C12-18-(even numbered, C18 unsaturated)-alkylamines acetates

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

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

Adequacy of study:

supporting study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Reference substance name:

C16-18-(even numbered)-alkylamines acetates

EC Number:

800-526-8

Cas Number:

1273322-45-4

Molecular formula:

R-NH2xHOOCCH3 R = alkyl, mainly C16-18-(even numbered)

IUPAC Name:

C16-18-(even numbered)-alkylamines acetates

Test material form:

other: solid wax

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

- Semi barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Free access to autoclaved hay and to Altromin 3122 maintenance diet for guinea pigs (lot no. 1725), rich in crude fibre
- Free access to tap water (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept in groups in Terluran - cages on Altromin saw fibre bedding (lot no. 300312)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least five days) under laboratory conditions

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: physiological saline 0.9% NaCl (AlleMan Pharma, lot no. 111036, expiry date: 09/2014) for induction I vaseline (Euro OTC Pharma, lot no. 1009040-01, expiry date: 09/2013) for induction II and for the challenge

Induction 2

Concentration / amount:

For the intradermal injection (induction - first stage), 0.05 g of the test item were dissolved in physiological saline 0.9% NaCl
to gain a final volume of 10 mL of a 0.5% solution (w/v).
For the topical application (induction – second stage), 0.3 g of the test item were suspended in vaseline to gain a final volume
of 10 g of a 3% suspension (w/w).
For the topical application (challenge), 0.05 g of the test item were suspended in vaseline to gain a final volume of 10 g
of a 0.5% suspension (w/w).

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: physiological saline 0.9% NaCl (AlleMan Pharma, lot no. 111036, expiry date: 09/2014) for induction I vaseline (Euro OTC Pharma, lot no. 1009040-01, expiry date: 09/2013) for induction II and for the challenge

Challenge 2

Concentration / amount:

For the intradermal injection (induction - first stage), 0.05 g of the test item were dissolved in physiological saline 0.9% NaCl
to gain a final volume of 10 mL of a 0.5% solution (w/v).
For the topical application (induction – second stage), 0.3 g of the test item were suspended in vaseline to gain a final volume
of 10 g of a 3% suspension (w/w).
For the topical application (challenge), 0.05 g of the test item were suspended in vaseline to gain a final volume of 10 g
of a 0.5% suspension (w/w).

No. of animals per dose:

10 test animals, 5 control animals and 9 animals for the dose range finding study

Details on study design:

Induction: First Stage, Intradermal Injection
Three pairs of intradermal injections of 0.1 mL volume were given in the shoulder region which was cleared of hair by clipping so
that one of each pair lies on each side of the midline.
Test Group: Day 0
Injection 1: a 1:1 mixture (v/v) FCA/physiological saline 0.9% NaCl
Injection 2: a 0.5% concentration of the test item in physiological saline 0.9% NaCl
Injection 3: a 0.5% concentration of the test item formulated in a 1:1 mixture (v/v) FCA/physiological saline 0.9% NaCl
Control Group: Day 0
Injection 1: a 1:1 mixture (v/v) FCA/physiological saline 0.9% NaCl
Injection 2: 100% physiological saline 0.9% NaCl
Injection 3: a 50% (v/v) formulation of physiological saline 0.9% NaCl in a 1:1 (v/v) mixture FCA/physiological saline 0.9% NaCl
Injections 1 and 2 were given close to each other and nearest to the head, while injection 3 was given toward the caudal part of the test area.

Induction: Second Stage, Topical Application
Test Group and Control Group: Day 6
Approximately twenty-four hours before the topical application the test area was close clipped.
Test Group: Day 7
The test item was suspended in vaseline at a concentration of 3.0%. A patch was fully loaded with 0.5 g of the prepared test item.
Then it was applied to the test area and held in contact with the help of an occlusive dressing for 48 hours.
Control Group: Day 7
A patch was fully loaded with 0.5 g of vaseline. Then it was applied to the test area and held in contact with the help
of an occlusive dressing for 48 hours.

Observation
Test Group and Control Group
Approximately 24 hours after removing the patch, the challenge area was cleared of hair by the use of a depilatory cream.
Approximately 24 and 48 hours after removing the patch the skin reaction was observed and recorded according to the grades shown below.
Additionally all animals were observed for signs of toxicity at least once daily during the test period.

Challenge controls:

Challenge: Topical Application
The flanks of treated and control animals were cleared of hair by close-clipping.
Test Group and Control Group: Day 20
The test item was suspended in vaseline at a concentration of 0.5%. A patch, loaded with 0.5 g of the prepared test item was applied to
the left flank of the animals and a patch loaded with 0.5 g of the vehicle to the right flank (intraspecific control). The patches were held in
contact with the help of an occlusive dressing for 24 hours.
The application area was not rinsed.

