N,N,N',N',N'',N''-hexamethyl-4,4',4''-methylidynetrianiline

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See additional information on materials and methods

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

yes

Remarks:

See additional information on materials and methods

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See additional information on materials and methods

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

N,N,N',N',N'',N''-hexamethyl-4,4',4''-methylidynetrianiline

EC Number:

210-043-9

EC Name:

N,N,N',N',N'',N''-hexamethyl-4,4',4''-methylidynetrianiline

Cas Number:

603-48-5

Molecular formula:

C25H31N3

IUPAC Name:

4-{bis[4-(dimethylamino)phenyl]methyl}-N,N-dimethylaniline

Test material form:

solid: particulate/powder

Details on test material:

- Purity: 99.4%

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Inc., Fredrick, MD
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Preliminary test: 10 weeks old and Main test: 9-11 weeks old
- Weight at study initiation: 18.0-22.8 g
- Housing: The animals were individually housed in plastic solid bottom cages during the dosing and resting phase of the study. After final weighing until sacrifice, animals were housed in their respective dose groups in plastic cages with bedding. Bedding in the plastic, solid bottom cages was changed at least once per week. Enrichment (e.g., nesting material) was placed in each cage.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7-21 days
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-28ºC
- Humidity (%): 45-63%
- Air changes (per hr): 12 or 13
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Study design: in vivo (LLNA)

Vehicle:

other: 1% Pluronic® L92

Concentration:

5, 10, and 25%

No. of animals per dose:

Preliminary irritation study: 2 per group
LLNA study: 5 per group

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The test substance, as received, was a solid. Solubility testing conducted by PSL indicated that the test substance was suspendable in 1% Pluronic® L92
- Irritation: No irritation at concentrations up to 25%
- Systemic toxicity: No signs of toxicity were observed
- Ear thickness measurements: Not examined
- Erythema scores: No irritation was observed

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: Any test substance that produces an SI > 3 in the LLNA is normally considered “positive” for dermal sensitization potential

TREATMENT PREPARATION AND ADMINISTRATION: Concentrations of 5%, 10% and 25% were selected for the main test based on results of the preliminary screening test. Dilutions of the test substance were prepared as w/w mixtures in 1% Pluronic® L92. The vehicle control, 1% Pluronic® L92, and a single concentration of a 25% w/w mixture of HCA in 1% Pluronic® L92 were also prepared. All dosage preparations were freshly prepared on the day of application. Beginning on Day 1, a quantity of 25 µL of the appropriate test substance concentration, the positive control substance, or the vehicle alone was applied to the dorsum of both ears of each mouse once per day for three consecutive days (Days 1, 2, and 3) using a micropipette. During application, the material was gently spread as evenly as possible over the dorsal surface of the ear using the tip of the pipette.

Positive control substance(s):

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

Statistics:

Significance was judged at p < 0.05. The treated groups and negative vehicle control group were compared using a One-Way Analysis of Variance (ANOVA), followed by comparison of the treated groups to control by Dunnett’s t-test for multiple comparisons (INSTAT Biostatistics, Graph Pad Software, San Diego, CA). Outlier analysis was conducted using methods described in Grubbs (1969). Procedures for Detecting Outlying Observations in Samples. Technometrics, 11(1):1-21.

Results and discussion

Positive control results:

Very slight to well defined erythema (scores of 1 or 2) were evident at seven positive control sites on Day 2 and at all sites on Days 3 and 6. Slight oedema (score of 1) was present at three sites on Day 3 and nine sites Day 6. Desquamation was present at five sites on Day 3 and all sites on Day 6. The SI for the positive control was 6.04.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.15

Test group / Remarks:

5% test substance

Results 2

Key result

Parameter:

SI

Value:

0.96

Test group / Remarks:

10% test substance

Results 3

Key result

Parameter:

SI

Value:

1.45

Test group / Remarks:

25% test substance

Cellular proliferation data / Observations:

No dermal irritation was observed for any of the vehicle or test substance group sites.

