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Description of key information

Skin Sensitisation: Not sensitising in the LLNA.

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:
13 May 2015 to 15 June 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD, EU & US EPA test guidelines in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Species: Mouse, CBA/J strain, inbred, SPF-Quality. Recognized by the international guidelines as the recommended test system (e.g. OECD, EC, EPA). Source: Janvier, Le Genest-Saint-Isle, France
Number of animals: 20 females (nulliparous and non-pregnant), five females per group (main study only).
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 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.
Reliability check: The results of a reliability test with three concentrations of Hexylcinnamaldehyde (CAS No. 101-86-0) in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously and using the same materials, animal supplier, animal strain and essential procedures. For both scientific and animal welfare reasons, no concurrent positive control group was included in the study.
An extensive data base is available with reliability checks performed each half year during at least the recent 9 years showing reproducible and consistent positive results.

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: the photoperiod was between 7:00 and 19:00 hrs daily. Any variations to these conditions were recorded in the raw data and had no effect on the outcome of the study.

Accommodation
Animals were group housed in labelled 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.
Vehicle:
methyl ethyl ketone
Concentration:
5, 10 or 25% w/w
No. of animals per dose:
Three experimental groups of five female CBA/J mice and one group of five female mice in the control (vehicle) group.
Details on study design:
Pre-screen test
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
Two test substance concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration that could technically be applied.
The test system, procedures and techniques were identical as those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.

Main study
Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the pre-screen test.
One group of five animals was treated with vehicle.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance, 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 substance.

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) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

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 a numerical scoring system (see Any other information). Furthermore, a description of all other (local) effects was recorded.
Necropsy: No necropsy for gross macroscopic examination was performed according to protocol.

Electronic data capture
Observations/measurements in the study were recorded electronically using the following programs: REES Centron Environmental Monitoring system version SQL 2.0 (REES scientific, Trenton, NJ, USA); Quantasmart 2.03 (PerkinElmer Life Sciences, Boston, MA, USA): LSC software.

Interpretation
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 substance 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 (2011) (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 substances and mixtures, including all amendments.
Consideration was given to the EC3 value (the estimated test substance concentration that will give a SI =3).
Positive control substance(s):
other: For both scientific and animal welfare reasons, no concurrent positive control group was included in the study.
Statistics:
No data specified in the study report.
Positive control results:
For both scientific and animal welfare reasons, no concurrent positive control group was included in the study.
An extensive data base is available with reliability checks performed each half year during at least the recent 9 years showing reproducible and consistent positive results.
Key result
Parameter:
SI
Value:
1.1 - 2.7
Parameter:
other: disintegrations per minute (DPM)
Value:
752 - 1 914

Body weights and skin reactions

TS1(%)

Animal

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

bw (g)2

Erythema3

Erythema

Erythema

Erythema

Erythema

Erythema

bw (g)

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

25

1

2

22

22

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0s

0s

0s

0s

23

22

50

3

4

21

21

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0sk

0sk

0sk

0sk

20

22

s. Scaliness. k. Scabs

1TS = test substance (% w/w)

2Body weight (grams)

3Grading erythema and eschar formation (Left = dorsal surface of left ear; right = dorsal surface of right ear): 0 = No erythema

Note: White staining of the dorsal surface of the ears by test substance remnants was noted for both animals treated at 50% throughout the observation period. The staining did not hamper the scoring of the ears. Bald spots behind the ears were noted for all animals on Day 6.

 

Ear thickness measurements

TS1(%)

Animal

Day 1

Day 3

Day 6

Left

Right

Left

Right

Left

Right

(mm)

(mm)

(mm)

%2

(mm)

%2

(mm)

%2

(mm)

%2

25

1

2

0.220

0.225

0.225

0.225

0.225

0.215

2

-4

0.220

0.220

-2

-2

0.225

0.230

2

2

0.235

0.230

4

2

50

3

4

0.220

0.220

0.220

0.220

0.225

0.225

2

2

0.215

0.220

-2

0

0.235

0.240

7

9

0.240

0.240

9

9

Left (mm) = thickness of left ear in millimetres; right (mm) – thickness of right ear in millimetres.

1TS = test substance (& w/w)

2Percent increase compared to Day 1 pre-dose value.

