N-Phenyl-diethanolamine, reaction products with formaldehyde

Endpoint summary

Description of key information

Skin sensitisation, OECD 429 (LLNA), van Huygevoort (2020): Sensitising (Category 1)

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:

19 August 2020 - 22 September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was conducted in accordance with the relevant OECD test guideline and in accordance with GLP. All validity criteria were met.

Qualifier:

according to guideline

Guideline:

other: EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay"

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France (Inbred, SPF-Quality)
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: Not known
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: 22.3 - 28.9 g
- 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 (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. These housing conditions were maintained unless deemed inappropriate by the Study Director and/or Clinical Veterinarian.
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
- Water (e.g. ad libitum): Municipal tap-water was freely available to each animal via water bottles.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.
- Indication of any skin lesions: None reported. 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Daily mean temperature: 22 - 23°C
- Humidity (%): Daily mean relative humidity: 42 - 69 %
- Air changes (per hr): >= 10, with 100 % fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle
- IN-LIFE DATES: From: 19 August 2020 To: 21 September 2020

For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA), except when interrupted by study procedures/activities.

Vehicle:

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

Remarks:

Source: Merck, Darmstadt, Germany and Acros Organics, Geel, Belgium.

Concentration:

10, 25 and 50 %

Test item concentrations selected for the main study were based on the results of a pre-screen test. At a 25 and 50 % test item concentration, no signs of systemic toxicity were noted and only very slight irritation were observed in all animals between Days 1 and 5. Therefore, a 50 % concentration was selected as highest concentration for the main study.

No. of animals per dose:

5 females per dose

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The acetone/olive oil vehicle was selected on the basis of maximising the solubility based on trial preparations performed at Charles River Den Bosch and on information provided by the Sponsor.
- Irritation: Very slight irritation were observed in all animals between Days 1 and 5
- Systemic toxicity: At 25 and 50 % test item concentration, no signs of systemic toxicity were noted.
- Ear thickness measurements: A maximum percentage increase (compared to Day 1 pre-dose ear thickness values) of 7 % was observed at 25 and 50 % test item concentration
- Erythema scores: A maximum score of 1 (very slight erythema [barely perceptible]) was observed at 25 and 50 % test item concentration

Pre-screen test details:
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 item 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 to the main study except that the animals were approx. 11 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.

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

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 cage during observation, unless necessary for identification or confirmation of possible findings.

Post-dose observations: Post-dose observations were performed once daily on Days 1-6 (on Days 1-3 between 3 and 4 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.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
Distintegrations Per Minute (DPM) values will be presented for each animal and for each dose group. A mean Stimulation Index (SI) will be 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 item should be regarded as a skin sensitizer.
The EC3 value (the estimated item concentration that will give a SI=3) may be determined if possible, based on the dose response relationship or calculated using linear interpolation.
SI ≥ 3 = Category 1 skin sensitiser:
EC3 value ≤ 2%: sub-category 1A
EC3 value > 2%: sub-category 1B
SI < 3 = Not a skin sensitiser

TREATMENT PREPARATION AND ADMINISTRATION:
Test item dosing formulations (w/w) were homogenised to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item.
The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle and test item, and no correction was made for the purity/composition of the test item, since the test method requires a logical concentration range rather than specific dose levels.

INDUCTION (Days 1, 2 & 3):
The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, 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 was administered instead of the test item.
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.
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 (maze size: 200 μm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200 g for 10 minutes at 4 ºC. 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 (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).

Positive control substance(s):

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

Positive control results:

The positive control was not run concurrently with the study, however 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.

A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by Charles River Den Bosch. In this study, performed in May 2020, females of the CBA/J mouse strain (Janvier, Le Genest-Saint- Isle, France) were checked for sensitivity to alpha- hexylcinnamaldehyde, technical grade (HCA). The females were approx. 10 weeks old at commencement of the study. The study was based on the OECD Guideline No. 429, EC No 440/2008, Part B.42 and EPA, OPPTS 870.2600 “Skin Sensitization”. Alpha- Hexylcinnamaldehyde, technical grade (CAS no. 101-86-0) was fabricated under lot no. MKCD3159 (Sigma- Aldrich, Steinheim, Germany).
The SI values calculated for the test item concentrations 5, 10 and 25 % were 2.4, 2.9 and 4.5, respectively. An EC3 value of 10.9 % 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 Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Key result

Parameter:

SI

Value:

1

Variability:

± 0.2

Test group / Remarks:

Vehicle control

Key result

Parameter:

SI

Value:

4.7

Variability:

± 1.0

Test group / Remarks:

10 %

Key result

Parameter:

SI

Value:

13

Variability:

± 2.4

Test group / Remarks:

25 %

Key result

Parameter:

SI

Value:

36.6

Variability:

± 2.9

Test group / Remarks:

50 %

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION: Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50 % were 1987, 5527 and 15600 DPM, respectively. The mean DPM/animal value for the vehicle control group was 426 DPM.

EC3 CALCULATION: The EC3 value (the estimated test item concentration that will give a SI =3) was established to be between > 0 and 10 %.

