Reactions mass of N-{2-[(4-bromo-6-substituted-2-alkyl-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl)diazenyl]-[alkyl(2-methoxyalkyl)amino]phenyl}acetamide) and N-{2-[(4-bromo-6-substiuted-2-alkyl-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl)diazenyl]-[alkyl(2-hydroxy-3-phenoxypropyl)amino]phenyl}acetamide

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:

Testing was conducted between the 28th February and 26th March 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline 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:

CBA

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatization period of at least five 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 eight to twelve weeks old.

The animals were individually 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 Harlan Laboratories UK Ltd., 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 approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve 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.

Justification:
Mice are the preferred species of choice since quantitative methods have been developed for the measurement of skin sensitization responses in the mouse and are specified in the appropriate test guidelines.

Vehicle:

dimethylformamide

Concentration:

Based on the preliminary screening test, the dose levels selected for the main test were 10%, 5% and 2.5% w/w in dimethyl formamide

No. of animals per dose:

Groups of four mice were treated with the test item at concentrations of 10%, 5% or 2.5% w/w in dimethyl formamide.

Details on study design:

RANGE FINDING TESTS:
Preliminary Screening Test
Using available information regarding the irritancy potential of the test item, a preliminary screening test was performed using three mice, one mouse per test item concentration. The mice were treated by daily application of 25 µL of the test item at concentrations of 50%, 25% or 10% w/w in dimethyl formamide, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the Draize scale for Erythema. Any clinical signs of toxicity, if present, were also recorded. The body weight of each mouse was recorded on Day 1 (prior to dosing) and on Day 6.

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.

MAIN STUDY:
Test Item Administration:
Groups of four mice were treated with the test item at concentrations of 10%, 5% or 2.5% w/w in dimethyl formamide. 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.

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. 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 ten 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 eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, re suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by ß scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty 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 twenty 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:
Introduction:
A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitizer. The methodology for the LLNA is detailed in the OECD Guideline for the Testing of Chemicals No. 429 and Method B.42 of Commission Regulation (EC) No. 440/2008.

Methods:
A group of five animals was treated with 50 µL (25 µL per ear) of α Hexylcinnamaldehyde, tech., 85% as a solution in dimethyl formamide at a concentration of 15% v/v. A further control group of five animals was treated with dimethyl formamide alone.

Positive control substance(s):

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

Positive control results:

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:

Concentration (% v/v) in dimethyl formamide Stimulation Index Result
15 7.91 Positive
Conclusion
α Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Estimation of the Proliferative Response of Lymph Node Cells: 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 dimethyl formamide Stimulation Index Result 2.5 1.11 Negative 5 1.20 Negative 10 1.43 Negative

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Estimation of the Proliferative Response of Lymph Node Cells The radioactive disintegrations per minute per lymph node and the stimulation index are given in the table below. Concentration (% w/w) in dimethyl formamide dpm dpm/Node a Stimulation Index b Result Vehicle 18658.79 2332.35 na na 2.5 20763.55 2595.44 1.11 Negative 5 22468.32 2808.54 1.20 Negative 10 26760.95 3345.12 1.43 Negative dpm = Disintegrations per minute a = Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes) b = Stimulation Index of 3.0 or greater indicates a positive result na = Not applicable

Preliminary screening study:

Dark blue colored staining on the ears and fur was noted in animals treated with the test item at concentrations of 25% or 10% w/w in dimethyl formamide.  No signs of systemic toxicity or visual local skin irritation were noted. No irritation indicated by an equal to or greater than 25% increase in mean ear thickness was noted in the animal treated with the test item at a concentration of 10% w/w in dimethyl formamide. A greater than 25% increase in mean ear thickness was noted in the animals treated with the test item at concentrations of 50% or 25% w/w in dimethyl formamide.

Clinical observations, body weight and mortality data are given in Table 1 and local skin irritation is given in Table 2. The ear thickness measurements and mean ear thickness changes are given in Table 3. Please see tables 1 to 3 below.

