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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 November 2000 to 30 December 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Magnusson and Kligman
Deviations:
not specified
GLP compliance:
no
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 3-Octadecyloxypropyl-N,N,N-trimethylammonium chloride
- Molecular formula (if other than submission substance): Not applicable
- Molecular weight (if other than submission substance): Not applicable
- Smiles notation (if other than submission substance): Not applicable
- InChl (if other than submission substance): Not applicable
- Structural formula attached as image file (if other than submission substance): Not applicable
- Substance type: white solid
- Physical state: solid
- Analytical purity: 93.6%
- Impurities (identity and concentrations): Not reported
- Composition of test material, percentage of components: Not reported
- Isomers composition: Not reported
- Purity test date: Not reported
- Lot/batch No.: HM-375
- Expiration date of the lot/batch: Not reported
- Radiochemical purity (if radiolabelling): Not applicable
- Specific activity (if radiolabelling): Not applicable
- Locations of the label (if radiolabelling): Not applicable
- Expiration date of radiochemical substance (if radiolabelling): Not applicable
- Stability under test conditions: Not reported.
- Storage condition of test material: Room temperature protecting from light.
- Other: Not reported.

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Crj:Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: Five weeks old.
- Weight at study initiation: 277 - 315 g
- Housing: The animals were housed in a stainless wire hanger cage (36 cmD X 25 cmW X 18 cmH) in a barrier-system-animal room.
- Diet (e.g. ad libitum): Commercial diet (RC4) ad libitum
- Water (e.g. ad libitum): Mains water ad libitum (Kaizu-cho, Kaizu-gun, Gifu, Japan)
- Acclimation period: One week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Room temperature of 20 - 26 deg C.
- Humidity (%): Relatively 40 - 70%
- Air changes (per hr): 12 - 18 exchanges/hour
- Photoperiod (hrs dark / hrs light): Light for 12 hours followed by 12 hours darkness.

IN-LIFE DATES: From: Day 0 To: Day 25

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: see 'concentration'
Concentration / amount:
(intradermal induction - induction Day 0)
Test group: area (1) Physiological saline + Freund's complete adjuvant (FCA), area (2) 0.01% of the substance as suspension in physiological saline, area (3) 0.01% of the substance + FCA

Control group: area (1) Physiological saline + FCA, area (2) physiological saline, area (3) Physiological saline + FCA

(Patch induction - induction Day 7)
Test group: 10% as as suspension in water for injection
Control group: water for injection

(Challenge - induction Day 21)
0.5, 0.3, 0.1, 0.05, 0.03 and 0.01% as suspension in water.

* Test condition of each treatment group is summarised in Table 1 in 'any other information on materials and methods incl. tables'. Please refer area (1), area (2) and area (3) in the figure, as attached.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: see 'concentration'
Concentration / amount:
(intradermal induction - induction Day 0)
Test group: area (1) Physiological saline + Freund's complete adjuvant (FCA), area (2) 0.01% of the substance as suspension in physiological saline, area (3) 0.01% of the substance + FCA

Control group: area (1) Physiological saline + FCA, area (2) physiological saline, area (3) Physiological saline + FCA

(Patch induction - induction Day 7)
Test group: 10% as as suspension in water for injection
Control group: water for injection

(Challenge - induction Day 21)
0.5, 0.3, 0.1, 0.05, 0.03 and 0.01% as suspension in water.

* Test condition of each treatment group is summarised in Table 1 in 'any other information on materials and methods incl. tables'. Please refer area (1), area (2) and area (3) in the figure, as attached.
No. of animals per dose:
Ten animals for test article and 5 animals for vehicle control.
Details on study design:
RANGE FINDING TESTS:
Intradermal – Four guinea pigs were injected intradermally at dorsal skin with 0.1 mL of test substance suspended in physiological saline (3, 1, 0.5, 0.3, 0.1, 0.05, 0.01 and 0.005%). Skin reaction was observed 24, 48 and 72 hours later. In the results, blackish change, ulcer or necrosis of skin was noted at 0.05% and more. In addition, moderate erythema was noted between 0.01 and 0.005%. From these results, 0.01% was selected for main study sensitising concentration.
Dorsal skin was applied with a 1.5 x 1.5 cm square lint, which was impregnated with 0.06mL of test substance suspended in physiological saline (10, 5, 3, 1 and 0.5%) or test substance dissolved in 50% ethanol, by closed patch method same as induction exposure and observed reaction. In the results, very slight erythema was noted at 1% and more in physiological saline suspension and at 10% in 50% ethanol solution. On the other hand, there were no signs of erythema or edema at 0.5% in physiological saline suspension. From these results, for the main study, water for injection was selected as vehicle, 10% was selected as induction concentration and 0.5, 0.3, 0.1, 0.05, 0.03 and 0.01% were selected as challenge concentration.