CLINICAL OBSERVATIONS: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals.
The majority of auricular lymph nodes were considered normal in size, except for the nodes in all animals treated at 50 %, which were slightly enlarged.
No macroscopic abnormalities of the surrounding area were noted for any of the animals.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness): The slight irritation of the ears as shown by all animals treated at 25 and 50 % on Days 1-3 was considered not to have a toxicologically significant effect on the activity of the nodes.

Table 1. Main Study: Relative Size Lymph Nodes, Radioactivity Counts (DPM) and Stimulation Index (SI)

Group	Test item (%)	Animal	Size nodes*		DPM^ / animal	Mean DPM ± SEM^#	Mean SI ± SEM
			Left	Right
1	0	1	n	n	218	426 ± 70	1.0 ± 0.2
		2	n	n	366
		3	n	n	633
		4	n	n	400
		5	n	n	512
2	10	6	n	n	1580	1987 ± 427	4.7 ± 1.0
		7	n	n	1610
		8	n	n	1917
		9	n	n	1196
		10	n	n	3633
3	25	11	n	n	4043	5527 ± 1036	13.0 ± 2.4
		12	n	n	4191
		13	n	n	7327
		14	n	n	8656
		15	n	n	3419
4	50	16	+	+	18864	15600 ± 1255	36.6 ± 2.9
		17	+	+	12628
		18	+	+	17946
		19	+	+	15507
		20	+	+	13053

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

^DPM = Disintegrations per minute

#SEM = Standard error of the mean

 

Table 2. Main Study: Body Weights and Skin Reactions

Group	Test item (%)	Animal	Day 1			Day 2		Day 3		Day 4		Day 5		Day 6
			Body weight (g)	Erythema*		Erythema		Erythema		Erythema		Erythema		Erythema		Body weight (g)
				Left	Right	Left	Right	Left	Right	Left	Right	Left	Right	Left	Right
1	0	1	22.4	0	0	0		0		0		0		0		21.4
		2	28.9	0	0	0		0		0		0		0		28.2
		3	25.3	0	0	0		0		0		0		0		24.4
		4	22.3	0	0	0		0		0		0		0		21.6
		5	23.9	0	0	0		0		0		0		0		23.8
2	10	6	25.5	0	0	0		0		0		0		0		25.8
		7	24.6	0	0	0		0		0		0		0		26.5
		8	24.1	0	0	0		0		0		0		0		23.9
		9	25.8	0	0	0		0		0		0		0		26.9
		10	24.2	0	0	0		0		0		0		0		23.6
3	25	11	26.6	1	1	1		1		0		0		0		27.3
		12	24.1	1	1	1		1		0		0		0		24.3
		13	25.4	1	1	1		1		0		0		0		24.9
		14	24.0	1	1	1		1		0		0		0		25.8
		15	24.5	1	1	1		1		0		0		0		24.5
4	50	16	25.7	1	1	1		1		0		0		0		27.0
		17	24.1	1	1	1		1		0		0		0		25.6
		18	23.1	1	1	1		1		0		0		0		23.9
		19	23.1	1	1	1		1		0		0		0		24.4
		20	23.4	1	1	1		1		0		0		0		25.1

* 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)

 

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on the conditions of this study, the test item was concluded to cause sensitisation to the skin (Stimulation Index > 3) at the concentrations tested (10, 25 and 50 %).

Executive summary:

A study was performed in accordance with OECD 429 (2010), in order to determine the sensitisation potential of the test item to the skin.

Test item concentrations selected for the main study were based on the results of a pre-screen test. At a 25 and 50 % test item concentration, no signs of systemic toxicity were noted and only very slight irritation were observed in all animals between Days 1 and 5. 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 item 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.

A concurrent positive control was not performed, however a 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.

The majority of auricular lymph nodes were considered normal in size, except for the nodes in all animals treated 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 item concentrations 10, 25 and 50 % were 1987, 5527 and 15600 DPM, respectively. The mean DPM/animal value for the vehicle control group was 426 DPM. The SI values calculated for the test item concentrations 10, 25 and 50 % were 4.7, 13.0 and 36.6, respectively. The results showed a dose response and indicates that the test item is able to elicit a SI ≥ 3. The EC3 value (the estimated test item concentration that will give a SI =3) was established to be between > 0 and 10 %. This range does not allow for sub-categorisation to 1A or 1B, therefore Category 1 is concluded.

Based on the SI values (SI = 4.7 at lowest concentration tested: 10 %), the test item would be classified as a Skin Sensitiser Category 1 in accordance with  Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

OECD 429 (LLNA)

A study was performed in accordance with OECD 429 (2010), in order to determine the sensitisation potential of the test item to the skin.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 10, 25 or 50 % w/w (decided based on pre-screen results) 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.

A concurrent positive control was not performed, however a 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.

The majority of auricular lymph nodes were considered normal in size, except for the nodes in all animals treated at 50 %, which were slightly enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals. SI values calculated for the test item concentrations 10, 25 and 50 % were 4.7, 13.0 and 36.6, respectively. The results showed a dose response and indicates that the test item is able to elicit a SI ≥ 3. The EC3 value was established to be between > 0 and 10 %.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The SI values (SI = 4.7 at lowest concentration tested: 10 %) obtained from the OECD 429 results in a classification of Skin Sensitiser Category 1 in accordance with Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures. The results do not allow for sub-categorisation.