Reaction products of tetraethylenepentamine and maleic anhydride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-04-25 to 2014-06-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented study performed according to OECD TG 429 and EU Method B.42, in compliance with GLP. No deviations were noted.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

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)

Deviations:

no

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

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): XTA 823; the undiluted test item refers to a 49.8% aqueous solution of XTA 823- Substance type: yellow slightly viscous liquid- Physical state: liquid- Analytical purity: 49.8%- Purity test date: no data- Lot/batch No.: BLW060113- Expiration date of the lot/batch: no data- Stability under test conditions: no data- Storage condition of test material: at room temperature in the dark- other: - Production date: 2014-02-10

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS- Source: 18 female CBA/Ca (CBA/CaOlaHsd) strain mice (16 for main study, 2 for preliminary screening test), supplied by Harlan Laboratories UK Ltd., Oxon, UK. - Age at study initiation: 8 - 12 weeks- Weight at study initiation: 15 - 23 grams- Housing: individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. - Diet (e.g. ad libitum): ad libitum, 2014C Teklad Global Rodent diet - Water (e.g. ad libitum): ad libitum, tap water- Acclimation period: at least five daysENVIRONMENTAL CONDITIONS- Temperature (°C): 19-25°C- Humidity (%): 30-70%- Air changes (per hr): approximately 15 changes per hour - Photoperiod (hrs dark / hrs light): 12/12, controlled by a time switch (light period from 06.00 to 18.00)

Study design: in vivo (LLNA)

Vehicle:

other: 1% pluronic L92 in distilled water

Concentration:

100% (undiluted test item) - 50% active ingredient50% v/v - 25% active ingredient25% v/v - 12.5% active ingredientvehicle only (1% pluronice L92 in distilled water)

No. of animals per dose:

preliminary screening test: 1 mouse/dosemain study: 4 mice/group; 4 groups

Details on study design:

RANGE FINDING TESTS:- 2 mice (1 per test item concentration) were treated by daily application of 25 µl to the dorsal surface of each ear for 3 consecutive days (days 1, 2, 3).- Concentration: undiluted test item (100%, 50% active ingredient) ; test item at a concentration of 50% v/v (25% active ingredient) in 1% pluronic L92 in distilled water- Observations (clinical signs, local skin irritation): twice daily on days 1, 2 and 3; once daily on days 4, 5 and 6- Body weight: on day 1 (prior to dosing) and on day 6- Irritation: scored daily according to the following scale: no erythema = 0; very slight erythema (barely perceptible) = 1; well-defined erythema = 2; moderate to severe erythema = 3; severe erythema (beef redness) to eschar formulation preventing grading of erythema = 4.- 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 thicknes changes were calculated between time periods days 1 and 3 and days 1 and 6. A mean ear thickness of equal or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation. MAIN STUDYTEST ITEM TREATMENT- Groups of 4 mice were treated with one of the 3 concentrations. 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 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 4 mice received the vehicle alone in the same manner.³H-METHYL THYMIDINE ADMINISTRATION- 5 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: 80µCi/ml, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20µCi to each mouse.TERMINAL PROCEDURES- Termination: 5 hours following the administration of ³HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the 4 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 suspensions: 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 rinced 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 resuspended 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) fo 10 minutes, re-suspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). ³HTdR incorporation was measured by beta-scintillation counting. The "Poly Q(TM)" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately 20 minutes. The purpose of this period 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 desintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).TREATMENT PREPARATION:- For the purpose of the study, the test item was used undiluted and freshly prepared as a solution in 1% pluronic L92 in distilled water. - 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.

Positive control substance(s):

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

Statistics:

No statistical analysis performed.

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:25% (v/v) in 1% pluronic L92 in distilled water: 8.79alpha-hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the condition of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: based on active ingredient 12.5% - 1.38 25% - 1.86 50% - 3.07

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

vehicle: 19867.74 dpm; 2483.47 dpm/node 25% (v/v) in 1% pluronic L92 in distilled water (12.5% active ingredient): 27332.94 dpm; 3416.62 dpm/node 50% (v/v) in 1% pluronic L92 in distilled water (25% active ingredient): 36896.75; 4612.09 dpm/node 100% (undiluted test item) (50% active ingredient): 60923.81 dpm; 7615.48 dpm/node

Any other information on results incl. tables

PRELIMINARY SCREENING TEST

- Fur loss was noted on the ears of the animal treated with the test item at a concentration of 50% v/v. A small amount of pale yellow colored residual test item around the edge of the ears was noted in animals treated with the undiluted test item or the test item at a concentration of 50% v/v.

- No signs of systemic toxicity, visual skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.

- Based on this information the undiluted test item (100%) and test item at concentrations of 50% and 25% v/v were selected for the main test.

MAIN STUDY

Clinical observations and mortality data:

- Fur loss was noted on the ears of one animal treated with the test item at a concentration of 50% v/v and all animals treated with the undiluted test item (100%). A small amount of pale yellow colored residual test item around the edge of the ears was also noted in animals treated with the undiluted test item.

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

Body weight:

- 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.

CALCULATION OF EC3 VALUE

a = 100

b = 3.07

c = 50

d = 1.86

EC₃ = 50 + [[(3 -1.86)/(3.07 -1.86)] x (100 -50)] = 97

The concentration of test item expected to caues a 3 fold increase in ³HTdR incorporation (EC₃ value) was calculated to be 97%.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Conclusions:

The test item (as a 49.8% solution) was considered to be a sensitizer under the conditions of the test.