Reaction products of amines, polyethylenepoly-, triethylenetetramine fraction and 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 04 August 2017 and 04 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 429 using the Skin Sensitisation: Local Lymph Node Assay method and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

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:

EC No. 440/2008

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:

3,3'-[propane-2,2-diylbis(benzene-4,1-diyloxy)]bis(1-{[2-({2-[(2-aminoethyl)amino]ethyl}amino)ethyl]amino}propan-2-ol)

Molecular formula:

C33H60N8O4

IUPAC Name:

3,3'-[propane-2,2-diylbis(benzene-4,1-diyloxy)]bis(1-{[2-({2-[(2-aminoethyl)amino]ethyl}amino)ethyl]amino}propan-2-ol)

Constituent 2

Reference substance name:

1,16-bis(4-{2-[4-(3-{[2-({2-[(2-aminoethyl)amino]ethyl}amino)ethyl]amino}-2-hydroxypropoxy)phenyl]propan-2-yl}phenoxy)-4,7,10,13-tetraazahexadecane-2,15-diol

Molecular formula:

C60H102N12O8

IUPAC Name:

1,16-bis(4-{2-[4-(3-{[2-({2-[(2-aminoethyl)amino]ethyl}amino)ethyl]amino}-2-hydroxypropoxy)phenyl]propan-2-yl}phenoxy)-4,7,10,13-tetraazahexadecane-2,15-diol

Constituent 3

Reference substance name:

3-(4-{2-[4-(3-{[2-({2-[(2-aminoethyl)amino]ethyl}amino)ethyl]amino}-2-hydroxypropoxy)phenyl]propan-2-yl}phenoxy)propane-1,2-diol

Molecular formula:

C27H44N4O5

IUPAC Name:

3-(4-{2-[4-(3-{[2-({2-[(2-aminoethyl)amino]ethyl}amino)ethyl]amino}-2-hydroxypropoxy)phenyl]propan-2-yl}phenoxy)propane-1,2-diol

Constituent 4

Reference substance name:

Polymeric adducts of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bis-oxirane and N1,​N2-​bis(2-​aminoethyl)​-​2-​ethanediamine

IUPAC Name:

Polymeric adducts of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bis-oxirane and N1,​N2-​bis(2-​aminoethyl)​-​2-​ethanediamine

Constituent 5

Reference substance name:

Unknown components

IUPAC Name:

Unknown components

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification: 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with triethylenetetramine
Batch: #01725
Purity: 100% UVCB
Physical state/Appearance: pale yellow paste
Expiry Date: 01 September 2021
Storage Conditions: room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd) strain

Sex:

female

Details on test animals and environmental conditions:

Animal Information
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatization period of at least 5 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 8 to 12 weeks old.

Animal Care and Husbandry
The animals were 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 Envigo RMS (UK) Limited, 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 at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 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.

Study design: in vivo (LLNA)

Vehicle:

other: 1% pluronic L92 in distilled water

Concentration:

For the purpose of the study, the test item was freshly prepared as a solution in 1% pluronic L92 in distilled water. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. The concentrations used were 25%, 10% or 5% w/w 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.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

No. of animals per dose:

Preliminary Screening Test:
1 mouse.

Main test:
Groups of four mice were treated with the test item at concentrations of 25%, 10% or 5% w/w in 1% pluronic L92 in distilled water

Details on study design:

Preliminary Screening Test
Using available information 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 25% w/w in 1% pluronic L92 in distilled water, 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 according to the scale included asTable 1. Any clinical signs of toxicity, if present, were also recorded. The body weight of the 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 Test
Test Item Administration
Groups of four mice were treated with the test item at concentrations of 25%, 10% or 5% w/w in 1% pluronic L92 in distilled water. 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 0.25 mL (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".
The results were also evaluated according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.
The results were also evaluated according to the Globally Harmonized Classification System.
The EC3 value was also calculated. The EC3 value is the concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation.
For chemicals, where the lowest concentration tested resulted in a stimulation index of greater than 3, an EC3 value is extrapolated from the two lowest doses utilized. The extrapolated EC3 value was calculated by log linear interpolation between the two points on a plane where the α axis represents the dose level and the γ axis represents the stimulation index. The point with the higher stimulation index was denoted (a, b) and the point with the lower stimulation index was denoted (c, d). The formula for the extrapolated EC3 value was as follows:
EC3 = 2^ {log2(c) + (3-d)/(b-d) x [log2(a) - log2(c)]}




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".

