Phenol, C14-18-alkyl derivs.

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27August 2014 to 15 September 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 3

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)

Test material

Test material information

Constituent 1

Reference substance name:

C14-16-18 Alkyl phenol

EC Number:

931-468-2

Cas Number:

1190625-94-5

Molecular formula:

For substance information - see Section 1.2

IUPAC Name:

C14-16-18 Alkyl phenol

Test material form:

other: slightly viscous liquid

Details on test material:

- Appearance: Clear amber liquid
- Storage conditions of test material: At room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Strain: CBA/J strain, inbred, SPF-Quality
- Age at study initiation: Approximately 10 to 12 weeks old
- Weight at study initiation: 20 to 25 g. Body weight variation was within ± 20 % of the sex mean.
- Housing: During acclimation, animals were group housed in cages (height 18 cm) containing sterilised sawdust as bedding material. Paper and shelters (disposable paper corner home) were supplied as cage-enrichment. Prior to the study, the animals were group housed with a sheet of paper instead of sawdust and cage enrichment.
- Diet: Free access to pelleted rodent diet
- Water: ad libitum access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24 °C
- Humidity: 40 to 70 % (relative)
- Air changes: at least 10 air changes/hour
- Photoperiod: 12-hour light/12-hour dark cycle

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

2.5, 5 and 10 % w/w

No. of animals per dose:

5 animals per dose

Details on study design:

RANGE FINDING TEST
A pre-screen test was conducted in order to select the highest test material concentration to be used in the main study. Initially, two test material concentrations were tested; 100 % and a 50 % concentration.
The test system, procedures and techniques were identical to 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. Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 (only animals 1 to 4) and 3, and on Day 6.
Animals were sacrificed after the final observation.
Based on the results of the initially treated animals, four additional animals were treated in a similar manner with two lower concentrations (25 and 10 %).

MAIN STUDY
INDUCTION - DAYS 1, 2 AND 3
The dorsal surface of both ears was topically treated (25 µL/ear) with the test material concentration. 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 material.

EXCISION OF THE NODES - DAY 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 µCi of ³H-methyl thymidine. After approximately 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) for each animal 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 200 x g for 10 minutes at 4 °C. To precipitate the DNA, the LNC were treated with 5 % trichloroacetic acid (TCA) and stored in the refrigerator until the next day.

RADIOACTIVITY MEASUREMENTS - DAY 7
Precipitates were recovered by centrifugation, re-suspended 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: Day 1 (pre-dose) and Day 6 (prior to necropsy)
- Clinical signs: Once daily on Days 1 to 6 (on Days 1 to 3 between 3 and 4 hours after dosing)
- Irritation: Once daily on Days 1 to 6 (on Days 1 to 3 within 1 hour after dosing) according to the numerical scoring system below. Furthermore, a description of all other (local) effects was recorded. Ear thickness measurements of treated animals were conducted using a digital thickness gauge prior to dosing on Days 1 and on Day 6.
- Necropsy performed: No

Grading for Irritation Reactions:
Erythema and eschar formation:
0 = No erythema
1 = Very slight erythema (barely perceptible)
2 = Well-defined erythema
3 = Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth)
4 = Severe erythema (beet redness) to eschar formation preventing grading of erythema

Positive control substance(s):

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

Statistics:

The EC3 values was determined using linear interpolation.

Results and discussion

Positive control results:

A reliability test with three concentrations of Hexylcinnamaldehyde in Acetone/Olive oil (4:1 v/v) was performed several months prior to the study using the same materials, animal supplier, animal strain and essential procedures as for the main test. For both scientific and animal welfare reasons, no concurrent positive control group was added to the study.
The positive control was tested at concentrations of 5, 10 and 25 %. The SI values calculated were 1.2, 1.4 and 4.7, respectively. An EC3 value of 17.3 % was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5 %. Based on the results, it was concluded that the LLNA as performed at the testing facility is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

7.6

Test group / Remarks:

10%

Results 2

Parameter:

SI

Value:

3.8

Test group / Remarks:

5%

Results 3

Parameter:

SI

Value:

2.2

Test group / Remarks:

2.5%

Results 4

Parameter:

SI

Value:

1

Test group / Remarks:

0%

Results 5

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test material concentrations 2.5, 5 and 10 % were 1809, 3072 and 6146 DPM, respectively. The mean DPM/animal value for the vehicle control group was 811 DPM.

