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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:
30 May 2012 to 30 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with OECD, EU and US EPA test guidance and in compliance with GLP. Justification for read across is appended below.
Justification for type of information:
Full read across justification as RAAF is listed in Section 13.
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
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
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.
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: A health inspection was performed prior to treatment, to ensure that the animals were in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality.
Reliability check: The results of a reliability test with three concentrations of Hexylcinnamaldehyde 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 is summarized in the appendix of this report. For both scientific and animal welfare reasons, no concurrent positive control group was added to 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.
Animal Husbandry Conditions
Environmental controls for the animal room were set to maintain 18 to 24°C (actual daily mean min max: 21.0 – 21.6°C), a relative humidity of 40 to 70% (actual daily mean min max: 42 -59%), approximately 15 room 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 (Litalabo, S.P.P.S., Argenteuil, France). 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. Certificates of analysis were examined and then retained in the WIL Research Europe archives.
Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Water: Free access to tap water.
Results of analysis for diet, sawdust, paper, shelters and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the WIL Research Europe archives.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10, 25 or 50% w/w
No. of animals per dose:
three experimental groups of five females
Details on study design:
Test substance preparation
Vehicle: Acetone/Olive oil (4:1 v/v) (Acetone p.a.: Merck, Darmstadt, Germany; Olive oil: Fagron, Nieuwerkerk a/d IJssel, The Netherlands).
Rationale: The vehicle was selected based on trial formulations performed at WIL Research Europe and on test substance data supplied by the sponsor.
Preparation: The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle and the purity of the test substance. Homogeneity was obtained to visually acceptable levels.

Pre-screen test: A pre-screen test was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation at the most (maximum grade 2) and/or an increase in ear thickness < 25%) and is the highest possible concentration that can technically be applied.
Two test substance concentrations were tested; an 80% and 40% concentration. The highest concentration was the maximum concentration as required in the test guidelines (maximized for solubility).
The test system, procedures and techniques were identical to those used in the main study except that assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected (in the range of 8 to14 weeks old). Each animal was treated with one concentration on three consecutive days. Animals were group housed in labelled Makrolon cages (MII type, height 14 cm).
Since test substance remnants were anticipated to hamper scoring on Day 2 (based on Day 1 observations), the ears were cleaned of residual test substance with tap water on Day 2 between 30 and 60 minutes prior to any further assessment. Scoring of the ears was conducted prior to dosing on Days 2 and 3.
Ear thickness measurements were conducted using a digital thickness gauge 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 concentration, 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 approximately five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) with 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: 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 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. Radioactive 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. Furthermore, a description of all other (local) effects was recorded.
Necropsy: No necropsy was conducted according to protocol.
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. The SI is the ratio of the DPM/group compared to DPM/vehicle control group.
Key result
Parameter:
SI
Value:
1.5
Test group / Remarks:
10% v/v group
Key result
Parameter:
SI
Value:
1
Variability:
25% v/v group
Key result
Parameter:
SI
Value:
0.8
Variability:
50% v/v group
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 623, 420 and 340 DPM respectively. The mean DPM/animal value for the vehicle control group was 422 DPM.

PRE-SCREEN TEST

Table 1: Body weights and skin reactions

TS1(%)

Animal

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

bw

Erythema3

Erythema

Erythema

Erythema

Erythema

Erythema

bw

(g)2

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

(g)

40

1

22

0F

0F

0

0

0

0

0

0

0

0

0

0

24

2

22

0F

0F

0

0

0

0

0

0

0

0

0

0

20

804

3

22

0F

0F

1

1

0

0

0

0

0

0

0

0

21

4

21

0F

0F

1

1

1

1

0

0

0

0

0

0

21

F. White test substance remnants, not hampering the scoring.

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; 1 = Very slight erythema (barely perceptible)

4Applied using a spatula, instead of using a pipette. Concentration could technically not be applied satisfactory on Day 2.

Note: On Day 2, the ears were cleaned using water to facilitate scoring.

 

Table 2: Ear thickness measurements

TS1(%)

Animal

Day 1

Day 3

Day 6

Left

Right

Left

Right

Left

Right

(mm)

(mm)

(mm)

%2

(mm)

%

(mm)

%

(mm)

%

40

1

0.240

0.240

0.245

2

0.230

-4

0.245

2

0.240

0

2

0.230

0.235

0.230

0

0.220

-6

0.240

4

0.245

4

80

3

0.235

0.235

0.220

-6

0.235

0

0.245

4

0.245

4

4

0.240

0.235

0.250

4

0.245

4

0.245

2

0.245

4

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

1TS = test substance (% w/w).

