Reaction products of 4,4'-methylenebis(cyclohexylamine) 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

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

Qualifier:

according to guideline

Guideline:

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

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'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 4,4'-methylenebis(cyclohexylamine)

EC Number:

500-103-5

EC Name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 4,4'-methylenebis(cyclohexylamine)

Cas Number:

38294-67-6

Molecular formula:

C

IUPAC Name:

Reaction products of 4,4'-methylenebis(cyclohexylamine) and 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Test material form:

liquid: viscous

Details on test material:

Sample ID :PACM BADGE adduct
Chemical Name :4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 4,4'-methylenebis(cyclohexylamine)
Physical State liquid
CAS # 38294-67-6
EU # 500-103-5
Manufacturer Air Products and Chemicals, Inc.
Batch # / Lot # 20160226-PDU-PACM 828

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Age and body weight Young adult animals (approx. 10 weeks old) were selected.
Body weight variation was within +/- 20% of the sex mean.
Identification Tail mark with a marker pen.
Health inspection At least prior to dosing. It was ensured that the animals were
healthy and that the ears were intact and free from any
abnormality.
Animal Husbandry
Conditions
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative
humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle.
Any variations to these conditions were maintained in the raw data and had no effect on the
outcome of the study.
Accommodation
Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing
sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH
+ CO. KG, Rosenberg, Germany). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill
Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404,
Datesand Ltd, USA) were supplied as cage-enrichment. The acclimatization period was at
least 5 days before the start of treatment under laboratory conditions. On Day 6, the animals
were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and
cage enrichment.
Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest,
Germany).
Water
Free access to tap water.
Diet, water, bedding and cage enrichment evaluations for contaminants and/or nutrients were
performed according to facility standard procedures. There were no findings that could
interfere with the study.

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

Group animal numbers induction (test item; % w/w)
1 01 - 05 0 (Dimethyl suplhoxide)
2 06 - 10 0.5
3 11 - 15 1.0
4 16 - 20 2.5

No. of animals per dose:

5

Details on study design:

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at
approximately the same time on each day. The concentrations were stirred with a magnetic
stirrer immediately prior to dosing.
The control animals were treated in the same way as the experimental animals, except that the
vehicle was administered instead of the test item.
Excision of the Nodes - Day 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline
(PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine
(PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of
Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular)
lymph node of each ear was excised. The relative size of the nodes (as compared to normal)
was estimated by visual examination and abnormalities of the nodes and surrounding area
were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.
Tissue Processing for Radioactivity - Day 6
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was
prepared in PBS by gentle separation through stainless steel gauze (diameter: 125 μm). LNC
were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To
precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA)
Radioactivity Measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to
10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US)
as the scintillation fluid. Radioactivity measurements were performed using a Packard
scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a
maximum of 5 minutes whichever came first. The scintillation counter was programmed to
automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations
Per Minute (DPM).
Observations
Mortality/Viability Twice daily.
Body weights On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours
after dosing).
Irritation Once daily on Days 1-6 (on Days 1-3 within 1 hour after
dosing) according to the following numerical scoring system.
In addition, a description of all other (local) effects was
recorded.
Grading Irritation Reactions:
Erythema and eschar formation:
No erythema ..... ..................................................................................................................................................... 0
Very slight erythema (barely perceptible) .............................................................................................................. 1
Well-defined erythema ........................................................................................................................................... 2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth) … ............................... 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema ........................................... 4
Necropsy No necropsy for gross macroscopic examination was performed
according to study plan.

Positive control substance(s):

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

Statistics:

DPM values are presented for each animal and for each dose group. A Stimulation Index (SI)
is calculated for each group using the individual SI values. The individual SI is the ratio of
the DPM/animal compared to the DPM/vehicle control group mean.
If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.
The results were evaluated according to the Globally Harmonized System of Classification
and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments)
and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16
December 2008 on classification, labelling and packaging of items and mixtures, including
all amendments.
Classification of results

UN-GHS 2015; EC-CLP 2008 EC Hazard statement
SI < 3 No sensitizer
---------------------------------------------------------------------------------------
SI ≥ 3 Cat 1 Skin sensitizer
EC3 value ≤ 2%: sub-category 1A
EC3 value > 2%: sub-category 1B

Results and discussion

Positive control results:

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local
Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for
testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

2.7

Test group / Remarks:

Group dosed with 0.5% solution of test item in DMSO

Results 2

Parameter:

SI

Value:

3.6

Test group / Remarks:

Group dosed with 1.0 % solution of test item in DMSO

Cellular proliferation data / Observations:

Pre-screen Test
At a concentration of 10%, hunched posture and piloerection were noted for both animals
between Days 1 and 3.Very slight to well-defined erythema was noted for both animals
throughout the observation period and scaliness and/or scabs were noted for both animals
between Days 2 and 6. Variations in ear thickness during the observation period were larger
than 25% from Day 1 pre-dose values.
At a concentration of 5%, hunched posture and piloerection were noted for both animals on
Day 3.Very slight erythema was noted for both animals between Days 1 and 3 and scaliness
and/or scabs were noted for both animals between Days 2 and 6. Variations in ear thickness
during the observation period were less than 25% from Day 1 pre-dose values.
Based on these results, the highest test item concentration selected for the main study was a
2.5% concentration.
Main Study
Skin Reactions / Irritation
Hardened ears were noted for three animals treated at 2.5% on Days 2 and/or 6. Based on
these findings the experimental group treated at 2.5% was not used for interpretation of the
study.
Very slight erythema was noted for one animal treated at 1% on Days 2 and 3 and scaliness
was noted for all animals treated at 1% on Day 6.
Systemic Toxicity
No mortality occurred and no clinical signs of systemic toxicity were observed in the
remaining animals of the main study. Body weights and body weight gain of experimental
animals remained in the same range as controls over the study period.
Macroscopic Examination of the Auricular Lymph Nodes and Surrounding
Area
The majority of auricular lymph nodes were considered normal in size, except for one of the
nodes of one animal treated at 1%.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Results of Local Lymph Node Assay study with 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 4,4'-methylenebis(cyclohexylamine) indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 0.7% was calculated. Based on the results, test item is considered to be sensitizer.

Executive summary:

Results of Local Lymph Node Assay  study with 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 4,4'-methylenebis(cyclohexylamine) indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 0.7% was calculated. Based on the results, test item is considered to be sensitizer.