Reaction mass of 1,5-naphthylene diisocyanate and Amines, hydrogenated tallow alkyl

Endpoint summary

Description of key information

The skin sensitisation potential of one NDI category substance, Reaction product of 1,5-naphthylene diisocyanate and cyclohexylamine, was assessed. The KeratinoSens^TM assay was inconclusive based on negative results at concentrations with sufficient cell viability, while due to precipitation and cytotoxicity not all concentrations could be tested up to and including 1000 µM (Woutersen 2018). Performance of a U-Sens™ assay was considered to give no additional information, as a negative result will not yield a final conclusion and a positive result may indicate the test item is a skin sensitiser, but still no final conclusion on the potency could be derived. A DPRA assay could not be performed on this substance as it is outside the applicability domain.

Therefore, no conclusion on skin sensitisation properties and potency could be drawn from the in vitro test performed and an LLNA assay, according to the OECD 429 guideline, was commissioned. Mean DPM/animal values for the experimental groups treated with test item concentrations of 5, 10 and 20% were 1245, 938 and 853 DPM, respectively. The mean DPM/animal value for the vehicle control group was 644 DPM. The SI values calculated for the test item concentrations of 5, 10 and 20% were 1.9, 1.5 and 1.3, respectively (van Sas 2019). Since there was no indication that the test item elicits a SI ≥3 when tested up to 20%, the test item was not considered to be a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 January 2018 to 02 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: KeratinoSens Assay

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD 442D).

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switserland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

Cell Culture;
Basic medium; Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
Maintenance medium; Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/mL).
Exposure medium; Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25).

In the main experiments the test item was suspended in dimethyl sulfoxide (DMSO) at 50 mM. From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0, 0.98, 0.49 and 0.24 µM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. In the first experiment precipitation was observed at the start of the incubation period at concentrations of 31 µM and upwards and at the end of the incubation period at concentrations of 125 µM and upwards. In the second experiment precipitation was observed at the start and end of the incubation period at concentrations of 125 µM and upwards.

Environmental conditions
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 74 – 98 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.8 – 36.4 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Experimental Design
For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+12 in experiment 1 and P+7 in experiment 2.

For the treatment of cells, the medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37±1.0°C in the presence of 5% CO2. In total 2 valid experiments were performed.

To measure the Luciferase Activity, the Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).

For the cytotoxicity assessment, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

The following parameters are calculated in the KeratinoSensTM test method:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
• The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

Positive control results:

Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.89 and the EC1.5 22 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.41 and the EC1.5 73 µM.

Run / experiment:

other: other: Experiment 1

Parameter:

other: Imax [442D] (migrated information)

Value:

1.11

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

Precipitation was observed at the start of the incubation period at concentrations of 31 µM and upwards and at the end of the incubation period at concentrations of 125 µM and upwards

Run / experiment:

other: other: Experiment 2

Parameter:

other: Imax [442D] (migrated information)

Value:

1.12

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

Precipitation was observed at the start and end of the incubation period at concentrations of 125 µM and upwards.

Run / experiment:

other: other: Experiment 1 and 2

Parameter:

other: IC30 [442D] (migrated information)

Value:

0.24

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

The test item precipitated at dose levels of 125 µM and upwards in experiment 1 and 2, respectively.

Run / experiment:

other: other: Experiments 1 and 2

Parameter:

other: IC50 [442D] (migrated information)

Value:

0.24

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

The test item precipitated at dose levels of 125 µM and upwards in experiment 1 and 2, respectively.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The test item showed toxicity (IC30 and IC50 values of < 0.24 µM in both experiments). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.11-fold and 1.12-fold in experiment 1 and 2 respectively. The test item is classified as inconclusive in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations < 1000 µM.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (11 % and 8.0 % in experiment 1 and 2, respectively).
- Acceptance criteria met for positive control: The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration. The EC1.5 of the positive control was between 5 and 125 µM (22 µM and 73 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.89- and 2.41-fold in experiment 1 and 2, respectively).
- Acceptance criteria met for variability between replicate measurements: The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (11% and 8.0 6% in experiment 1 and 2, respectively).

Table 1: Overview Luminescence Induction and Cell Viability of Reaction mass of aniline and m-tolylidene diisocyanate in Experiment 1 and 2

Concentration (µM)	0.24	0.49	0.98	2.0	3.9	7.8	16	31	63	125	250	500
Exp. 1 luminescence	0.98	0.85	0.92	1.01	1.05	1.03	1.03	1.07	1.06	1.11	1.00	1.02
Exp. 1 viability (%)	21.9	24.2	27.4	25.4	29.4	27.9	27.9	29.3	34.3	24.9	22.7	30.0
Exp. 2 luminescence	0.88	0.93	1.00	1.01	1.04	1.04	1.09	1.12	1.07	1.06	1.12	1.10
Exp. 2 viability (%)	34.0	37.6	34.9	36.6	34.2	36.3	40.6	39.4	36.7	35.2	35.3	34.4