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

 

MAIN STUDY

 

Table 3: Body weights and skin reactions

Group

TS1(%)

Animal

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Bw

Erythema3

Erythema

Erythema

Erythema

Erythema

Erythema

Bw

(g)2

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

(g)

1

0

1

21

0

0

0

0

0

0

0

0

0

0

0

0

20

2

22

0

0

0

0

0

0

0

0

0

0

0

0

21

3

21

0

0

0

0

0

0

0

0

0

0

0

0

20

4

22

0

0

0

0

0

0

0

0

0

0

0

0

21

5

22

0

0

0

0

0

0

0

0

0

0

0

0

22

2

10

6

21

0

0

0

0

0

0

0

0

0

0

0

0

20

7

22

0

0

0

0

0

0

0

0

0

0

0

0

23

8

21

0

0

0

0

0

0

0

0

0

0

0

0

20

9

21

0

0

0

0

0

0

0

0

0

0

0

0

20

10

23

0

0

0

0

0

0

0

0

0

0

0

0

23

3

25

11

20

0F

0F

0F

0F

0F

0F

0

0

0

0

0

0

21

12

23

0F

0F

0F

0F

0F

0F

0

0

0

0

0

0

23

13

23

0F

0F

0F

0F

0F

0F

0

0

0

0

0

0

23

14

22

0F

0F

0F

0F

0F

0F

0

0

0

0

0

0

20

15

22

0F

0F

0F

0F

0F

0F

0

0

0

0

0

0

21

4

50

16

22

0F

0F

1F

1F

1F

1F

0F

0F

0

0

0

0

22

17

23

0F

0F

1F

1F

1F

1F

0F

0F

0

0

0

0

23

18

23

0F

0F

1F

1F

1F

1F

0F

0F

0

0

0

0

22

19

22

0F

0F

1F

1F

1F

1F

0F

0F

0

0

0

0

23

20

24

0F

0F

1F

1F

1F

1F

0F

0F

0

0

0

0

24

1TS = test substance (% w/w).

2Body weight (grams).

3Grading erythema nd eschar formation (Left = dorsal surface of left ear; right = dorsal surface of right ear): 0 = No erythema; 1 = Very slight erythema (barely perceptible)

F. White test substance remnants on the dorsal surface of the ears, not hampering the scoring.

 

Table 4: 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

n

n

479

422 ±74

1.0 ± 0.2

2

n

n

402

3

n

n

357

4

n

n

212

5

n

n

662

2

10

6

n

n

455

623±97

1.5±0.3

7

n

n

932

8

n

n

715

9

n

n

627

10

n

n

386

3

25

11

n

n

346

420 ±124

1.0 ± 0.3

12

n

n

428

13

n

n

250

14

n

n

889

15

n

n

189

4

50

16

n

n

194

340±71

0.8±0.2

17

n

n

364

18

n

n

291

19

n

n

252

20

n

n

601

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:
Read across is deemed appropraite on the basis of structural analogy. Based on these results, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines.
Executive summary:

Read across is deemed appropraite on the basis of structural analogy. Assessment of Contact Hypersensitivity to 6,6’-di-tert-butyl-4,4’-butylidenedi-m-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" and EPA, OPPTS 870.2600 (2003) “Skin Sensitization”. The study was performed in accordance with the Principles of Good Laboratory Practice (GLP).

 

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

 

In the main study, three experimental groups of five female CBA/J mice were treated with substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)).

Three days after the last exposure, all animals were injected with3H-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.

 

RESULTS

The slight irritation of the ears as shown by animals treated at 50% was considered not to have a toxicologically significant effect on the activity of the nodes. No irritation was seen at 25 and 10%. White test substance remnants were seen in the animals treated at 25 and 50%, not hampering the scoring of the ears.

Body weights and body weight gain of experimental animals were in range with controls.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 623, 420 and 340 DPM respectively. The mean DPM/animal value for the vehicle control group was 422 DPM.

 

CONCLUSION

The SI values calculated for the substance concentrations 10, 25 and 50% were 1.5, 1.0 and 0.8 respectively.

Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 50%, 6,6’-ditert-butyl-4,4’-butylidenedi-m-cresol was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 50%.

The six-month reliability check with Alpha-hexylcinnamicaldehyde 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, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-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) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.

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

Results are available in vivo for read across purposes from an equivalent substance. In the interests of animal welfare, given the known acute toxicity effects, it was deemed appropriate to assess the effects of the substance via application of read across for in vivo results. Results indicate that this category of substances are not sensitisers. No classification is applicable.

Migrated from Short description of key information:

The read across substance, 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol is not regarded as a skin sensitizer according to the recommendations made in the test guidelines.

Justification for selection of skin sensitisation endpoint:

Endpoint conclusion determined in experimental conditions utilising OECD, EU & US EPA test guidance.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

For this category of substance it is agreed that there is no risk for respiratory sensitisation for workers exists at high exposure. The following should be noted:

1) For the substance no history of respiratory problems, such as occupational asthma, is associated with the manufacture and use of these types of substance.

2) Due to the low vapour pressure of the substance, no inhalation hazard is proposed for the substance.

The potential to cause respiratory sensitisation is not considered to be applicable for this substance.

Justification for selection of respiratory sensitisation endpoint:

The potential to cause respiratory sensitisation is not considered to be applicable for this substance.

Justification for classification or non-classification

The above studies have all been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the study was conducted to GLP, although is read across to an analogue. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds.

The above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008). No classification for sensitising effects is therefore required.