[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 August - 04 October 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

GLP study conducted to current guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

Reaction products of 2,3; 2,4; 2,5 and 2,6 mixed xylidenes, C7 and C8 linear substituted naphthols and diazonium salts

Molecular formula:

Not available: variable

IUPAC Name:

Reaction products of 2,3; 2,4; 2,5 and 2,6 mixed xylidenes, C7 and C8 linear substituted naphthols and diazonium salts

Test material form:

solid: particulate/powder

Details on test material:

Batch No. SR164:1
Appearance Dark red solid (see certificate of analysis)
Test substance storage Ambient conditions but protected from temperatures below 5°C

Stability under storage Stable under recommended storage conditions.
Manufacturing Date 12 July 2022
Expiry date 12 July 2027

Specific details on test material used for the study:

No further details in the study report

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Species: Mouse
Strain: CBA/J
Condition: Inbred, SPF-Quality
Source: Janvier, Le Genest-Saint-Isle, France
Number of Animals: 20 females (nulliparous and non-pregnant). Five females per group.
Age at the Initiation of Dosing: Young adult animals (approximately 11 weeks old) were selected.
Weight at the Initiation of Dosing: 24.0 to 29.4 g.
Note: For pre-screen details see section 4.8.2 pre-screen test

Justification for Test System and Number of Animals
The CBA/J mouse was chosen as the animal model for this study as recognized by international guidelines as a recommended test system (e.g., OECD, FDA, MHLW). The test method and number of animals were based on the test guidelines.
The results of a reliability test with three concentrations of Alpha-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 are summarized in Appendix 4 of this report. 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.
The study plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of Appendix 1 of project license AVD2360020172866 approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).

Animal Identification
At study assignment, each animal was identified using a tail mark with indelible ink.

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

Selection, Assignment, Replacement, and Disposition of Animals
Animals were assigned to the study at the discretion of the coordinating biotechnician, with all animals within ± 20% of the sex mean body weights. Animals in poor health or at extremes of body weight range were not assigned to the study.
Before the initiation of dosing, a health inspection was performed, and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.
The disposition of all animals was documented in the study records.

Husbandry
Housing
On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized wooden fibers as bedding material equipped with water bottles. The rooms in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly labeled.

Environmental Conditions
Target temperatures of 20 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 22°C with an actual daily mean relative humidity of 44 to 64%. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap-water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment
Animals were socially housed for psychological/environmental enrichment and were provided with materials such as devices for hiding in and paper, except when interrupted by study procedures/activities. Results of analysis for contaminants were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants that would interfere with the objectives of the study.

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

50, 25, 10, 0 % w/w
Dose route and dose concentrations used are in compliance with the OECD test guideline for LLNA studies.
At a 25% and 50% test material concentration, no signs of systemic toxicity were noted, no irritation was observed, and ear thickness increase did not exceed 25% on Day 6 and therefore a 50% concentration was selected as highest concentration for the main study.
Red test material remnants were present on the dorsal surface of the ears of all animals at 25% and 50% on Days 1-6, which did not hamper scoring of the skin reactions.

No. of animals per dose:

5

Details on study design:

A pre-screen test was conducted in order to select the highest test material 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 material concentrations were tested: a 25% and 50% concentration. The highest concentration was the highest concentration that could be prepared homogeneously. The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 10 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult females 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 material concentration per group. The highest test material concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.

Allocation
See any other information on materials and methods

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test material, at approximately the same time on each day. The formulations were stirred with a magnetic stirrer until dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle only was administered.

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 euthanized according to laboratories Standard Operating Procedures. 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 PBS with 0.5% Bovine Serum Albumin (PBS/BSA) .

Tissue Processing for Radioactivity - Day 6
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS/BSA by gentle separation through stainless steel gauze (maze size: 200 µm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS/BSA 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 scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). 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).

In - Life Procedures, Observations, and Measurements
Mortality/Moribundity Checks
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations
Post-dose Observations
Post-dose observations were performed once daily on Days 1-6 (on Days 1-3 at least 3 hours after dosing).
All the animals were examined for reaction to dosing. The onset, intensity and duration of these signs was recorded (if appropriate), particular attention being paid to the animals during and for the first hour after dosing.

Body Weights
Animals were weighed individually on Day 1 (pre-dose) and 6 (prior to necropsy).

Irritation
Erythema and eschar formation observations were performed 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.

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

Premature Terminal Procedures
No early necropsy was performed since all animals of the main study survived until the end of the observation period.

ANALYSIS
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented. 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 material 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 (2017) (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.
The EC3 value (the estimated test material concentration that will give a SI =3) was determined, using linear interpolation.