Based on this information the dose levels selected for the main test were 10%, 5% and 2.5% w/w in dimethyl formamide.

Table 1     Clinical Observations, Body Weight and Mortality Data – Preliminary Screening Test

Concentration (%w/w) in dimethyl formamide	Animal Number	Body Weight (g)		Day
				1		2		3		4	5	6
		Day 1	Day 6	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
50	S-1	18	20	0	0	0	0	0	0	0	0	0
25	S-2	18	18	0	0Fs	0Fs	0Fs	0	0Fs	0Fs	0Fs	0Fs
10	S-3	18	18	0	0	0	0Fs	0	0Fs	0	0	0

0=          No signs of systemic toxicity

Fs =        Dark blue colored staining on the ears and fur

Table 2     Local Skin Irritation – Preliminary Screening Test

Concentration (%w/w) in dimethyl formamide	Animal Number	Local Skin Irritation
		Day 1		Day 2		Day 3		Day 4		Day 5		Day 6
		left	right	left	right	left	right	left	right	left	right	left	right
50	S-1	0	0	0	0	0	0	0	0	0	0	0	0
25	S-2	0	0	0	0	0	0	0	0	0	0	0	0
10	S-3	0	0	0	0	0	0	0	0	0	0	0	0

 

 

Table 3     Measurement of Ear Thickness and Mean Ear Thickness Changes – Preliminary Screening Test

Concentration (%w/w) in dimethyl formamide	Animal Number	Ear Thickness Measurement (mm)
		Day 1		Day 3		Day 6
		pre‑dose		post dose
		left	right	left	right	left	right
50	S-1	0.21	0.21	0.32	0.32	0.21	0.23
overall mean (mm)		0.21		0.32		0.22
overall mean ear thickness change (%)		na		52.38		4.76
Concentration (%w/w) in dimethyl formamide	Animal Number	Ear Thickness Measurement (mm)
		Day 1		Day 3		Day 6
		pre‑dose		post dose
		left	right	left	right	left	right
25	S-2	0.20	0.22	0.28	0.29	0.25	0.27
overall mean (mm)		0.21		0.29		0.26
overall mean ear thickness change (%)		na		35.71		23.81
Concentration (%w/w) in dimethyl formamide	Animal Number	Ear Thickness Measurement (mm)
		Day 1		Day 3		Day 6
		pre‑dose		post dose
		left	right	left	right	left	right
10	S-3	0.21	0.23	0.23	0.24	0.22	0.22
overall mean (mm)		0.22		0.24		0.22
overall mean ear thickness change (%)		na		6.82		0.00

Main Test:

 Clinical Observations and Mortality Data:

Dark blue colored staining on the ears and fur was noted in animals treated with the test item at a concentration of 10% w/w in dimethyl formamide.

 

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

The test item was considered to be anon-sensitizerunder the conditions of the test.

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

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

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 or excessive local irritation were noted at aconcentration of10% 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 dimethyl formamide at concentrations of 10%, 5% or 2.5% w/w. A further group of four animals was treated with dimethyl formamide 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 dimethyl formamide	Stimulation Index	Result
2.5	1.11	Negative
5	1.20	Negative
10	1.43	Negative

 

Conclusion:

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The 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 or excessive local irritation were noted at aconcentration of10% 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 dimethyl formamide at concentrations of 10%, 5% or 2.5% w/w. A further group of four animals was treated with dimethyl formamide 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 dimethyl formamide	Stimulation Index	Result
2.5	1.11	Negative
5	1.20	Negative
10	1.43	Negative

 

Conclusion:

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

Migrated from Short description of key information:
Determination of the skin sensitization potential of FAT 41044/ TE in the CBA/Ca strain mouse following topical application to the dorsal surface of
the ear.

Justification for selection of skin sensitisation endpoint:
Only this study is available.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to CLP regulation (Regulation EC No. 1272/2008) and DSD (Directive 67/548/EEC).