guinea

pigs the partially unsaturated IQAC, DMS quaternised does not have to be classified and labelled as a skin sensitiser.

Reference 8

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study report which meets basic scientific principles

Qualifier:

equivalent or similar to guideline

Guideline:

other: Maximization test according B. Magnusson and A.M. Kligmann

GLP compliance:

not specified

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A valid GPMT conducted according to guideline is available, which is reliable without restrictions and adequate for classification and labelling purposes. Potency estimation is not mandatory when existing guideline and GLP conforming data are available, which were conducted before the new annex of the REACH Regulation entered into force. Moreover, no indication for skin sensitisation was observed in this study, thus, no dose response information is needed. For this reason and for reasons of animal welfare no additional LLNA was conducted.

Species:

guinea pig

Strain:

Pirbright-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Guinea pigs strain Pirbright (Hoe:DHPK (SPF-LAC.) / Boe.
- Age at study initiation: no data
- Weight at study initiation: ca. 400 g
- Housing: 2 animals / cage (Makrolon III)
- Diet: ad libitum, standard diet for guinea pigs, Ssniff-G
- Water: ad libitum, tap water
- Acclimation period: 7 d


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 45 - 55
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): artificial light, 120 Lux, 4000 °K, 12 hrs dark / 12 hrs light, from 7:00 to 19:00


IN-LIFE DATES: From: To:

Route:

intradermal and epicutaneous

Vehicle:

water

Concentration / amount:

induction:
- intradermal: 5 % in Aqua dest (plus peanut oil by use of FCA)
- epicutaneous: 5 % in petrolatum after pretreatment with 10 % Na-lauryl-sulphate in white Vaseline

Challenge (3 concentrations per animal):
- 10 % in Aqua dest.
- 5 % in Aqua dest.
- 2.5 % in Aqua dest.

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

induction:
- intradermal: 5 % in Aqua dest (plus peanut oil by use of FCA)
- epicutaneous: 5 % in petrolatum after pretreatment with 10 % Na-lauryl-sulphate in white Vaseline

Challenge (3 concentrations per animal):
- 10 % in Aqua dest.
- 5 % in Aqua dest.
- 2.5 % in Aqua dest.

No. of animals per dose:

15 animals (female and male)

Details on study design:

RANGE FINDING TESTS:
Testing of dermal compatibility with 5 % a.i. aq., 10 % aq., and 2.5 % a.i. aq. test substance with 2 animals/concentration

MAIN STUDY
CONCENTRATIONS AND TEST FORMULATIONS
Intradermal application:
1. 0.05 ml FCA (1 : 1 in Aqua dest.)
2. 0.05 ml test substance (5 % in Aqua dest.)
3. 0.05 ml test substance (5 %) in FCA (1 : 1 in peanut oil)

Topical application:
1. Na-lauryl-sulphate 10 % in white Vaseline (petrolatum)
2. test substance, 5 % in petrolatum

Challenge retesting:
1. test substance 10 % in Aqua dest.
2. test substance 5 % in Aqua dest.
3. test substance 2.5 % in Aqua dest.

Negative control group: Aqua dest
Positive control group: Methyl metacrylate

A. INDUCTION EXPOSURE
Combined intradermal and epicutaneous application, for intradermal induction 3 injections pairs, in total 6 injections, epicutaneous induction treatment between first and second set of intradermal injections.
An area of 4 x 6 cm over the shoulders was clipped short with electric clippers.

A.1 Intradermal
1. 0.05 ml FCA (1 : 1 in Aqua dest.)
2. 0.05 ml test substance (5 % in Aqua dest.)

A.2 Epicutaneous
One day after this intradermal induction treatment the left part of the clipped area was pretreated with 10 % Na-lauryl-sulphate in white Vaseline, to induce a slight irritation (no wrapping).
6 - 8 h later test substance, 5 % in petrolatum was dermally applied for topical induction treatment (site was covered with gaze and wrapped with Elastoplast).

A.3 Intradermal
On the day after epicutaneous induction (48 h after the first intradermal injections) the bandage was removed and the second part of the intradermal induction treatment was performed by intradermal application of
0.05 ml test substance (5 %) in FCA (1 : 1 in peanut oil).

The control substances were used accordingly the test substance.

