bis({9-[2-(carboxy-κO)phenyl]-6-(bis(tallow alkyl hydrogenated)amino)-3H-polyheterocycl-3-ylidene}-3-bis(tallow alkyl hydrogenated)iminium) zinc(2+) dichloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 January - 01 February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and EC guidelines and according to principles of GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Details on test material:

-Substance type: dark purple waxy solid
- Physical state: solid
- Stability under storage conditions: stable
- Storage condition of test material: at room temperature in the dark

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.
Source: Charles River France, L’Arbresle Cedex, France.
Number of animals: 20 females (nulliparous and non-pregnant), five females per group.
Age and bodyweight: Young adult animals (approx. 8-9 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean.
Weight at study initiation: 19-22 grams

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 ± 3.0ºC (actual range: 19.3 – 22.9ºC), a relative humidity of 40-70% (actual range: 17 - 80%) and 12 hours artificial fluorescent light and 12 hours darkness per day.
Accommodation
Individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material. Paper was supplied as cage-enrichment.
The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on Day 3.

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 (MIII type; height 18 cm).

Diet
Free access to pelleted rodent diet.

Water
Free access to tap water.

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

10%, 25%, 50%

No. of animals per dose:

5

Details on study design:

Three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Methyl ethyl ketone).
Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

Positive control substance(s):

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

Statistics:

not applcable

Results and discussion

Positive control results:

The SI values calculated for the substance concentrations 5, 10 and 25% were 1.4, 1.2 and 5.1 respectively. An EC3 value of 16.9% 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 13.1, 15.6, 14.1, 13.8, 13.9, 16.0 and 11.9%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 0% (vehicle) 1.0 ± 0.1 10% 3.5 ± 0.3 25% 7.2 ± 1.2 50% 24.7 ± 3.8

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: 0% (vehicle) 451 ± 20 10% 1577 ± 101 25% 3255 ± 501 50% 11132 ± 1652

Any other information on results incl. tables

Purple staining by the test substance prevented scoring for erythema. No oedema was observed in any of the animals examined. The ears were sticky and rigid due to the test substance.

One auricular lymph node of one control animal, one node of three animals at 10% and all nodes of animals at 25% and 50% were considered enlarged in size. The remaining nodes of the control animals and animals at 10% were considered normal in size.

The largest auricular lymph nodes were found in the highest dose group.

No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

These results show that the test substance elicits an SI ≥ 3. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between 0 and 10% (and expected to be in the vicinity of 10%).