N,N-dibutylbutan-1-aminium bis[2-(hydroxy-kO)benzoato(2-)-kO]borate(1-)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 May to 21 May 2013 experimental in-life

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline (OECD 429) compliant study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Pooled treatment group approach

Deviations:

yes

Remarks:

chemical analysis of the formulated test substance was not performed

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-dioxo-4H,4'H-2,2'-spirobi[[1,3,2]benzodioxaborinin]-2-uide; tributylazanium

EC Number:

700-906-2

Cas Number:

22450-96-0

Molecular formula:

C26H36BNO6

IUPAC Name:

4,4'-dioxo-4H,4'H-2,2'-spirobi[[1,3,2]benzodioxaborinin]-2-uide; tributylazanium

Constituent 2

Reference substance name:

SABoTBA

IUPAC Name:

SABoTBA

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Tri-n-butylammonium borodisalicylate was received on 4 March 2013
- Name of test material (as cited in study report): SABoTBA
- Purity: 98.77%
- Purity test date: 08 April 2014
- Lot/batch No.: 12342
- Expiration date of the lot/batch: Stable under recommended storage conditions
- Storage condition of test material: Room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River UK.
- Age at study initiation: 8 - 12 weeks at start of treatment
- Weight at study initiation: 17.3 - 21.9 g
- Housing: in pairs
- Diet (e.g. ad libitum): Rat and Mouse No. 1 maintenance diet, Ad-libitum
- Water (e.g. ad libitum): potable water supplied in polycarbonate bottles fitted with sipper tubes, ad-libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 40 - 70%
- Air changes (per hr): monitored but actual number not included in the study report.
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: From: To: 08 May to 20 May 2013

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

10, 25 and 50% w/v in the vehilce. Higher concentrations were not possible to formulate as the material was too bulky.

No. of animals per dose:

4 in main study

Details on study design:

RANGE FINDING TESTS:
- Preliminary invstigations were performed on 2 animals/treatment and treated at 25 and 50% w/v SABoTBA.
- Compound solubility: NA, formulation was a suspension.
- Irritation: no erythema noted. No effect of treatment on ear thickness.
- Lymph node proliferation response: not performed.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA pooled treatemnt group approach
- Criteria used to consider a positive response: SI of 3 or greater.

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION
Formulations were freshly prepared on each day of treatment using acetone/olive oil. A positive control group was similarly treated with 25% v/v hexylcinnamic aldehyde in acetone: olive oil (4:1 v/v).
The test item was prepared for administration in the main study at 10, 25 and 50% v/v in acetone/olive oil. The mice were treated by daily application of 25 µL of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test
substance was applied to the dorsal surface of each ear using an automatic micropipette and was spread over the entire dorsal surface of the ear using the tip of the pipette. Further groups of four mice received the vehicle alone or the positive control substance in the same manner.
Five days following the first topical application of test substance (Day 6) all mice were injected via the tail vein with 250 μ/L of phosphate buffered saline containing 3H-methyl Thymidine (Tritiated 3H-methyl thymidine (3HTdR) at 80 μCi/mL was used in the study) giving a nominal 20 μ Ci to each mouse. The injection into the tail vein was carried out using a plastic syringe and needle after the mouse had been heated in a warming chamber. Five hours later the animals were euthanased by carbon dioxide asphyxiation and the draining auricular lymph nodes were recovered from each animal. The nodes from mice subjected to the same treatment were pooled and suspensions of the cellular components of the lymph nodes were prepared in 5% w/v trichloroacetic acid and processed through a scintillation counter.
Test results are expressed in terms of Stimulation Indices, the ratios of the mean scintillation count per group obtained from the test groups relative to the corresponding mean scintillation count from controls. The threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation is 3.0.
RECOVERY OF LYMPH NODES
Once death had been confirmed for each mouse the auricular lymph nodes were excised and pooled nodes of all mice from each dose group were placed in 1 mL phosphate buffered saline.
PREPARATION FOR SCINTILLATION COUNT
A single cell suspension of lymph node cells (LNC) was prepared by gentle mechanical disaggregation through a stainless steel gauze (200 mesh size). The pooled LNC were then washed by adding 10 mL PBS, pelleted at 190 x g for 10 minutes and resuspended. The cells were washed twice again and resuspended in 3 mL trichloroacetic acid (TCA: 5%) following the final wash.
SCINTILLATION COUNTING
After overnight incubation (minimum of 18 hours) with 5% TCA at 4°C, the precipitate was recovered by centrifugation and resuspended in 1 mL 5% TCA and transferred to 10 mL Ultima gold scintillation fluid on Day 7. The 3HTdR incorporation was measured by β-scintillation counting. The proliferative response of LNC was expressed as radioactive disintegrations per minute per lymph node (dpm/node).
Results for each treatment group were expressed as the Stimulation Index (SI). This was derived by dividing the mean dpm/mouse for each treated group and the positive control group by the mean dpm/mouse in the vehicle control group.
If the SI is 3 or more, the test substance is regarded as a skin sensitizer.
The positive control group is expected to give an SI of 3 or more to demonstrate the validity of the study.
The Estimated Concentration of Three (EC3) is the concentration of test substance which would result in a SI of 3 and, where data permits, this value was calculated (Ryan 2007). As all concentrations tested resulted in an SI of less than 3 then the EC3 was reported as greater than the highest concentration tested 50% w/v.