The results were also evaluated according to the Globally Harmonized Classification System.
The EC3 value was also calculated. The EC3 value is the concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation. The equation used for the calculation of EC3 is:
EC3 = c + [[(3-d)/(b-d)] x (a-c)]

Positive control substance(s):

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

Results and discussion

Positive control results:

Current Positive Control Study for the Local Lymph Node Assay

Introduction
The Report has been re issued to amend the positive control identification.
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. The study described in this document is based on these test methods but has been refined in order to reduce the number of animals required. The reduced LLNA (rLLNA) has been endorsed by the non Commission members of the European Centre for the Validation of Alternative Methods (ECVAM) Scientific Advisory Committee (ESAC) at its 26th meeting held on 26 – 27 April 2007 at ECVAM, Ispra, Italy.

Test item: α Hexylcinnamaldehyde, tech., 85%
Study number: MN81RV
Study dates: 09 November 2016 to 15 November 2016

Methods
A group of five animals was treated with 50 µL (25 µL per ear) of α Hexylcinnamaldehyde, tech., 85% as an emulsion in 1% pluronic L92 in distilled water at a concentration of 25% v/v. A further control group of five animals was treated with 1% pluronic L92 in distilled water alone.

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
1% pluronic L92 in distilled water Stimulation Index Result
25 6.12 Positive

Conclusion
α Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

4.31

Test group / Remarks:

5% w/w in 1% pluronic L92 in distilled water

Remarks on result:

other: Positive

Results 2

Parameter:

SI

Value:

5.28

Test group / Remarks:

10% w/w in 1% pluronic L92 in distilled water

Remarks on result:

other: Positive

Results 3

Parameter:

SI

Value:

5.41

Test group / Remarks:

25% w/w in 1% pluronic L92 in distilled water

Remarks on result:

other: Positive

Any other information on results incl. tables

 Preliminary Screening Test

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Fur loss was noted on days 2 to 6.

Based on this information the dose levels selected for the main test were25%,10% and5% w/win1% pluronic L92 indistilled water.

 

Main Test

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 1% pluronic L92 indistilled water	Stimulation Index	Result
5	4.31	Positive
10	5.28	Positive
25	5.41	Positive

 

Clinical Observations and Mortality Data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Fur loss was noted during the test in animals treated with the test item at concentrations of 10% or 25% w/win1% pluronic L92 indistilled water

 

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 EC₃Value

aEC₃= c + [[(3-d)/(b-d)] x (a-c)]

a	=	10
b	=	5.28
c	=	5
d	=	4.31
EC3= 5 + [[(3 -4.31)/(5.28 - 4.31)] x (10 - 5)] = 2

The concentration of test item expected to cause a 3 fold increase in³HTdR incorporation (EC₃value) was calculated to be 2%.

a=  lowest concentration giving stimulation index >3

b = actual stimulation index caused by ‘a’

c =  highest concentration failing to produce a stimulation index of 3

d = actual stimulation index caused by ‘c’

 

Applicant's summary and conclusion

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Remarks:

Criteria used for interpretation of results: GHS

Conclusions:

The test item was considered to be a sensitizer under the conditions of the test.
The test item was also classified as a contact sensitizer (Category 1A) according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. It is reasonable to assume that the Signal Word “Warning” and the Hazard Statement “H317: May cause an allergic skin reaction” are therefore required.
The test item was classified as a contact sensitizer (Category 1A) according to the Globally Harmonized System of Classification and Labelling of Chemicals.

Executive summary:

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 were noted at a concentration of25% w/w, in acetone/ olive oil 4-1 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 asolutionin1% pluronic L92 indistilled waterat concentrations of25%,10% or5% w/w. A further group of four animals was treated with1% pluronic L92 indistilled wateralone.

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 1% pluronic L92 indistilled water	Stimulation Index	Result
5	4.31	Positive
10	5.28	Positive
25	5.41	Positive

 

The concentration of test item expected to cause a 3 fold increase in³HTdR incorporation (EC₃value) was calculated to be2%.

Conclusion

The test item was considered to be asensitizerunder the conditions of the test.

The test item was also classified as a contact sensitizer (Category 1A) according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. It is reasonable to assume that the Signal Word “Warning” and the Hazard Statement “H317: May cause an allergic skin reaction” are therefore required.

The test item was classified as a contact sensitizer (Category 1A) according to the GloballyHarmonized Systemof Classification and Labelling of Chemicals.