Positive control substance(s):

yes

Remarks:

performed periodically every 6 months

Results and discussion

Positive control results:

The recent reliability check was performed in March/April 2012. The raw data of this study are kept in the BSL archives (BSL Project ID 120793).
The reliability checks are audited by the QA-unit periodically.
Positive-control substance: mercaptobenzothiazole, purity > 98%, Fluka Chemica, Lot No. 41107195, expiry date: 20/01/2014
Concentrations: 2% induction I phase (in cottonseed oil)
25% induction II phase (in vaseline)
15% challenge (in vaseline)
The sensitisation rate after application of the positive-control substance mercaptobenzothiazole (15% in vaseline) was 100%,
confirming the reliability of the test system.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

5

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: 5.0. Clinical observations: none.

Results 2

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: none.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.5 %

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0.5 %. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: none.

Results 4

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.5%

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0.5%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: none.

Any other information on results incl. tables

Based on the results of the preliminary test, a concentration of 0.5% was chosen for the intradermal application of the main test

and a concentration of 3.0% was selected for the dermal induction. These concentrations caused slight signs of irritation, without leading to systemic effects.

A concentration of 0.5% was found to be the highest dose which did not cause any signs of irritation after a topical treatment over

a period of 24 hours and therefore was chosen for the challenge application in the main test.

 

Main Test

Signs of irritation during the induction:

Intradermal Induction I (24-hour reading):

Injection site 1: erythema grade 1 in 5/5 control and 10/10 test animals, oedema grade 1 in 5/5 control and 10/10 test animals

Injection site 2:  erythema grade 1 in 10/10 test animals, no oedema was observed in any animal, necrosisø 1 mm in 4/10 test animals,
necrosisø 2 mm in 4/10 test animals

Injection site 3: erythema grade 1 in 1/5 control and 7/10 test animals, oedema grade 1 in 6/10 test animals

Intradermal Induction I (48-hour reading):

Injection site 1:erythema grade 1 in 5/5 control and 10/10 test animals, oedema grade 1 in 5/5 control and 1/10 test animals

Injection site 2: erythema grade 1 in 10/10 test animals, no oedema was observed in any animal, necrosisø 1 mm in 4/10 test animals,
necrosisø 2 mm in 4/10 test animals

Injection site 3: erythema grade 1 in 8/10 test animals, oedema grade 1 in 6/10 test animals

Dermal Induction II (48-hour exposure, occlusive):

Immediately after removing the patch: no signs of irritation in any of the test or control animals.

24 hours after removing the patch:  no signs of irritation in any of the test or control animals.

Challenge Exposure (24-hour exposure, occlusive):

24 hours after removing the patch: erythema grade 1 in 1/10 test animals, scratches in 1/5 control animals

48 hours after removing the patch: erythema grade 1 in 1/10 test animals

No oedema was observed in any animal at any observation time.

There was evidence of sensitisation and the percentage of sensitised animals was 10%.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the present study it can be stated that the test item C16-18-(even numbered)-alkylamines acetates showed indications of sensitization reactions in only 1 animal (incidence = 10%).
According to the EC criteria for classification and labelling requirements for dangerous substances and preparations
(Guidelines in Commission Directive 2001/59/EC [5]) labelling is not necessary, as the percentage of sensitised animals was less than 30%.

Executive summary:

In a GLP-compliant OECD TG 406 skin sensitization study according to Magnusson-Kligmann (guinea pig - maximisation test, GPMT), 10 female guinea pigs + 5 control animals (strain Dunkin-Hartley) were treated with the registration substance using physiological saline and vaseline as vehicles for intradermal induction and topical induction / challenge respectively. Based on the results of a pretest, a test substance concentration of 0.5% was used for intradermal induction (0.1 mL), followed by a 3% concentration at epidermal induction. Challenge was performed using a 0.5% substance concentration. For epidermal treatments, patches were loaded with 0.5 mL. In contrast to results of the pretest, no dermal irritation was observed after epidermal induction. After challenge treatment, 1/10 animals showed grade 1 erythema at the 24h and 48 h readings (positive rate = 10%). One control animal showed scratches after 24 h. No oedema was observed in any animal at any observation time. Considering the reported data of this sensitization test it can be stated that the test item caused only slight reactions at the tested concentration in 1 out of 10 animals. According to the EC criteria the registration substance does not classify as skin sensitizer as the sensitization rate was 10%.