 

Body weights and skin reactions

Group

TS1(%)

Animal

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

bw (g)2

Erythema3

Erythema

Erythema

Erythema

Erythema

Erythema

bw (g)

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

1

0

1

2

3

4

5

21

22

17

22

22

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

21

21

17

22

22

2

5

6

7

8

9

10

19

20

22

25

22

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

19

21

23

24

23

3

10

11

12

13

14

15

18

25

19

21

20

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

19

24

20

22

20

4

25

16

17

18

19

20

21

23

20

22

19

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

21

24

21

23

20

1TS = test substance (% w/w)

2Body weight (grams)

3Grading erythema and eschar formation (Left = dorsal surface of left ear; right = dorsal surface of right ear): 0 = No erythema

Note: Scaliness (Days 4, 5 and 6) and bald spots around the ears (Days 3, 4, 5 and 6) were noted for all animals treated at 25%.

 

Relative size lymph nodes, radioactivity counts (DPM) and Stimulation Index (SI)

Group

TS1(%)

Animal

Size nodes2

DPM3/ animal

Mean

DPM ± SEM4

Mean

SI ± SEM

Left

Right

1

0

1

2

3

4

5

N

N

N

N

N

N

N

N

N

N

791

569

694

563

956

715 ± 74

1.0 ± 0.1

2

5

6

7

8

9

10

N

+

+

+

+

N

+

+

+

+

367

619

705

811

1259

752 ± 146

1.1 ± 0.2

3

10

11

12

13

14

15

+

+

+

+

+

+

+

+

+

+

1649

1910

1738

1510

1167

1595 ± 125

2.2 ± 0.2

4

25

16

17

18

19

20

+

+

+

+

+

+

+

+

+

+

2961

1983

1554

1567

1506

1914 ± 275

2.7 ± 0.5

1TS = test substance (% w/w)

2Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +, ++ or +++: degree of enlargement, n: considered to be normal).

3DPM = Disintegrations per minute

4SEM = Standard Error of the Mean

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Since there was no indication that the 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] elicits a SI ≥ 3 when tested up to 25%. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 25%.
Based on these results, 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines.
Executive summary:

Assessment of skin sensitization to 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] in the Mouse (Local Lymph Node Assay).

The study was carried out based on the guidelines described in:

OECD, Section 4, Health Effects, No.429 (2010),

EC, No 440/2008; B42: "Skin Sensitization: Local Lymph Node Assay"

EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

 

Test substance concentrations selected for the main study were based on the results of a pre-screen test.

 

In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 5, 10 or 25% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Methyl ethyl ketone).

 

Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal.

After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

 

No erythema of the ears was observed in any of the animals examined. Scaliness and/or bald spots around the ears were noted for all animals treated at 25% on Days 3, 4, 5 and 6.

 

The majority of auricular lymph nodes were considered enlarged, except for the nodes in one animal treated at 5% which were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

 

Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 752, 1595 and 1914 DPM, respectively. The mean DPM/animal value for the vehicle control group was 715 DPM. The SI values calculated for the substance concentrations 5, 10 and 25% were 1.1, 2.2 and 2.7, respectively.

 

Since there was no indication that the 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] elicits a SI ≥ 3 when tested up to 25%. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 25%.

 

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

 

Based on these results, 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Assessment of skin sensitization to 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] in the Mouse (Local Lymph Node Assay).

In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 5, 10 or 25% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Methyl ethyl ketone).

Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal.

After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

No erythema of the ears was observed in any of the animals examined. Scaliness and/or bald spots around the ears were noted for all animals treated at 25% on Days 3, 4, 5 and 6.

The majority of auricular lymph nodes were considered enlarged, except for the nodes in one animal treated at 5% which were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 752, 1595 and 1914 DPM, respectively. The mean DPM/animal value for the vehicle control group was 715 DPM. The SI values calculated for the substance concentrations 5, 10 and 25% were 1.1, 2.2 and 2.7, respectively.

Since there was no indication that the 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] elicits a SI ≥ 3 when tested up to 25%. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 25%.

Based on these results, 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines.


Migrated from Short description of key information:
Skin sensitisation: Not sensitising in the mouse local lymph node asay (LLNA).

Justification for selection of skin sensitisation endpoint:
Endpoint derived from laboratory study conducted to OECD Guideline 429, EU Method B.42 and US EPA Standard 870.2600.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The test substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).

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