Registration Dossier

Administrative data

Description of key information

A Local Lymph Node Assay was performed on BMS 296796-02 in 2000 and 2016 according to OECD methods and in accordance with GLP. This studies are both considered key studies with an assigned a reliability rating of 1 (reliable without restriction). Both reported SI values < 3 indicated BMS 296796 -02 is not a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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.
Reference:
Composition 0
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 information:
Composition 1
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.
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
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.
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.

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.

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.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March - April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
The assay has undergone extensive inter-laboratory validation and has been shown to reliably
detect test items that are moderate to strong sensitizers.
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were 8 to 12 weeks old.

The animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) 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 fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness. The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
used at request of sponsor
Concentration:
Preliminary screening: 25% w/w on one mouse
Main test: 25%, 10% or 5% w/w
No. of animals per dose:
5 mice per dose
Details on study design:
The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of five mice received the vehicle alone in the same manner.

Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse.
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Termination: Five hours following the administration of 3HTdR all mice were euthanatized by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes.
Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures were used.
Positive control results:
A group of five animals was treated with 50 μL (25 μL per ear) of α-Hexylcinnamaldehyde, tech., 85% as a solution in acetone/olive oil 4:1 at a concentration of 25% v/v. A further control group of five animals was treated with acetone/olive oil 4:1 alone. The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group was 6.08. The positive control substance is considered a sensitiser under conditions of the test.
Key result
Parameter:
SI
Value:
ca. 0.87
Test group / Remarks:
5% w/w in acetone/olive oil 4:1
Key result
Parameter:
SI
Value:
ca. 0.61
Test group / Remarks:
10% w/w in acetone/olive oil 4:1
Key result
Parameter:
SI
Value:
ca. 0.62
Test group / Remarks:
25% w/w in acetone/olive oil 4:1

Detailed results on clinical observations, body weight, mortality, individual disintegrations and Stimulation Index can be found in the attached "BMS-296796 -02 LLNA Tables.pdf"

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be a non-sensitizer under the conditions of the test.
The test item does not meet the criteria for classification according to the Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.
The test item does not meet the criteria for classification according to the Globally Harmonized Classification System.
Executive summary:

Introduction

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 μL (25 μL per ear) of the test item as a solution/suspension in acetone/olive oil 4:1 at concentrations of 25%, 10% or 5% w/w. A further group of five animals was treated with acetone/olive oil 4:1 alone.

Results

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

 Concentration (%w/w) in acetone/olive oil 4:1 Stimulation Index (SI)  Result 
5 0.87 Negative
10 0.61 Negative
25 0.62

Negative

Conclusion

The test item was considered to be a non-sensitizer under the conditions of the test.

The test item does not meet the criteria for classification according to the Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

The test item does not meet the criteria for classification according to the Globally Harmonized Classification System.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

For a Local lymph Node Assay the sensitisation results 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.

 

For the 2000 study, 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.

For the study completed in 2016, the concentration of test material in the vehicle at 5% resulted in a stimulation index (SI) of 0.87, the concentration at 10 % resulted in a SI of 0.61 and the concentration at 25 % resulted in a SI of 0.62. A stimulation index of less than 3 was recorded for all three concentrations of the test material and therefore considered negative results.

Conclusion

The test item was considered to be a non-sensitizer under the conditions of the test.

The test item does not meet the criteria for classification according to the Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

The test item does not meet the criteria for classification according to the Globally Harmonized Classification System.

All results are considered valid and the material was not classified as a skin sensitiser.

Justification for classification or non-classification

The results of this study are considered valid as the key study was assigned a reliability rating of 1, 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 classified as a non-sensitiser. Therefore, BMS 296796-02 did not meet the criteria as a sensitizer for classification according to the EU regulations.