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REACH

4,4',4''-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]

EC number: 217-420-7 | CAS number: 1843-03-4

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

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:

06 June 2012 to 18 June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed to OECD, EU & US EPA test guidance 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.
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.
Environmental controls for the animal room were set to maintain 18 to 24 °C, a relative humidity of 40 to 70%, 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.

Vehicle:

methyl ethyl ketone

Concentration:

0, 5, 10, 25 % w/w

No. of animals per dose:

5 animals per group

Details on study design:

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 (see section 6.7) 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; a 25% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids, 50% for solids).
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 labeled Makrolon cages (MII type, height 14 cm). 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

Positive control substance(s):

not specified

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.
If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.

Parameter:

Remarks on result:

other: see Remark

Remarks:

The SI values calculated for the substance concentrations 5, 10 and 25% were 4.2, 6.0 and 13.7 respectively. These results indicate that the test substance could elicit an SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of below 5% was established.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Pre-screen test: At a 25% test substance concentration no signs of systemic toxicity were noted and only very slight irritation was observed. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values at 25% test substance concentration. White test substance remnants were present on the dorsal surface of the ears of both animals at 25 and 50% (throughout the observation period), which did not hamper scoring of the skin reactions. Based on these results, the highest test substance concentration selected for the main study was a 25% concentration. Main study Skin reactions / Irritation: The very slight irritation of the ears as shown by all animals treated at 25% was considered not to have a toxicologically significant effect on the activity of the nodes. Test substance remnants were present on the dorsal surface of the ears of all test substance treated animals (throughout the observation period), which did not hamper scoring of the skin reactions. Systemic toxicity: No mortality occurred and no clinical signs of systemic toxicity were observed in the 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. The body weight loss noted for some animals across the dose groups was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent. Macroscopy of the auricular lymph nodes and surrounding area: All auricular lymph nodes of the animals at 25% and of two animals at 10% were considered larger in size as compared to the control group. No macroscopic abnormalities of the surrounding area were noted in any of the animals. Radioactivity measurements: Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 1487, 2142 and 4875 DPM respectively. The mean DPM/animal value for the vehicle control group was 356.

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 (g)²

Erythema³

Erythema

Bw (g)

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

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)

2 = Well-defined erythema

F = White test substance remnants on the dorsal surface of the ears, which did not hamper the scoring of skin reactions.

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)	%
25	1	0.230	0.225	0.265	15	0.260	16	0.265	15	0.255	13
	2	0.230	0.225	0.265	15	0.265	18	0.265	15	0.265	18
50	3	0.225	0.225	0.325	44	0.310	38	0.310	38	0.310	38
	4	0.225	0.230	0.270	20	0.270	17	0.305	36	0.315	37

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 (g)²

Erythema³

Erythema

Bw (g)

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

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

2 Body weight (grams).

3 Grading 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)

F = Test substance remnants on the dorsal surface of the ears, which did not hamper scoring of skin reactions.

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
Group	TS¹(%)	Animal	Left	Right	DPM₃/animal	Mean DPM±SEM⁴	Mean SI±SEM
1	0	1	n	n	476	356 ± 31	1.0 ± 0.1
		2	n	n	307
		3	n	n	335
		4	n	n	349
		5	n	n	311
2	5	6	n	n	1583	1487 ± 187	4.2 ± 0.6
		7	n	n	1286
		8	n	n	1099
		9	n	n	2168
		10	n	n	1298
3	10	11	+	+	2011	2142 ± 561	6.0 ± 1.7
		12	n	n	3874
		13	n	n	2809
		14	+	+	1363
		15	n	n	653
4	25	16	+	+	7666	4875 ± 1137	13.7 ± 3.4
		17	+	+	2158
		18	+	+	3358
		19	+	+	7514
		20	+	+	3681

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:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

According to the recommendations made in the test guidelines, 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] would be regarded as skin sensitizer.

Executive summary:

Assessment of Contact Hypersensitivity to 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-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" EPA, OPPTS 870.2600 (2003) “Skin Sensitization”. Study performed in compliance with GLP.

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/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 with³H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal.

After precipitating theof 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.

The very slight irritation of the ears as shown by all animals treated at 25% was considered not to have a toxicologically significant effect on the activity of the nodes.

Test substance remnants were present on the dorsal surface of the ears of all test substance treated animals (throughout the observation period), which did not hamper scoring of the skin reactions.

No mortality occurred and no clinical signs of systemic toxicity were observed in the 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. The body weight loss noted for some animals across the dose groups was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.

All auricular lymph nodes of the animals at 25% and of two animals at 10% were considered larger in size as compared to the control group. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 1487, 2142 and 4875 DPM respectively. The mean DPM/animal value for the vehicle control group was 356.

The SI values calculated for the substance concentrations 5, 10 and 25% were 4.2, 6.0 and 13.7 respectively.

These results indicate that the test substance could elicit an SI≥3. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of below 5% was established.

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:

- according to the recommendations made in the test guidelines, 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] would be regarded as skin sensitizer.

- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011), 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] should be classified as skin sensitizer (Category 1).

- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] should be classified as skin sensitizer (Category 1) and labeled as H317: May cause an allergic skin reaction.

Endpoint conclusion

Endpoint conclusion:: adverse effect observed (sensitising)
Additional information:: Migrated from Short description of key information:
Substance was found to be sensitisting (SI = >3).

Justification for selection of skin sensitisation endpoint:
Study performed to OECD, EU & US EPA test guidance.

Justification for classification or non-classification

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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