B. CHALLENGE EXPOSURE - Closed Patch-Test
- No. of exposures: 3 different concentrations/animal, highest concentration at the most caudal region
- Day(s) of challenge: 14 days after last induction treatment
- Exposure period: 24 h
- Test groups: 0.04 ml of test substance in 3 different concentrations was applied to small Hansaplast strips (Beiersdorf, Hamburg, Germany) and applied on the clipped area. Covering with gauze wrap and Elastoplast.
- Control groups: same procedure as with test group, also three concentrations tested with positive control substance
- Site: clipped left site
- Evaluation (hr after challenge): 24 h, 48 h

C. SCORING SYSTEM:
No skin reaction: score 0
Discrete or patchy erythema: score 1
Moderate or confluent erythema: score 2
Intense erythema and swelling: score 3

Positive control substance(s):

yes

Remarks:

Methyl metacrylate (Merck-Schuchard, Hohenbrunn, Germany), tested in parallel

Positive control results:

Methylmethacrylat: 15/15 animals were positive at both scorings.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

2.5, 5, 10 %

No. with + reactions:

0

Total no. in group:

15

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 2.5, 5, 10 %. No with. + reactions: 0.0. Total no. in groups: 15.0. Clinical observations: none.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

2.5, 5, 10 %

No. with + reactions:

0

Total no. in group:

15

Clinical observations:

none

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 2.5, 5, 10 %. No with. + reactions: 0.0. Total no. in groups: 15.0. Clinical observations: none.

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

2.5 %

No. with + reactions:

12

Total no. in group:

15

Clinical observations:

skin reaction score 1 or 2

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 2.5 %. No with. + reactions: 12.0. Total no. in groups: 15.0. Clinical observations: skin reaction score 1 or 2.

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

5, 10 %

No. with + reactions:

15

Total no. in group:

15

Clinical observations:

skin reaction score 1 or 2

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 5, 10 %. No with. + reactions: 15.0. Total no. in groups: 15.0. Clinical observations: skin reaction score 1 or 2.

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

2.5 %

No. with + reactions:

11

Total no. in group:

15

Clinical observations:

skin reaction score 1

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 2.5 %. No with. + reactions: 11.0. Total no. in groups: 15.0. Clinical observations: skin reaction score 1.

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

5 %

No. with + reactions:

14

Total no. in group:

15

Clinical observations:

skin reaction score 1 or 2

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 5 %. No with. + reactions: 14.0. Total no. in groups: 15.0. Clinical observations: skin reaction score 1 or 2.

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

10 %

No. with + reactions:

15

Total no. in group:

15

Clinical observations:

skin reaction score 1 or 2

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 10 %. No with. + reactions: 15.0. Total no. in groups: 15.0. Clinical observations: skin reaction score 1 or 2.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

15

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0 %. No with. + reactions: 0.0. Total no. in groups: 15.0. Clinical observations: none.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

15

Clinical observations:

none

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0 %. No with. + reactions: 0.0. Total no. in groups: 15.0. Clinical observations: none.

Preliminary range finding test

No primary dermal irritation

Main study

- Induction: not reported, however, to create a local irritation during induction phase, animals were treated with 10 % Na-lauryl-sulphate in white Vaseline

- Challenge

-- Test animal: at both scorings all scores were zero for all sites treated with the test item concentrations of 2.5, 5 or 10%. The sensitisation rate at 24 h and at 48 h was 0 %.

-- Negative control (Aqua dest.): at both scorings all scores were zero

-- Positive control (Methylmethacrylat): --- 24 h scoring: 12/15, 15/15 and 15/15 of skin areas treated with methylmethacrylate concentrations of 2.5%, 5% and 10 %, respectively, showed positive reactions score 1 or 2 --- 48 h scoring: 11/15 skin areas treated with a methylmethacrylate concentration of 2.5 % showed positive reactions score 1; 14/15 and 15/15 of skin areas treated with 5% and 10 %, respectively, showed positive reactions score 1 or 2

Discussion

Test performance was almost similar to OECD guideline 406. Deviations to this guideline are the combined intradermal and epicutaneous induction treatment instead of consecutive intradermal, epicutaneous induction treatment and the challenge treatment with simultaneous application of three different test item concentrations, no test item negative control (induction treatment without test item, challenge with test item). These deviations are not expected to have an impact on study result. Test concentration used for induction was non-irritating and thus not the lowest concentration which cause mild-to moderate skin irritations as requested by the guideline. However in course of the induction treatment, epicutaneous application test sites was pretreated with 10 % sodium lauryl sulphate in order to create a local irritation. According OECD guideline 406 sodium lauryl sulphate is recommended if the substance is not a skin irritant. It can be assumed that the promoter substance sodium lauryl sulphate has compensated the missing irritative effect of the test item induction concentration. Therefore, the induction treatment used in this test is evaluated as sufficient to induce a potential allergic sensitisation reaction and the test is judged as valid.