Positive control substance(s):

other: None

Results and discussion

Positive control results:

None

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.4

Variability:

Standard Error of the Mean
± 0.3

Test group / Remarks:

10%

Results 2

Key result

Parameter:

SI

Value:

5.5

Variability:

Standard Error of the Mean
± 0.6

Test group / Remarks:

25 %

Results 3

Key result

Parameter:

SI

Value:

13.6

Variability:

Standard Error of the Mean.
± 1.8

Test group / Remarks:

50 %

Cellular proliferation data / Observations:

Pre - Screen Test

At a 25% and 50% test material concentration, no signs of systemic toxicity were noted, no irritation was observed, and ear thickness increase did not exceed 25% on Day 6 and therefore a 50% concentration was selected as highest concentration for the main study.
Red test material remnants were present on the dorsal surface of the ears of all animals at 25% and 50% on Days 1-6, which did not hamper scoring of the skin reactions.

Main Study
Skin Reactions / Irritation

No irritation was observed in any of the animals.
At 10, 25 and 50%, red test material remnants were present on the dorsal surface of the ears of all animals on Day 1-6, which hampered scoring of skin reactions from Day 3 onwards in the animals dosed at 50%. The remnant of test material at 50% did not hamper the scoring of skin reactions in the pre-screen test, which is contrary to that seen in the main study.
However, the difference is considered to be minor and does not impact on the conclusion of the study.

Systemic Toxicity

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals. Red coloration of the skin, fur and/or paws were observed in all test material-treated animals from Day 1 onwards. This finding was considered related to the color of the test material and therefore not considered a sign of systemic toxicity.
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Macroscopic Examination of the Lymph Nodes and Surrounding Area

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size, except for one node in two animals at 25% and one or both nodes in all animals at 50%, which were slightly enlarged.
No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Radioactivity Measurements and SI Values
(see any other information on results)
Mean DPM/animal values for the experimental groups treated with test material concentrations 10, 25 and 50% were 1850, 4211 and 10458 DPM, respectively. The mean DPM/animal value for the vehicle control group was 769 DPM. The SI values calculated for the test material concentrations 10, 25 and 50% were 2.4, 5.5 and 13.6, respectively.

Any other information on results incl. tables

Pre-Screen Test: Body Weights and Skin Reactions

 

TI ^1 (%)

Animal

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

bw (g) ^2

Erythema^3 left        right

Erythema left        right

Erythema left        right

Erythema left       right

Erythema left        right

Eryt left

hema right

bw (g)

25

81

25.4

0f

0f

0f

0f

0f

0f

0f

0f

0f

0f

0f

0f

26.5

82

26.1

0f

0f

0f

0f

0f

0f

0f

0f

0f

0f

0f

0f

26.0

50

83

24.7

0f

0f

0f

0f

0f

0f

0f

0f

0f

0f

0f

0f

24.9

84

26.8

0f

0f

0f

0f

0f

0f

0f

0f

0f

0f

0f

0f

25.7

¹ TI = test material (% 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

f Red staining of test material remnants on the dorsal surface of the ears did not hamper scoring of erythema.

 

Pre-Screen Test: Ear Thickness Measurements

		Day 1		Day 3				Day 6
TI ^1 (%)	Animal	Left (mm)	Right (mm)	Left (mm)	%^2	Right (mm)	% ^2	Left (mm)	%^2	Right (mm)	% ^2
25	81	0.215	0.220	0.230	7	0.235	7	0.215	0	0.215	-2
	82	0.210	0.215	0.230	10	0.230	7	0.220	5	0.215	0
50	83	0.220	0.220	0.240	9	0.235	7	0.220	0	0.225	2
	84	0.220	0.210	0.240	9	0.230	10	0.225	2	0.215	2

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

¹ TI = test material (% w/w).

² Percent increase compared to Day 1 pre-dose value. A 25% value is used as the threshold for selection for use in the main study.

 

Main Study: Body Weights and Skin Reactions

 

Group

TI ^1 (%)

Animal

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

bw (g) ^2

Erythema^3 left    right

Erythema left           right

Erythema left           right

Erythema left              right

Erythema left              right

Eryth left

ema right

bw (g)

