Isobutyl 4-chloro-3,5-diaminobenzoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-06-06 - 2016-06-28 (Experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitization: Local Lymph Node Assay" (adopted 22 July 2010)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Method B42 Skin Sensitization (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Isobutyl 4-chloro-3,5-diaminobenzoate

EC Number:

251-311-5

EC Name:

Isobutyl 4-chloro-3,5-diaminobenzoate

Cas Number:

32961-44-7

Molecular formula:

C11H15ClN2O2

IUPAC Name:

2-methylpropyl 3,5-diamino-4-chlorobenzoate

Test material form:

solid: particulate/powder

Details on test material:

Storage Conditions: room temperature in the dark
Stability in Solvent: stable in water, not quantified

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/Ca (CBA/CaOlaHsd) strain mice

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 15 to 23 g
- Housing: The animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. On receipt the animals were randomly allocated to cages. 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. 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.
- Diet (e.g. ad libitum): food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) ad libitum
- Water (e.g. ad libitum): mains tap water ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: none stated

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): The lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.

Study design: in vivo (LLNA)

Vehicle:

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

Remarks:

This vehicle was chosen as it produced the highest concentration that was suitable for dosing.

Concentration:

50% (pre-test)
10%, 25%, 50% (main test)

No. of animals per dose:

four

Details on study design:

PRE-SCREEN TESTS:
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test item at a concentration of 50% w/w in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The body weight was recorded on Day 1 (prior to dosing) and on Day 6.

- Compound solubility: For the purpose of the study, the test item was freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the highest concentration that was suitable for dosing.
- Ear thickness measurements: The thickness of each ear was measured using a Mitutoyo 547 300S gauge (Mitutoyo Corporation), pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.
- Erythema scores:
Scale for Erythema
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to eschar formation preventing grading of erythema 4

MAIN STUDY
3H-Methyl Thymidine Administration
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.

Observations
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).

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.
Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells 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 cell 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 pooled 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, re suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by  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).

Data Evaluation
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitizer".


ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: random
- Criteria used to consider a positive response: The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitizer".

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of four mice were treated with the test item at concentrations of 50%, 25% or 10% w/w in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. 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 four mice received the vehicle alone in the same manner.

Positive control substance(s):

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

Results and discussion

Positive control results:

The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:
Treatment Stimulation Index Result
5% v/v in acetone/olive oil 4:1 1.42 Negative
10% v/v in acetone/olive oil 4:1 1.53 Negative
25% v/v in acetone/olive oil 4:1 3.71 Positive
The concentration of α Hexylcinnamaldehyde, tech., 85% expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 20%.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.85

Test group / Remarks:

50% test item

Remarks on result:

other: negative

Results 2

Parameter:

SI

Value:

2.65

Test group / Remarks:

25% test item

Remarks on result:

other: negative

Results 3

Parameter:

SI

Value:

2.7

Test group / Remarks:

10% test item

Remarks on result:

other: negative

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
Concentration (%w/w) in acetone/olive oil 4:1 Stimulation Index Result
10 2.70 Negative
25 2.65 Negative
50 2.85 Negative

CLINICAL OBSERVATIONS: There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHTS: Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

see also "any other information on results incl. tables"

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The study was conducted under GLP according to OECD guideline 429 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the sensitizing potential of Isobutyl 4-chloro-3,5-diaminobenzoate in mice. All animals survived throughout the test period without showing any clinical signs of toxicity or local irritation. Calculated SI values in treated groups remained under the value of 3, which is the threshold to consider the substance a sensitizer.
Isobutyl 4-chloro-3,5-diaminobenzoate was so identified as a non-sensitizing agent in the Local Lymph Node Assay. Hence, no classification as skin sensitizer is triggered.

Executive summary:

A study according to OECD TG 429 under GLP was performed to assess the skin sensitization potential of Isobutyl 4-chloro-3,5-diaminobenzoate in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% 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 four animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 50%, 25% or 10% w/w. A further group of four animals was treated with acetone/olive oil 4:1 alone.

 

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	Result
10	2.70	Negative
25	2.65	Negative
50	2.85	Negative

 

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