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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 December 2000- 12 December 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was completed in 2000 according to an OECD guideline and in accordance with GLP. The material is well characterized.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
other: first stage in the assessment of skin sensitization (OECD guideline for testing chemicals, No 406 1992 "Skin Sensitization".)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test material is a beige solid which was received at testing laboratory on 27 October 2000 and stored at room temperature in the dark.

In vivo test system

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
male
Details on test animals and environmental conditions:
At the start of the study the mice were in the weight range of 19 to 22 grams and were six to eight weeks old. Free access to mains drinking water and food was allowed throughout the study. The temperature and relative humidity were set to achieve limits of 19 to 25 C and 30 to 70% respectively. The rate of air exchange was at least 15 changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours of darkness. All doses were freshly made for day 0, 1, 2 administration.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Treated with 50 ul ( 25 ul per ear) of the test solution in acetone/olive oil(4:1) at concentrations of 0.1%, 1% and 10% w/w for 3 consecutive days
No. of animals per dose:
Three groups of four animals each were treated at each concentration group and a further group of four animals were treated as a control group with acetone/olive oil (4:1) alone.
Details on study design:
The 3 groups were treated for three days with the test formulation by using a micropipette whose tip is used to spread the formulation over the dorsal surface of each ear. On Day 5 all mice were injected with 250 ul of a phosphate buffered saline solution containing 3H-methyl thymidine (3HTdR ) to the tail vein giving a total of 20 uCi to each mouse. All animals were observed on Day 0, 1, and 2 for the remainder of the study. Any signs of toxicity or ill health were noted. Body weights of each mouse were recorded before dosing and before termination. Five hours after administration of 3HTdR all mice were killed by carbon dioxide asphyxiation and their auricular lymph nodes were excised and pooled for each experimental group. A 1 ml of phosphate buffered saline was added to each set of lymph nodes. Preparation of a single cell suspension was completed for the pooled lymph nodes cells following standard procedure. Determination of the 3HTdR incorporation was completed by measuring radioactive disintegration for each experimental group lymph node cells by using a beta-scintillation counter. Note that injection of one control and one animal in the 1 % w/v group was unsuccessful and they were removed from the study.
Positive control substance(s):
other: 1-chloro-2,4-dinitrobenzene

Results and discussion

Positive control results:
Positive controls data using 1-chloro-2,4-dinitrobenzene in acetone/olive oil 4:1 at concentrations of 0.1%, 0.25% and 0.5% w/v was generated to support the study. The ratio of test to control lymphocyte proliferation that is greater than 3.0 indicates a positive result.  Therefore the positive control test substance was considered to be a sensitizer.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Concentration of test material in the vehicle at 0.1% resulted in a test/control ratio of 0.89 which is a negative result. Concentration of test material in the vehicle at 1.0 % resulted in a test/control ratio of 0.74 which is a negative result. Concentration of test material in the vehicle at 10 % resulted in a test/control ratio of 1.76 which is a negative result. A test/control ratio of less than 3 was recorded for the three concentrations of the test material (0.1%, 1.0 % and 10 % w/v).
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Concentration of test material in the vehicle at 0.1 % resulted in a mean disintegration of 1061.42 dpm which is a negative result. Concentration of test material in the vehicle at 1 % resulted in a mean disintegration of 657.36 dpm which is a negative result. Concentration of test material in the vehicle at 10 % resulted in a mean disintegration of 2091.46 dpm which is a negative result.

Any other information on results incl. tables

Clinical Observations and Mortality Data: There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the study. Bodyweight changes of the test animals between Day 0 and Day 5 were comparable to those observed in the corresponding control group animals over the same period. No evidence of skin irritation was noted at the treatment site of test or control animals.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
At all three concentration tested, BMS 296796-02 was not a moderate to strong sensitiser and did not meet the criteria as a sensitizer for classification according to the EU regulations.
Executive summary:

For a Local Lymph Node Assay the sensitisatoin redsults are determined by the Stimulation Index (SI) which is the ratio of 3HTdR incorporation into the lymph node cells of the test material nodes relative to that recorded for the control nodes. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a non-sensitiser.

The concentration of test material in the vehicle at 0.1% resulted in a stimulation index (SI) of 0.89, the concentration at 1% resulted in a SI of 0.74 and the concentration at 10% resulted in a SI of 1.76. A stimulation index of less than 3 was recorded for all three concentrations of the test material and therefore considered negative results.

The results of this study are considered valid as the key study was assigned a reliability rating of 2, conducted in accordance with GLP and followed an OECD guideline. A stimulation index of less than 3 was recorded for all three concentrations of the test material and was classiffied as a non-sensitiser. Therefore, BMS 296796 -02 did not meet the criteria as a sensitiser for classification according to the EU regulations.