1

0

1

29.4

0

0

0

0

0

0

0

0

0

0

0

0

28.8

2

25.4

0

0

0

0

0

0

0

0

0

0

0

0

26.3

3

27.1

0

0

0

0

0

0

0

0

0

0

0

0

27.6

4

25.5

0

0

0

0

0

0

0

0

0

0

0

0

26.1

5

24.0

0

0

0

0

0

0

0

0

0

0

0

0

25.2

2

10

6

27.3

0fa

0f

0fa

0f

0fa

0f

0fa

0f

0fa

0f

0fa

0f

27.6

7

26.3

0fa

0f

0fa

0f

0fa

0f

0fa

0f

0fa

0f

0fa

0f

27.4

8

25.3

0fa

0f

0fa

0f

0fa

0f

0fa

0f

0fa

0f

0fa

0f

25.6

9

25.7

0fa

0f

0fa

0f

0fa

0f

0fa

0f

0fa

0f

0fa

0f

25.4

10

28.6

0fa

0f

0fa

0f

0fa

0f

0fa

0f

0fa

0f

0fa

0f

29.6

3

25

11

27.3

0fa

0f

0fa

0f

0fa

0f

0fa

0f

0fa

0f

0fa

0f

27.1

12

24.6

0fa

0f

0fa

0f

0fa

0f

0fa

0f

0fa

0f

0fa

0f

26.8

13

28.8

0fa

0f

0fa

0f

0fa

0f

0fa

0f

0fa

0f

0fa

0f

28.5

14

27.2

0fa

0f

0fa

0f

0fa

0f

0fa

0f

0fa

0f

0fa

0f

27.8

15

26.5

0fa

0f

0fa

0f

0fa

0f

0fa

0f

0fa

0f

0fa

0f

26.6

4

50

16

27.6

0fa

0f

0fa

0f

0fa

0f

NSa

NS

NSa

NS

NSa

NS

30.6

17

24.7

0fa

0f

0fa

0f

0fa

0f

NSa

NS

NSa

NS

NSa

NS

26.9

18

27.0

0fa

0f

0fa

0f

0fa

0f

NSa

NS

NSa

NS

NSa

NS

28.7

19

29.1

0fa

0f

0fa

0f

0fa

0f

NSa

NS

NSa

NS

NSa

NS

27.7

20

25.0

0fa

0f

0fa

0f

0fa

0f

NSa

NS

NSa

NS

NSa

NS

25.6

¹ TI = test material (% 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

NS No scoring possible due to red staining of the ears.

f Red staining of test material remnants on the dorsal surface of the ears did not hamper scoring for erythema. a Red colorization of the skin, fur and/or paws.

 

Main Study: Relative Size Lymph Nodes, Radioactivity Counts (DPM) and Stimulation Index (SI)

			Size Nodes 2
Group	TI^1 %	Animal	Left	Right	DPM^3 / Animal	Mean DPM ± SEM ^4		Mean SI ± SEM
1	0	1	n	n	549	769	± 94	1.0	± 0.1
		2	n	n	758
		3	n	n	576
		4	n	n	945
		5	n	n	1015
2	10	6	n	n	1356	1850	± 242	2.4	± 0.3
		7	n	n	1865
		8	n	n	2360
		9	n	n	1257
		10	n	n	2412
3	25	11	n	n	3304	4211	± 473	5.5	± 0.6
		12	n	n	3387
		13	+	n	5815
		14	n	n	3830
		15	+	n	4718
4	50	16	+	n	6753	10458	± 1384	13.6	± 1.8
		17	+	n	8557
		18	+	+	9864
		19	n	+	12722
		20	+	+	14396

¹ TI = test material (% w/w).

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

³ DPM = Disintegrations per minute.

⁴ SEM = Standard Error of the Mean.

Applicant's summary and conclusion

Interpretation of results:

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

Remarks:

H317: May cause an allergic skin reaction.

Conclusions:

These results show that the test material elicits a SI ≥ 3. The data showed a dose-response and
an EC3 value (the estimated test material concentration that will give a SI =3) of 12.9% was
calculated.
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 (see Appendix 4).
Based on these results:
• According to the recommendations made in the test guidelines (including all
amendments), Red HF2 would be regarded as skin sensitizer.
• According to the Globally Harmonized System of Classification and Labelling of
Chemicals (GHS) of the United Nations (2017) (including all amendments), Red HF2
should be classified as skin sensitizer (Category 1B).
• According to the Regulation (EC) No 1272/2008 on classification, labelling and
packaging of materials and mixtures (including all amendments), Red HF2 should be
classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic
skin reaction.

Executive summary:

The objective of this study was to evaluate whether Red HF2 induces skin sensitization in mice after three epidermal exposures of the animals under the conditions described in the report.
The study was carried out based on the guidelines described in:
• OECD, Section 4, Health Effects, No.429 (2010).
• EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay".
• EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.
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.
Test material concentrations selected for the main study were based on the results of a prescreen test. At a 25% and 50% test material concentration in Acetone/Olive oil (4:1 v/v), no signs of systemic toxicity were noted, and no skin irritation was observed, and therefore a
50% concentration was selected as highest concentration for the main study.
In the main study, three experimental groups of five female CBA/J mice were treated with test material 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 the vehicle alone (Acetone/Olive oil (4:1 v/v)). 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.
All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size, except for one node in two animals at 25% and one or both nodes in all animals at 50%, which were slightly enlarged. 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 10, 25 and 50% were 1850, 4211 and 10458 DPM, respectively. The mean DPM/animal value for the vehicle control group was 769 DPM. The SI values calculated for the test material concentrations 10, 25 and 50% were 2.4, 5.5 and 13.6, respectively.
These results show that the test material elicits a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test material concentration that will give a SI =3) of 12.9% was calculated.
Based on these results:
• According to the recommendations made in the test guidelines (including all amendments), Red HF2 would be regarded as skin sensitizer.
• According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments), Red HF2 should be classified as skin sensitizer (Category 1B).
• According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of materials and mixtures (including all amendments), Red HF2 should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.