Any other information on results incl. tables

Pre-screen test

Ptosis and piloerection were noted for all animals at 50 and 100 % on Days 2 and/or 3. Additionally, hunched posture and lean appearance were noted for the animals at 100 % between Days 4 and 6. One animal at 100 % was found dead on Day 5. Moderate to severe erythema was noted for all animals at 50 and 100 % and variations in ear thickness during the observation period were more than 25 % from Day 1 pre-dose values.

At 10 and 25 %, no irritation or clear signs of systemic toxicity were observed in any of the animals examined. Scaliness was noted for both animals at 25 % on Day 6. Variations in ear thickness were less than 25 % from Day 3 pre-dose values for the animals at 10 % but more than 25 % for the animals treated at 25 %.

Based on these results, the highest test material concentration selected for the main study was a 10 % concentration.

 

Main study

- Skin reactions / Irritation

No irritation of the ears was observed in any of the animals examined. Variations in ear thickness during the observation period were less than 25 % from Day 1 pre-dose values.

- Systemic toxicity

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.

- Macroscopy of the auricular lymph nodes and surrounding area

The majority of auricular lymph nodes of the animals treated at 2.5 and 5 % were considered normal in size, except for the nodes of two animals at 2.5 % and one animal at 5 % which were considered enlarged. The lymph nodes of most animals treated at 10 % appeared enlarged, except for the nodes of one animal which were considered normal in size.

No macroscopic abnormalities of the surrounding area were noted for any of the animals.

 

Table 1: Relative size lymph nodes, radioactivity counts (DPM) and Stimulation Index (SI)

Test Material Concentration (% w/w)	Animal Number	Size of Nodes¹		DPM²/animal	Mean DPM ± SEM³	Mean SI ± SEM
		Left	Right
0	1	N	N	702	811 ± 100	1.0 ± 0.2
	2	N	N	642
	3	N	N	1187
	4	N	N	675
	5	N	N	851
2.5	6	N	N	1171	1809 ± 341	2.2 ± 0.5
	7	+	+	2956
	8	N	N	1371
	9	+	+	2229
	10	N	N	1320
5	11	+	+	4840	3072 ± 574	3.8 ± 0.8
	12	N	N	1908
	13	N	N	1857
	14	N	N	3846
	15	N	N	2908
10	16	+	+	3826	6146 ± 1107	7.6 ± 1.7
	17	N	N	6461
	18	+	+	3604
	19	+	+	7356
	20	+	+	9481

¹Relative size of auricular lymph nodes. N = normal; + = enlarged

²DPM = Disintegrations per minute

³SEM = Standard error of the mean

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test material was determined to be a sensitiser (category 1B).

Executive summary:

An assessment of contact hypersensitivity to the test material was made in the mouse. A Local Lymph Node Assay (LLNA) study was carried out in accordance with the standardised guidelines OECD 429, EU Method B.42 and EPA OPPTS 870.2600 under GLP conditions.

Test material 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 material concentrations of 2.5, 5 or 10 % w/w in Acetone:Olive oil (4:1 v/v) on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone.

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 irritation of the ears was observed in any of the animals examined. Variations in ear thickness during the observation period were less than 25 % from Day 1 pre-dose values.

The majority of auricular lymph nodes of the animals treated at 2.5 and 5 % were considered normal in size, except for the nodes of two animals at 2.5 % and one animal at 5 % which were considered enlarged. The lymph nodes of most animals treated at 10 % appeared enlarged, except for the nodes of one animal 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 material concentrations 2.5, 5 and 10 % were 1809, 3072 and 6146 DPM, respectively. The mean DPM/animal value for the vehicle control group was 811 DPM. The SI values calculated for the material concentrations 2.5, 5 and 10 % were 2.2, 3.8 and 7.6, respectively.

These results indicate that the test material could elicit an SI value = 3. The data showed a dose-response and an EC3 value (the estimated test material concentration that will give an SI = 3) of 3.8 % was calculated.

A six-month reliability check with Alpha-hexylcinnamaldehyde carried out several months prior to the study indicated that the Local Lymph Node Assay as performed at the testing facility is an appropriate model for testing for contact hypersensitivity.

Under the conditions of this study, the test material was determined to be a sensitiser.