INTRADERMAL INDUCTION
On the day before intra-dermal induction (induction -1 day), intradermal induction area of neck
dorsal skin (Figure, broken line) and its surrounding were shaved and clipped with an electric shaver and electric clipper. On next day (induction 0 day), 2 areas of (1, (2) and (3) shown in the figure were injected intradermally with 0.1 mL/area of follows.
Area (1) in all groups was injected with FCA emulsified liquid in physiological saline. Areas (2) and (3) in the test article group were injected with 0.01% of the test substance suspended in physiological saline and 0.01% FCA emulsified liquid, respectively. Areas (2) and (3) in the vehicle control group were injected physiological saline and FCA emulsified liquid in physiological saline, respectively.

PATCH INDUCTION
On induction 6 day, patch induction area (Figure, broken line) and its surrounding were shaved
and clipped with an electric shaver and electric clipper.
On induction 7 day, patch induction area was applied with square lint (size of 2 X 4cm), which was impregnated with 0.2 mL of follows. In the test article group, 10% of the test substance suspended in physiological saline was used. In the vehicle control group, water for injection was used. The lint was covered with a dental damp-proof rubber sheet and fixed by swaddling with an adhesive elastic bandage. The dressing was left for 48 hours.

CHALLENGE
On induction 20 day, dorsal skin was shaved and clipped with an electric shaver and electric
clipper. On next day (induction 21 day), in both test article and vehicle control group, dorsal
challenge area (Figure, solid line) was applied a 1.5 X 1.5cm square lint, which was impregnated with 0.06 mL of the test substance suspended in physiological saline (0.5, 0.3, 0.1, 0.05, 0.03 and 0.01 %). The lint was covered with a dental damp-proof rubber sheet and fixed by swaddling with an adhesive elastic bandage. The dressing was left for 24 hours. To avoid deviation of the skin reaction, the challenge sites were rotated among the animals.

OBSERVATIONS AND JUDGEMENTS
Skin reaction was observed 24, 48 and 72 hours later from removing test article and scored
according to the criteria in Table 2 in 'any other information on materials and methods incl. tables'. The mean score of each group at each time point (sum of the total score of the reaction in each group/number of animals in each group) were calculated. In addition, all animals were photographed.


Challenge controls:
Conditions for test group and control group are identical (0.5, 0.3, 0.1, 0.05, 0.03 and 0.01%. as suspension in water).
Positive control substance(s):
not specified

Study design: in vivo (LLNA)

Statistics:
Not reported.

Results and discussion

Positive control results:
Not applicable.

In vivo (non-LLNA)

Results
Reading:
other: Challenge
Hours after challenge:
24
Group:
other: test and vehicle control groups
Dose level:
0.5, 0.3, 0.1, 0.05, 0.1, 0.03 and 0.01%
No. with + reactions:
0
Total no. in group:
15
Clinical observations:
There were no signs of erythema or oedema at each challenge area in both test article and vehicle control groups.
Remarks on result:
other: see Remark
Remarks:
Reading: other: Challenge. . Hours after challenge: 24.0. Group: other: test and vehicle control groups. Dose level: 0.5, 0.3, 0.1, 0.05, 0.1, 0.03 and 0.01%. No with. + reactions: 0.0. Total no. in groups: 15.0. Clinical observations: There were no signs of erythema or oedema at each challenge area in both test article and vehicle control groups..

Any other information on results incl. tables

There were no erythema or oedema at each challenge area in both test article and vehicle control group. Tables of results, individual data and reference are as attached.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
There was no evidence of reactions indicative of skin sensitisation to the notified chemical under the conditions of the test. The test material does not meet the criteria for classification according to EU classification system.
Executive summary:

From these results of preliminary study,

water for injection as vehicle, 10% as induction concentration and 0.5, 0.3, 0.1, 0.05, 0.03 and 0.01% as challenge concentration were selected for the main study.

There were no signs of erythema or oedema at each challenge area in both test article and vehicle control groups. The test material does not meet the criteria for classification according to EU classification system.