Positive control substance(s):

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

Results and discussion

Positive control results:

The SI for the positive control substance hexyl cinnamic aldehyde (HCA) was 5.6 which demonstrates the validity of this study.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

10 % SABoTBA / 4 animals

Results 2

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

25 % SABoTBA / 4 animals

Results 3

Key result

Parameter:

SI

Value:

1.5

Test group / Remarks:

50 % SABoTBA / 4 animals

Any other information on results incl. tables

Preliminary investigations were performed at 25 and 50% w/v with 2 mice per concentration to establish the highest concentration of test substance which did not lead to systemic toxicity or excessive local irritation. The results of preliminary investigations indicated that 50% w/v would be a suitable high concentration for use on the main study.

Main Study

Group dpm/node and Stimulation Index

Group	Concentration	dpm	Number of lymph nodes per animal	Dpm/node	Stimulation index #	Result + positive, - negative
3	AOO	357.10	8.0	44.64	n/a	n/a
4	10% w/v SABoTBA	395.20	8.0	49.40	1.1	-
5	25 w/v SABoTBA	364.80	8.0	45.60	1.0	-
6	50 w/v SABoTBA	520.50	8.0	65.06	1.5	-
7	HCA 25% v/v	1985.10	8.0	248.14	5.6	+

# Stimulation Index of 3 or more indicates a positive result

dpm Disintegrations per minute less background count of 35.80

AOO Acetone:olive oil (4:1 v/v) (vehicle control)

HCA Hexyl cinnamic aldehyde (positive control)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

SABoTBA is not regarded as a potential skin sensitizer.

Executive summary:

The study was performed to assess the skin sensitization potential of SABoTBA using the local lymph node assay (LLNA) in accordance with OECD Guideline 429. Preliminary investigations were performed at 25 and 50% w/v with 2 mice per concentration to establish the highest concentration of test substance which did not lead to systemic toxicity or excessive local irritation. The results of preliminary investigations indicated that 50% w/v would be a suitable high concentration for use on the main study.

The study comprised three treated groups, each comprising four female mice receiving the test substance at concentrations of 10, 25 or 50% w/v. Similarly constituted groups received the vehicle Acetone: olive oil (4:1 v/v) or positive control substance (25% v/v hexyl cinnamic aldehyde). The mice were treated by daily application of 25 µL of the appropriate concentration or control (vehicle or positive), to the dorsal surface of both ears for three consecutive days.

The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of 3H-methyl Thymidine (3HTdR) by β-scintillation counting of LNC suspensions. The response was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio), termed as Stimulation Index (SI).

The test substance is regarded as a sensitizer if at least one concentration of the chemical has a SI of three or more.

Results

The SI obtained for 10, 25 and 50% w/v were 1.1, 1.0 and 1.5 respectively which indicates that SABoTBA did not show the potential to induce skin sensitization.

The SI for the positive control substance hexyl cinnamic aldehyde was 5.6, which demonstrates the validity of this study.