Custom Yellow #2

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 April - 11 may 2009

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 GLP principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Description: Bright yellow powder
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable

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: 25 females (nulliparous and non-pregnant), five females per group.
Age and bodyweight: Young adult animals (approx. 12 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean.
Weight at study initiation: 23-27 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: 21.2 – 24.0ºC), a relative humidity of 40-70% (actual range: 39 - 56%) 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 (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) 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:

other: Ethanol/Water (7:3 (v/v))

Concentration:

5%, 10% and 25%

No. of animals per dose:

5

Details on study design:

Three groups of five experimental animals were treated with test substance concentrations of 5%, 10% or 25% on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Ethanol/Water 7:3 (v/v)) and five positive control animals were similarly treated, but with positive control substance.
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):

other: 0.5% 1-Chloro-2,4-Dinitrobenzene

Results and discussion

Positive control results:

The SI value calculated for 0.5% 1-Chloro-2,4-Dinitrobenzene in Ethanol/Water (7:3 (v/v)) was 65.5 and no systemic toxicity was observed for Ethanol/Water (7:3 (v/v)). Therefore, Ethanol/Water (7:3 (v/v)) was proved suitable as vehicle in the Local Lymph Node Assay performed at NOTOX.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 2 5% 0.9 ± 0.3 3 10% 0.9 ± 0.3 4 25% 1.0 ± 0.2 1 0% (Vehicle) 1.0 ± 0.3

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: 0% (Vehicle) 251 ± 51 5% 228 ± 50 10% 224 ± 55 25% 240 ± 22

Any other information on results incl. tables

Yellow test substance remnants prevented scoring for erythema. No oedema was observed in any of the test substance-treated animals examined. Slight to well-defined erythema was observed in all animals of the positive control group and no skin irritation was observed in the control animals. The irritation of the ears as shown by the animals was considered not to have a toxicologically significant effect on the activity of the nodes.

All nodes of the experimental and vehicle control groups were considered normal in size, except for one node at 10% which was considered reduced in size. All nodes of the positive control group were considered enlarged in size.

No macroscopic abnormalities of the surrounding area were noted.

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Reliability check:

The SI values calculated for the HCAs 5, 10 and 25% were 2.0, 2.2 and 4.2 respectively. An EC3 value of 16.0 % was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 2 and 20%. Theresults of the 6 monthly reliability checks of the recent years were 13.1, 15.6, 14.1, 13.8 and 13.9%.

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

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Since there was no indication that the test substance could elicit an SI >= 3 when tested up to 25%, it was established that the EC3 value (if any) exceeds 25%.