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

In this test almost similar to OECD guideline 406, none of the animals of the test group were observed with positive skin reactions after challenge treatment with the test substance patially unsaturated IQAC, DMS quaternised at concentrations of 2.5, 5, and 10 % in water. With methyl metacrylate, tested in parallel as positive control material, a clear positive result was achieved. The result of the positive control affirms the sensitivity and reliability of the experimental technique used.

Executive summary:

In a dermal sensitisation study with the partially unsaturated IQAC, DMS quaternised, 15 Pirbright-Hartley guinea pigs were tested using the maximisation method of Magnusson and Kligman. For the intradermal and epicutaneous induction procedure test substance concentrations of 5 % in water were used. Intradermal and epicutaneous induction was not consecutively but conjunctly performed. In course of the induction treatment, epicutaneous application test sites were pretreated with 10 % sodium lauryl sulphate in order to create a local irritation. The test article concentrations for the challenge procedure were 2.5, 5 and 10 %, applied simultaneously to distinct skin areas.

Test performance was almost similar to OECD guideline 406. Deviations to this guideline are the combined intradermal and epicutaneous induction treatment instead of consecutive intradermal, epicutaneous induction treatment and the challenge treatment with simultaneous application of three different test item concentrations, no test item negative control (induction treatment without test item, challenge with test item). These deviations are not expected to have an impact on study result. Test concentration used for induction was non-irritating and thus not the lowest concentration which cause mild-to moderate skin irritations as requested by the guideline. However in course of the induction treatment, epicutaneous application test sites was pretreated with 10 % sodium lauryl sulphate in order to create a local irritation. According OECD guideline 406 sodium lauryl sulphate is recommended if the substance is not a skin irritant. It can be assumed that the promoter substance sodium lauryl sulphate has compensated the missing irritative effect of the test item induction concentration. Therefore, the induction treatment used in this test is evaluated as sufficient to induce a potential allergic sensitisation reaction and the test is judged as valid.

None of the animals of the test group were observed with positive skin reactions after challenge treatment with the test substance at concentrations of 2.5, 5, and 10 % in water. With methyl metacrylate, tested in parallel as positive control material, a clear positive result was archived. The result of the positive control affirms the sensitivity and reliability of the experimental technique used.

In this study, the partially unsaturated IQAC, DMS quaternised is not a dermal sensitiser.

Reference 9

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

No experimental data on skin sensitisation are available for the target substance hydrogenated tallow/nortallow based IQACs. However, studies are available for source substances basically unsaturated fatty acid based IQACs. A detailed justification for read-across is attached to IUCLID section 13.

Nine sensitisation studies were performed. Among these the outcomes of three studies were rated as ambiguous. The other six were rated as “Not sensitising”. The studies were performed with IQACs based on partially unsaturated fatty acid species. The results rated as ambiguous were derived from 2 studies with palm oil and 1 with tallow fatty acids.

Of the studies rated as ambiguous, 2 were performed with the LLNA and 1 with the Magnuson-Kligman Maximization test. Of the results rated as “Not sensitising” one was obtained with a variant of the LLNA test, including a challenge test, 3 with the Buehler test and 2 with the standard Magnuson-Kligman Maximization test.

The reasons for rating the GPMT study as ambiguous were technical insufficiencies and lack of differentiation between irritation and sensitisation as the test group and the controls showed similar incidences of erythema and squamation (Evonik, 1983).

In a second study the concentration of the test substance was too high to clearly differentiate between irritation and allergic response in an LLNA (Evonik, 2008, report no.169300) and the interpretation of the result was further compromised due to the observed inconsistency in the results induced at high levels of irritation which masked the possibility to distinguish between a true immunologically mediated memory response and a non-immunologically mediated irritation effect.

The test was further found to be inconclusive due to the missing measurement of ear thickness on the day of the challenge application.

 

In another LLNA study (Evonik, 2008, report no 1133503) with a partially unsaturated IQAC, DMS quaternised, (palm oil based; 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. 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 results 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 stimulation indices (S.I.) of 17.20, 13.34, and 11.72 determined 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 questions whether the increased incorporation of 3H-TdR 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 (Evonik, 2008, report no. 1180400).

