reaction product of toluenediisocyanate ( reaction mass of isomers: 65 % 2,4- and 35 % 2,6-diisocyanate) and aniline (molarratio 1:2)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21/03/2018 - 30/04/18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EC No. 640/2012 Part B (Skin Sensitization: Local Lymph Node Assay) July 2012

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:

-

EC Number:

430-960-1

EC Name:

-

Molecular formula:

C21H20N4O2

IUPAC Name:

Reaction mass of aniline and m-tolylidene diisocyanate

Test material form:

solid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks old
- Weight at study initiation: 19.0 - 24.4 g
- Housing: Group housed in groups of 5
- Diet (e.g. ad libitum): Pelleted rodent diet
- Water (e.g. ad libitum): Tap water freely available
- Acclimation period: 5 days
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 41-51
- Air changes (per hr): Ten or greater
- Photoperiod (hrs dark / hrs light): 12 hour light: 12 hour dark

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

0, 2, 5, 10% w/w

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: 10%
- Irritation: Slight erythema
- Systemic toxicity: No signs
- Ear thickness measurements: Variation - 25%

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: SI > 3

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 dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day.

Positive control substance(s):

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

Statistics:

Stimulation index (SI) calculated for each group using individual SI values

Results and discussion

Positive control results:

An EC3 value of 19.2% was calculated using linear interpolation.
The calculated EC3 value was found to be within the acceptable range of 4.8 and 19.5%.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1

Variability:

0.1

Test group / Remarks:

Control

Remarks on result:

other: Mean SI group value used

Results 2

Parameter:

SI

Value:

1.3

Variability:

0.1

Test group / Remarks:

2%

Remarks on result:

other: Mean SI group value used

Results 3

Parameter:

SI

Value:

1.2

Variability:

0.2

Test group / Remarks:

5%

Remarks on result:

other: Mean SI group value used

Results 4

Parameter:

SI

Value:

1.4

Variability:

0.1

Test group / Remarks:

10%

Remarks on result:

other: Mean SI group value used

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION : Individual SI is the ratio of the DPM/animal compared to the DPM/control group mean

CLINICAL OBSERVATIONS: No clinical signs of systemic toxicity

BODY WEIGHTS: Body weights remained in same range

Any other information on results incl. tables

group	TS^1 (%)	animal	Size nodes^2		DPM^3/ animal	mean DPM ± SEM^4			mean SI ± SEM
			left	right
1	0	1	n	n	802	896	±	117	1.0	±	0.1
		2	n	n	717
		3	n	n	1151
		4	n	n	1193
		5	n	n	617
2	2	6	n	n	1232	1158	±	132	1.3	±	0.1
		7	n	n	984
		8	n	n	797
		9	n	n	1586
		10	n	n	1191
3	5	11	n	n	467	1110	±	197	1.2	±	0.2
		12	n	n	1403
		13	n	n	922
		14	n	n	1603
		15	n	n	1154
4	10	16	n	n	1256	1260	±	129	1.4	±	0.1
		17	n	n	1316
		18	n	n	1708
		19	n	n	1068
		20	n	n	954

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Test item was determined not to be a skin sensitiser.

Executive summary:

Three experimental groups of five female CBA/J mice were treated with test item concentrations of 2, 5 and 10% on three consecutive days by open application to the ears. Five vehicle control mice were also treated with the vehicle alone (dimethyl sulfoxide). Three days after the last exposure, all animals were injected with the ³H-methyl thymidine and after five hours the draining lymph nodes were excised and pooled for each animal. After precipitating the DNA of lymph node cells, radioactivity measurements were performed. The SI values calculated for the test item at concentrations of 2, 5 and 10% were 1.3, 1.2 and 1.4 respectively. There was no indication that the test item elicited a SI≥3 when tested up to 10%. Therefore, the test item was determined not to be a skin sensitiser.