Reaction product of M-Tolylidendiisocyanat (26471-62-5), p-Toluidine (106-49-0), and n-Octylamine (111-86-4)

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:

2018

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:

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of m-tolylidene diisocyanate and cyclohexylamine and cyclohex-1,2-ylenediamine and (Z)-octadec-9-enylamine

EC Number:

945-075-9

Molecular formula:

Mixture of C47H74N8O4 and C34H58N4O2

IUPAC Name:

Reaction product of m-tolylidene diisocyanate and cyclohexylamine and cyclohex-1,2-ylenediamine and (Z)-octadec-9-enylamine

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
- Microbiological status of animals, when known: SPF
- Age at study initiation: Young adult animals (approximately 10 weeks old)
- Weight at study initiation: 20.1 to 24.7 g
- Housing: Group housed (5 females) in polycarbonate cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24, daily mean temperature of 22°C
- Humidity (%): 42 to 61
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 1, 2, 5 % w/w

No. of animals per dose:

5 females

Details on study design:

- Vehicle choice: The vehicle, N,N-dimethylformamide, was selected on the basis of maximizing the solubility based on trial preparations performed at the laboratory.

PRE-SCREEN TESTS:
- Concentrations: 5, 10, 25 and 40 % w/w
- Method: 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 females per concentration were treated 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 prior to dosing and on Days 1 and 3, and 6.
- Ear thickness measurements, systemic toxicity and irritation: At a 40%, 25% and 10% test item concentration, variation in ear thickness during the observation period were more than 25% from Day 1 pre-dose values and/or clinical signs of systemic toxicity were noted. Therefore these concentrations did not meet the selection criteria. At a 5% test item concentration, no signs of systemic toxicity were noted and no irritation was observed and therefore this concentration was selected as highest concentration for the main study.

MAIN STUDY
- Name of test method: LLNA
- Criteria used to consider a positive response: SI > 3
- Treatment preparation: The dosing formulations were prepared daily, kept at room temperature and dosed within 4 hours after adding the vehicle to the test item. The concentrations were stirred with a magnetic stirrer immidiately prior to dosing.
- Treatment administration: The dorsal surface of both ears was topically treated with 25 microlitres/ear with the test item, at approximately the same time on each day on Days 1, 2 and 3. On Day 6, each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline containing 20 μCi of 3H-methyl thymidine. After five hours, all animals were euthanized by intraperitoneal injection (0.2 mL/animal) of Euthasol 20%. The draining (auricular) lymph node of each ear was excised and 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 and a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze. LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. The LNC were exposed to 5% trichloroacetic acid (TCA) and stored in the refrigerator until the next day. On Day 7, precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter, with counting time to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first.
- Observations: Animals were observed for general health/mortality and moribundity twice daily. Postdose observations were performed once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing) for reaction to dosing. Animals were weighed individually on Day 1 (predose) and 6 (prior to necropsy). Erythema and eschar formation observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing). No necropsy was performed, since all animals survived until the end of the observation period.

Positive control substance(s):

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

Statistics:

Disintegrations Per Minute (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 item may be regarded as a skin sensitizer. Consideration is also given to the EC3 value (the estimated test item concentration that will give a SI =3).

Results and discussion

Positive control results:

The SI values calculated for the item concentrations 5, 10 and 25% were 1.1, 2.0 and 5.5 respectively. An EC3 value of 14.3% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 13.4, 14.1, 17.3, 9.8, 17.8 and 19.2%.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1

Variability:

0.2

Test group / Remarks:

0 % w/w

Remarks on result:

other:

Remarks:

Mean SI value

Results 2

Parameter:

SI

Value:

1.9

Variability:

0.2

Test group / Remarks:

1 % w/w

Remarks on result:

other:

Remarks:

Mean SI value

Results 3

Parameter:

SI

Value:

0.6

Variability:

0.1

Test group / Remarks:

2 % w/w

Remarks on result:

other:

Remarks:

Mean SI value

Results 4

Parameter:

SI

Value:

0.5

Variability:

0.1

Test group / Remarks:

5 % w/w

Remarks on result:

other:

Remarks:

Mean SI value

Cellular proliferation data / Observations:

- Skin Reactions / Irritation: The very slight erythema of the ears as shown by the animals of the highest dose group was considered not to have a toxicologically significant effect on the activity of the nodes. White test item remnants were present on the dorsal surface of the ears of test item treated animals between Days 2 and 5, which did not hamper scoring of the skin reactions.
- Systemic Toxicity: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals. 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. No macroscopic abnormalities of the surrounding area were noted for any of the animals.
- Radioactivity Measurements and SI Values: Mean DPM/animal values for the experimental groups treated with test item concentrations 1, 2 and 5% were 343, 103 and 82 DPM, respectively. The SI values calculated for the test item concentrations 1, 2 and 5% were 1.9, 0.6 and 0.5, respectively.
- Vehicle control: The mean DPM/animal value for the vehicle control group was 178 DPM.
- Conclusion: Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 5%, the substance was not considered to be a skin sensitizer. It was established that the EC3 value exceeds 5% and, based on the absence of a dose response, it is not to be expected that higher concentrations would lead to an SI ≥ 3. Based on these results, the substance would not be regarded as a skin sensitizer.

Any other information on results incl. tables

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

group	TS^1 (%)	animal	Size nodes^2		DPM^3/ animal	mean DPM ± SEM^4			mean SI ± SEM
			left	right
1	0	1	n	n	137	178	±	29	1.0	±	0.2
		2	n	n	230
		3	n	n	153
		4	n	n	109
		5	n	n	261
2	2	6	n	n	352	343	±	37	1.9	±	0.2
		7	n	n	305
		8	n	n	315
		9	n	n	479
		10	n	n	262
3	5	11	n	n	80	103	±	20	0.6	±	0.1
		12	n	n	82
		13	n	n	83
		14	n	n	182
		15	n	n	87
4	10	16	n	n	107	82	±	21	0.5	±	0.1
		17	n	n	26
		18	n	n	111
		19	n	n	37
		20	n	n	129

¹  TS = test item (% 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:

GHS criteria not met

Conclusions:

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 5%, the substance was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 5%.

Executive summary:

The substance was administered to three experimental groups of five female mice at concentrations of 0 (vehicle control), 1, 2 or 5% w/w on three consecutive days, by open application on the ears. 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. No macroscopic abnormalities of the surrounding area were noted for any of the animals. There was no indication that the test item elicits a SI ≥ 3 when tested up to 5% and, based on the absence of a dose response, it is not to be expected that higher concentrations would lead to an SI ≥ 3, so it was established that the EC3 value exceeds 5%. Therefore, the substance was not considered to be a skin sensitizer.