N,N-dimethyl-alkyl-1-amines, reaction products with alkali hydroxide and chloroacetic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study was performed before LLNA was required as the first choice method for in-vivo testing.

Test material

Test material information

Constituent 1

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle, France
- Age at study initiation: 1 - 2 months old
- Weight at study initiation: males 332 - 401 g; females 335 - 396 g
- Housing: individually
- Diet (e.g. ad libitum): 106 pelleted diet (SAFE, Villemoisson, Epinay-sur-Orge, France), ad libitum
- Water (e.g. ad libitum): Millipore-filtered (0.22 micron) drinking water, ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 30 - 70
- Air changes (per hr): approx. 12
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

physiological saline

Induction 2

Concentration / amount:

Induction: 0.5, 1, 2.5, 5, 10 and 25 % (w/w) intradermal; 10, 25, 50 and 100 % (w/w) epicutaneous;
Challenge: 0.5, 1, 2.5, 5, 10, 25, 50 and 100 % (w/w)

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Challenge 2

Concentration / amount:

Induction: 0.5, 1, 2.5, 5, 10 and 25 % (w/w) intradermal; 10, 25, 50 and 100 % (w/w) epicutaneous;
Challenge: 0.5, 1, 2.5, 5, 10, 25, 50 and 100 % (w/w)

No. of animals per dose:

20 (10 males, 10 females); 10 control (5 males, 5 females)

Details on study design:

RANGE FINDING TESTS:
For both the preliminary and the main test, the application sites of all animals were clipped before intradermal injections and topical applications of the induction phase, clipped and shaved before topical applications of the challenge phase (each flank), shaved before the 48-hour reading of the challenge phase and before the 24-hour reading of the induction phase (preliminary test), and clipped before skin sampling (for the concerned animals).

By intradermal route: Concentrations of 0.5, 1, 2.5, 5, 10 and 25 % (w/w) were intradermally injected in a volume of 0.1 mL into the interscapular region, local reactions were evaluated approximately 24, 48 hours and 6 days after the injections.
By cutaneous route: Under the conditions of the induction phase concentrations of 10, 25, 50 and 100 % (w/w) were applied epicutaneously to the shaved skin on a filter paper (approx. 8 cm²) fully-loaded with the dosage preparation. The filter paper was held in place by means of an occlusive dressing for 48 hours, the cutaneous reactions were evaluated 24 and 48 hours after removal of the dressing.
Under the conditions of the challenge phase concentrations of 0.5, 1, 2.5, 5, 10, 25, 50 and 100 % (w/w) were loaded onto the filter paper of a Finn Chamber. The chamber was held in place by means of an occlusive dressing for 24 hours, cutaneous reactions were evaluated 24 and 48 hours after the removal of the dressings.

The following criteria were used for the selection of concentrations: they should be well-tolerated systemically and locally, intradermal injections should cause moderate irritant effects (no necrosis or ulceration of the skin), cutaneous application for induction should cause at most weak or moderate skin reactions or should be the maximal practicable concentration, cutaneous application for challenge should be the highest concentration which does not cause irritant effects.

As the test substance demonstrated irritant characteristics during the pre-test, a topical application of sodium lauryl sulfate was not necessary on day 7.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: on day 1 (intradermal) and 8 (epicutaneous, for 48 hours)
- Test groups: intradermal: FCA + saline; test substance + saline; test substance + FCA + saline; epicutaneous: test substance in purified water
- Control group: FCA + saline; Saline; Vehicle + FCA + saline
- Site: intradermal: into each side of the interscapular region (3 pairs of sites); epicutaneous: same sites
- Frequency of applications: 7 days
- Duration: day 1 to 10
- Concentrations: intradermal: 0.5 % (w/w) test substance; epicutaneous 25 % (w/w)


B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 22
- Exposure period: for 24 hours
- Test groups: test substance in purified water and vehicle, respectively
- Control group: test substance in purified water and vehicle, respectively
- Site: test substance: posterior right flank; vehicle: posterior left flank
- Concentrations: 1 % (w/w)
- Evaluation (hr after challenge): 24 and 48 hours after removal of the dressing

Challenge controls:

not performed

Positive control substance(s):

yes

Remarks:

Mercaptobenzothiazole

Results and discussion

Positive control results:

The sensitivity of the experimental technique is regularly assessed using a known moderate sensitizer. In a recent study performed under the experimental conditions of the testing laboratory, the strain of guinea pigs used showed a satisfactory sensitisation response in 100 % of the animals.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

1 %

No. with + reactions:

5

Total no. in group:

20

Clinical observations:

discrete erythema (grade 1), dryness of the skin in one animal

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 1 %. No with. + reactions: 5.0. Total no. in groups: 20.0. Clinical observations: discrete erythema (grade 1), dryness of the skin in one animal.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

1 %

No. with + reactions:

2

Total no. in group:

20

Clinical observations:

moderate erythema (grade 2), dryness of the skin in both animals

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1 %. No with. + reactions: 2.0. Total no. in groups: 20.0. Clinical observations: moderate erythema (grade 2), dryness of the skin in both animals.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

1 %

No. with + reactions:

3

Total no. in group:

10

Clinical observations:

discrete erythema (grade 1)

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 1 %. No with. + reactions: 3.0. Total no. in groups: 10.0. Clinical observations: discrete erythema (grade 1).

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

1 %

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

discrete erythema (grade 1), dryness of the skin

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 1 %. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: discrete erythema (grade 1), dryness of the skin.

Any other information on results incl. tables

In the control group, a discrete erythema was observed on the right flank (test item application) of 3/10 animals at the 24-hour reading and persisted in one of them, associated with a dryness of the skin, at the 48-hour reading.

In the treated group, on the right flank, a discrete erythema (grade 1) was noted in 5/20 animals at the 24-hour reading; at the 48-hour reading, a moderate erythema (grade 2) was observed in 2/20 animals. A dryness of the skin was also noted in 1/20 and 2/20 animals at the 24- and 48-hour readings, respectively.

Conclusion:

The higher number of animals demonstrating cutaneous reactions (5/20, corresponding to 25 %) in the 24-hour reading have to be ascribed to the irritant character of the test substance and must be considered as local irritation reaction. Even this number does not exceed the threshold of 30% positive reactions, demanded by the underlying OECD guideline for sensitisation tests with adjuvant, required to consider a test substance as sensitising.

After 48 hours, only 2/20 animals (10%) with cutaneous reactions remain, which have to be considered as sensitised.

Under the experimental conditions chosen, using the maximisation method of Magnusson and Kligman, the test item finally induced cutaneous reactions in 2/20 (10%) guinea pigs. According to the criteria of Council Directive 67/548/EEC (DSD) and the CLP regulation the test item does not need to be classified as sensitising to the skin.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

CLP: not classified
DSD: not classified