  

In the “NATIONAL INDUSTRIAL CHEMICALS NOTIFICATION AND ASSESSMENT SCHEME; FULL PUBLIC REPORT, Varisoft 3690” by the National Occupational Health and Safety Commission of Australia 1999 (NICNAS 1999) an Assessment of the sensitising potential of Varisoft 3690 (CAS-No. 72749-55-4; Oleic-acid based IQAC, DMS quaternised) has been presented. As reference compound Varisoft 475 (CAS-No. 68122-86-1; partially unsaturated IQAC, DMS quaternised (based on tallow fatty acids) was discussed. Three lots of the test sub stance were studied at 1% concentration in the Buehler test, in which two lots were evaluated as non-sensitising and one lot as sensitising. While the results on the two lots tested as non-sensitisers were reported in detail, conducted with concomitant controls and containing a challenge experiment, this was not the case with the third lot where the reported data were rather limited and did not include information on control animals. The same test substance tested at 5 % was evaluated as sensitising.

The test performed at 5% concentration was only submitted as an abstract so that the claim of a sensitising potential cannot be reconstructed.

Further, a Human Repeat Insult Patch Test conducted with a partially unsaturated IQAC, DMS quaternised (based on tallow fatty acids; Test lot with 90 % a.i.) was submitted as a summary of the test results. NICNAS cites 217 volunteers receiving 9 exposures to an induction concentration of 0.25 % aqueous partially unsaturated IQAC, DMS quaternised (tallow fatty acids, 0.3 mL/occluded patch) applied for a 24 hour period, three times a week during a three week induction period and after two weeks later a challenge dose of 0.25% aqueous solution of the test substance applied in a single 24-hour occluded patch. It is reported that none of the volunteers were sensitised to partially unsaturated IQAC, DMS quaternised (tallow fatty acids; 90 %).

NICNAS has evaluated the available information and state that based on these results, a skin sensitisation potential of oleic-acid based IQAC, DMS quaternised (CAS-no. 72749-55-4) cannot be discounted. The difficulties in discerning irritative from truly sensitising effects may not have been considered sufficiently in this context. The proof of evidence for a sensitising potential of this substance class is very limited and contradictory, so that no firm conclusion can be drawn.

In summary it can be concluded that the majority of the studies conducted with different representatives of the IQAC structural family and with due consideration of skin irritation as a potential confounder, have proven that these substances are not to be regarded as skin sensitisers.

No studies have been reported where a clear evidence for a skin sensitising potential could be demonstrated while excluding the confounding effect of significant skin irritation.

The OECD TG 429 refers to confounding factors - like irritation - believed to play a role in an observed increase of false positives in the LLNA when looking at e.g. Surfactants.

 

The majority of the studies were judged to be of good quality and reliability. The animal studies were either in full compliance or partly compliant with OECD guideline 429 (LLNA) or 406 (guinea pig maximization or Buehler test). One Maximisation Test and one LLNA were rated as ‘not reliable due to significant methodological deficiencies’ (Klimisch rating 3). While broadly compliant with the testing guidelines, a high level of irritation was observed in several studies in test and control animals. This could have masked a potential skin sensitisation response or impacted on the overall study outcomes. These studies were dismissed from final evaluation, because no reliable conclusion, with regard to classification and labelling, can be drawn from these studies.

Reliable studies, evaluated on the basis of the requirement for classification as skin sensitiser were negative with respect to a skin sensitising potential.

Discussion:

Following the Integrated Testing Strategy (ITS) according to Guidance on information requirements and chemical safety assessment, Endpoint specific guidance, Chapter R. 7.2.6 there is sufficient information available to proceed with classification and labelling using a weight of evidence approach for all IQACs studied.

It is considered appropriate to include all IQACs in the weight of evidence approach, the IQACs differing mainly in the fatty acid moiety.

In conclusion the weight of the evidence suggests that neither of the IQACs represents a skin sensitisation hazard to humans and hence there is no need to classify according to Directive 67/548/EEC as well as GHS Regulation EC No 1272/2008 and labelling is not required.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Due to the low vapour pressure of the IQACs, the absence of aerosols in car wash applications and the absence of indications for skin sensitisation, a respiratory sensitisation potential of the test substance can practically be excluded.

Short description of key information:
A respiratory sensitisation potential of the test substance can practically be excluded.

Justification for classification or non-classification

Based on the results of the dermal sensitisation studies according to OECD guidelines 406 and 429 there is no need for classification of IQACs according to EU criteria and GHS requirements with respect to skin sensitisation. On the basis of absence of skin sensitisation and the very low vapour pressure of the test substance family there is no need to classify the test substance with respect to respiratory sensitisation.