[1,1'-Biphenyl]-2-carboxylic acid, 4'-(bromomethyl)-, methyl ester

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 September 2013 - 23 April 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test method according to OECD Guideline 429. GLP study.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/J Rj

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: ELEVAGE JANVIER, Route des Chènes Secs B.P. 4105, 53940 LE GENEST-ST-ISLE, France
- Age at study initiation: 9 weeks old
- Weight at study initiation: 20.1-22.9 g
- Housing: Group caging / mice were provided with glass tunnel-tubes. Cage type: Type II. polypropylene / polycarbonate.
- Diet (e.g. ad libitum): ad libitum, ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice”
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: al leaste 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Vehicle:

other: N,N-dimethylformamide (DMF)

Concentration:

0.25, 0.1 and 0.05 % (w/v).

No. of animals per dose:

4 animals per dose.

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The solubility of the test item was examined in a short Preliminary Compatibility Test. Based on physical characteristics of the test item, 100% (w/v) concentration in DMF was achievable.
- Preliminary Irritation/Toxicity Test: Four preliminary irritation/toxicity tests were performed at of 100 and 50 % (w/v) (2 animals/dose), 25 and 10 % (w/v) (2 animals/dose), 5, 2.5, 1 and 0.5 % (w/v) (1 animal/dose) and 0.25, 0.1, 0.05 and 0.025 % (w/v) (1 animal/dose). The preliminary experiments were conducted in a similar experimental manner to the main study, but they were terminated on Day 6 with a body weight measurement and the radioactive proliferation assay was not performed. All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals. Based on the observations recorded in the preliminary tests, 100, 50, 25, 10, 5, 2.5, 1 and 0.5 % (w/v) doses were considered too high based on the increased ear thickness values indicating excessive local skin irritation. Therefore, 0.25, 0.1, and 0.05 % (w/v) doses were examined in the main test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
*That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in
control mice, as indicated by the stimulation index.
*The data are compatible with a conventional dose response, although allowance must be made for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
During the study, animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On day 6, each mouse received an injection of 250µl of sterile PBS (phosphate buffered saline) containing approx. 20 µCi of 3HTdR. Five hours later, the mice were euthanized and the auricular lymph nodes were extracted from the animals. A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and the samples were prepared to be examined in a β-scintillation counter.

OBSERVATIONS:
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection).

EVALUATION OF RESULTS:
Radioactive disintegrations per minute (DPM) was measured for each pooled group of nodes and corrected with the background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes). Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated.

Positive control substance(s):

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

Parameter:

SI

Remarks on result:

other: The stimulation index values were 62.3, 22.3 and 8.7 at concentrations of 0.25, 010 and 0.05 % (w/v), respectively. The calculated EC3 value was calculated to be 0.037% (w/v).

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Test item 0.25 % (w/v) in DMF: 91185.5 DPM Test item 0.10 % (w/v) in DMF: 32711.5 DPM Test item 0.05 % (w/v) in DMF: 12744.5 DPM

Clinical observation:

No mortality or systemic clinical signs were observed during the study. There were no indications of any irritancy at the site of application. The ear thickness values and the revealing ear punch weights were within the historical control range in all test item treated dose groups.

Body weight:

No treatment related effects were observed on body weights.

Proliferation assay:

Test Group Name	Measured DPM / group	DPM	Number lymph nodes	DPN	Stimulation Index
Background (5 % (w/v) TCA)	31/36	-	-	-	-
(-) control (DMF)	1498	1464.5	8	183.1	1.0
Test item 0.25 % (w/v) in DMF	91219	91185.5	8	11398.2	62.3
Test item 0.10 % (w/v) in DMF	32745	32711.5	8	4088.9	22.3
Test item 0.05 % (w/v) in DMF	12778	12744.5	8	1593.1	8.7
(+) control (25 % (w/v) HCA in DMF)	21454	21420.5	8	2677.6	14.6

The appearance of the lymph nodes was normal in the negative (vehicle) control group. Larger than normal lymph nodes were observed in the positive control group, in the 0.25% (w/v) dose group and for three animals in the 0.10% (w/v) group. Slightly enlarged lymph nodes were detected in the 0.05 % (w/v) group and for one animals in the 0.10% (w/v) group.

The stimulation index values were 62.3, 22.3 and 8.7 at concentrations of 0.25, 0.10 and 0.05% (w/v), respectively.

The calculated EC3 value was calculated 0.037% (w/v).

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item showed to have sensitisation potential (sensitizer) in the Local Lymph Node Assay. The stimulation index values were 62.3, 22.3 and 8.7 at concentrations of 0.25, 0.10 and 0.05% (w/v), respectively. The calculated EC3 value was calculated to be 0.037% (w/v).

Executive summary:

The skin sensitisation test following dermal exposure was performed according to OECD Guideline 429 and EU method B.42 (GLP study). Based on the results of the Preliminary Compatibility Test, the test item was formulated in N,N-dimethylformamide (DMF) at a highest achievable concentration of 100 % (w/v). The Preliminary Irritation / Toxicity Test was performed in CBA/J Rj mice using twelve doses: 100, 50, 25, 10, 5, 2.5, 1, 0.5, 0.25, 0.10, 0.05 and 0.025% (w/v) in DMF. The 0.25 % (w/v) was selected as top dose for the main test. Twenty female CBA/J Rj mice were allocated to five groups of four animals each in the main test. Three groups received the test item at 0.25, 0.10 and 0.05 % (w/v) concentrations, the negative control group received the vehicle (DMF) and the positive control group received 25 % (w/v) HCA (dissolved in DMF). The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI). No mortality or systemic clinical signs were observed during the study. There were no indications of any irritancy at the site of application. The ear thickness values and the revealing ear punch weights were 62.3, 22.3 and 8.7 at concentrations of 0.25, 0.10 and 0.05% (w/v), respectively. The calculated EC3 value was 0.037% (w/v). All validity criteria were fulfilled. In conclusion, the test item showed to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Key study: The skin sensitisation test following dermal exposure was performed according to OECD Guideline 429 and EU method B.42 (GLP study). Based on preliminary results, three groups (four animals in each) received test item formulated in DMF at 0.25, 0.10 and 0.05 % (w/v) concentrations. No mortality or systemic clinical signs were observed during the study. No treatment related effects were observed on body weight. There were no indications of any irritancy at the site of application. The stimulation index values were 62.3, 22.3 and 8.7 at concentrations of 0.25, 0.10 and 0.05 % (w/v), respectively. The calculated EC3 value was 0.037% (w/v). In conclusion, the test item showed to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.

Migrated from Short description of key information:
Key study: Test method OECD 429 and EU B.42. GLP study. The substance has sensitisation potential (sensitizer) in the Local Lymph Node Assay.

Justification for selection of skin sensitisation endpoint:
Only one study avalaible.

Justification for classification or non-classification

Based on the available results, the substance is classified as Skin Sensitizer Category 1A, H317 according to CLP Regulation (EC) no. 1272/2008.