Amides of alkyldiamine, fatty acids and carboxylic acids

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Remarks:

Existing study done in 2007

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 June 2007 to 13 August 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Justification for non-LLNA method:

Existing study done in 2007.

Test material

Reference

Name:

Unnamed

Type:

Constituent

Type:

impurity

Test material form:

solid: particulate/powder

Details on test material:

The test material is a UVCB substance.

Specific details on test material used for the study:

Identification AD-2000
Form White powder
Batch L-620150
Storage At room temp in the dark
Stability under storage condition Stable
Expiry date 1 January 2009

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Species: Mouse, CBA strain, inbred, SPF-Quality.
Number of animals: 20 females (nulliparous and non-pregnant), five females per group
Age and body weight: Young adult animals (approx. 9 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean.

Conditions
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 21.4
- 23.3°C), a relative humidity of 30-70% (actual range: 41 - 76%) and 12 hours artificial fluorescent light and 12 hours darkness per day.
Cleaning procedures in the room might have caused the temporary fluctuations above the
optimal maximum level of 70% for relative humidity. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

Accommodation
Individual housing in labeled Macrolon cages (MI type; height 12.5 em) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France).

Acclimatization period
The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. Accommodation was as described above except that the animals were group housed in Macrolon cages (Mill type; height 18 em). Paper (Enviro-dri, Wm. Lillico & Son (Wenham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment

Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiaten GmbH, Soest, Germany).

Water
Free access to tap water.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

10%, 25% and 50%

No. of animals per dose:

5

Details on study design:

Preliminary irritation study:

A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (grade 2) at the highest

A series of two test substance concentrations was tested, selected from the series: 100% (undiluted), 50%, 25%, 10%, 5%, 2.5%, 1% and if needed further lower concentrations using the same steps. The highest concentration, selected from this series, was the maximum concentration that could technically be applied.
The test system, procedures and techniques were identical to those used during Days 1 to 3 of the main study unless otherwise specified. Two young adult animals were selected (5..14 weeks old). Each animal was treated with one concentration on three consecutive days. Approximately
3-4 hours after the last exposure, the ear was cleaned of residual test substance with tap water and the irritation was assessed. Bodyweights were determined on Day 3. The animals were sacrificed after the final observation and no necropsy was performed.

Main study:

Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the preliminary irritation study.
One group of five animals was treated with vehicle.

Induction - Days 1, 2 and 3

The dorsal surface of both ears was epidermally treated (25 µl/ear) with the test substance concentration, at approximately the same time per day" The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing.

The control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle alone was administered.

Treatment - Day 6

All animals:
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline
(PBS) (Merck, Darmstadt, Germany) containing 20 µCi of 3H-methyl thymidine (GE Health care, Buckinghamshire, UK).

After approximately five hours, all animals were killed by intraperitoneal injection with pentobarbital Euthesate® (0.2 ml/animal) (Ceva Sante Animale BV, Naaldwijk, The Netherlands). The draining (auricular) lymph node of each ear was excised.
The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded" The nodes were pooled for each animal in approximately 3 mL PBS.

Tissue processing for radioactivity - Day 6

A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 µm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4° C. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) at 4° C during the night.

Radioactivity measurements - Day 7

Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinEimer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of± 0.2% or a maximum of 5 minutes whichever comes first The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

None

Results and discussion

Positive control results:

The Sl values calculated for the substance concentrations 5, 10 and 25% were 1.3, 1.5 and 5.5 respectively. An EC3 value of 15.6% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 2 and 20%. The results of the 6 monthly HCA reliability checks of the recent years were 7.3, 10.3, 9.5 and 13.1%.
Based on these results it was concluded that the Local Lymph Node Assay as performed in the present study is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.8

Variability:

+/- 0.5

Test group / Remarks:

Animal group with 10% test substance

Results 2

Key result

Parameter:

SI

Value:

1.3

Variability:

+/- 0.4

Test group / Remarks:

Animal group with 25% test substance

Results 3

Key result

Parameter:

SI

Value:

0.7

Variability:

+/- 0.2

Test group / Remarks:

Animal group with 50% test substance

Results 4

Key result

Parameter:

SI

Value:

1

Variability:

+/- 0.3

Test group / Remarks:

Animal group with 0% test substance

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was found not to be a skin sensitizer in an LLNA study.