Any other information on results incl. tables

TABLE 1: INDIVIDUAL DPM[1]VALUES

 

Group	Animal #	dpm	dpm minus background[2]	Group mean dpm	Std. Dev.	SI[3]	SI ≥ 3
Vehicle control	3601 3602 3603 3604 3605	3974.42 2690.29 2069.04 2458.30 2776.09	3922.30 2638.17 2016.92 2406.18 2723.97	2741.51	714.62	-----	-----
Positive Control	3606 3607 3608 3609 3610	16646.44 17782.60 13550.30 17385.71 17750.46	16594.32 17730.48 13498.18* 17333.59 17698.34	16570.98	1777.61	6.04	Yes
5% TS	3611 3612 3613 3614 3615	4759.20 4497.14 1457.88 3515.77 1801.68	4707.08 4445.02 1405.76 3463.65 1749.56	3154.21	1516.86	1.15	No
10% TS	3616 3617 3618 3619 3620	4228.21 1717.77 1140.26 3459.57 2887.66	4176.09 1665.65 1088.14 3407.45 2835.54	2634.57	1259.39	0.96	No
25% TS	3621 3622 3633 3634 3635	3953.37 4967.70 4547.26 2226.90 4389.58	3901.25 4915.58 4495.14 2174.78 4337.46	3964.84	1064.46	1.45	No

 

*The dpm value for Animal No. 3608 was determined to be an outlier, but was included in the average and the standard deviation calculation for the test group. There is no indication in the raw data that this animal was dosed incorrectly; therefore, this animal will not be excluded from the calculations.

 

TABLE 2: STIMULATION INDEX

 

Group	Group mean dpm	SI	Sensitization
Vehicle control	2741.51	-----	Not applicable
Positive control	16570.98**	6.04	Positive – valid study
5% Test substance	3154.21	1.15	Not a sensitizer
10% Test substance	2634.57	0.96	Not a sensitizer
25% Test substance	3964.84	1.45	Not a sensitizer

 

** Statistically significant difference from vehicle control at p < 0.01 by Dunnett’s Multiple Comparisons Test

[1]Disintegrations per minute

[2]Values analysed for outliers, Grubbs 1969 

[3]Stimulation Index = Average dpm of Test Substance/Average dpm of Vehicle

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was not a skin sensitizer when tested in the LLNA.

Executive summary:

A dermal sensitization test was conducted with mice to determine the potential for the test substance to produce sensitization after repeated topical applications (OECD Guideline 429, U.S. EPA Health Effects Test Guidelines, OPPTS 870.2600, and Official Journal of the European Communities. Methods for the Determination of Toxicity and Health Effects, Part B.42 (Skin Sensitization: Local Lymph Node Assay) Commission Regulation (EC) No. 440/2008).

Three concentrations of the test substance (5%, 10% and 25%) in 1% Pluronic® L92 Surfactant w/w in distilled water and the vehicle alone were topically applied to twenty healthy test mice (5 mice/group) for three consecutive days. Three days after the last application, the mice were given an IV injection containing 20 μCi of 3H-methyl thymidine. Approximately five hours later, all animals were euthanized via an overdose of inhaled Isoflurane and the draining (auricular) lymph nodes were harvested and prepared for analysis in a scintillation counter. The results are presented in disintegrations per minute per mouse (dpm/mouse). Each animal’s ears were also evaluated for erythema and oedema prior to each application and again on Day 6, prior to the IV injection. A positive control group (five animals) was maintained under the same environmental conditions and treated with a 25% w/w mixture of alpha-Hexylcinnamaldehyde, ≥ 95% (HCA) in 1% Pluronic® L92 in the same manner as the test animals.

Based on the results of this study, the test substance is not considered to be a contact dermal sensitizer at concentrations less than or equal to 25% in the LLNA. Proper conduct of the LLNA was confirmed via a positive response with 25% alpha-Hexyl-cinnamaldehyde, ≥ 95% (HCA), a moderate contact sensitizer.