Table 2: Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (µM)	7.8	16	31	63	125	250
Exp. 1 Luminesence	1.33	1.40	1.65***	1.85***	2.07***	2.89***
Exp. 1 viability (%)	96.7	95.9	104.8	102.1	105.2	107.5
Exp. 2 Luminesence	0.99	1.13	1.25	1.44	1.79***	2.41***
Exp. 2 viability (%)	114.6	107.7	121.6	126.4	133.3	130.3

*** p <0.001 Student's t-test

Table 3: Overview EC_1.5, I_max, IC₃₀ and IC₅₀ Values

	EC1.5 (µM)	Imax	IC30 (µM)	IC50 (µM)
Test Item; Experiment 1	NA	1.11	NA	NA
Test Item; Experiment 2	NA	1.12	NA	NA
Pos. Control; Experiment 1	22	2.89	NA	NA
Pos. Control; Experiment 2	73	2.41	NA	NA

NA = Not applicable

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item showed toxicity in a KeratinoSens assay (IC30 values and IC50 values of < 0.24 µM in both experiments). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.11-fold and 1.12-fold in experiment 1 and 2 respectively. The test item is classified as inconclusive in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations < 1000 µM.

Executive summary:

The skin sensitisation of the test item was assessed in a KerationSens Assay with procedure based on the OECD TG 442D guidance, in two independent experiments in triplicate. The test item was suspended in the vehicle solvent dimethyl sulfoxide (DMSO) prior to dilution with test media and the experiment was performed at final test concentrations of 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0, 0.98, 0.49 and 0.24 µM (final concentration DMSO of 1%).  In the first experiment precipitation was observed at the start of the incubation period at concentrations of 31 µM and upwards and at the end of the incubation period at concentrations of 125 µM and upwards. In the second experiment precipitation was observed at the start and end of the incubation period at concentrations of 125 µM and upwards. Alongside the test item a solvent (negative control) of DMSO and positive control of Ethylene dimethacrylate glycol (EDMG), for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted, so that the final concentration of the positive control ranges from 7.8 to 250 µM (final concentration DMSO of 1%).

The test item showed toxicity in a KeratinoSens assay (IC30 values and IC50 values of < 0.24 µM in both experiments).  No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments.  The maximum luciferase activity induction (Imax) was 1.11-fold and 1.12-fold in experiment 1 and 2 respectively.  The test item is classified as inconclusive in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations < 1000 µM.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 January 2019 to 15 January 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2012

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
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: Young adult animals (approximately 12 weeks old)
- Weight at study initiation: 20.2 to 24.7 g
- Housing: Group housed (5 females) in polycarbonate cages
- Diet: Pelleted rodent diet, provided ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22°C
- Humidity (%): 43 to 58%
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12h light/12h dark

Vehicle:

dimethylformamide

Concentration:

0, 5, 10, 20 % 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: 10 and 20 % w/w
- Method: The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 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 and treated with one concentration on three consecutive days. Animals were group housed in labelled 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.
- Systemic toxicity and erythema scores: At 10% and 20% test item concentrations, none to very slight erythema and no signs of toxicity were noted.
- Ear thickness measurements: Variations in ear thickness during the observation period were more than 25% from Day 1 pre-dose values for one ear of one animal at 20% on Day 3 and for both ears of one animal at 10% on Day 6. Since the results of the ear thickness measurements were not conclusive, 20% was selected as the highest concentration to be used in the main study and ear thickness measurements were added to 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.

- Treatment administration: The dorsal surface of both ears was topically treated (25 μL/ear or an equivalent amount when dosed with a spatula) with the test item, at approximately the same time on each day on Days 1, 2 and 3. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. 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% and 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 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: 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. Post dose 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). Ear thickness measurements were performed for all animals prior to dosing on Days 1 and 3, and on Day 6 using a digital thickness gauge (Kroeplin C110T-K). 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 were presented for each animal and for each dose group. A Stimulation Index (SI) was 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).

Positive control results:

The SI values calculated for the positive control concentrations of 5, 10 and 25% were 1.4, 1.3 and 3.0 respectively. An EC3 value of 24.7% was calculated using linear interpolation. It was noted that the EC3 value was just above the range of the 6 monthly HCA reliability checks of the recent years (14.1, 17.3, 9.8, 17.8, 19.2 and 14.3%) and above the acceptable range of anticipated variation as indicated in the study plan (between 4.8 and 19.5%). This indicates that the test system is appropriate for evaluating the sensitizing potential of test items, however, with a slightly lower sensitivity compared to previous years and compared to the acceptable range. This variation in sensitivity may have an impact on the conclusion of studies showing borderline results.

Parameter:

SI

Value:

1

Variability:

0.2

Test group / Remarks:

0 % w/w

Remarks on result:

other: Mean SI value

Parameter:

SI

Value:

1.9

Variability:

0.4

Test group / Remarks:

5 % w/w

Remarks on result:

other: Mean SI value

Parameter:

SI

Value:

1.5

Variability:

0.3

Test group / Remarks:

10 % w/w

Remarks on result:

other: Mean SI value

Parameter:

SI

Value:

1.3

Variability:

0.1

Test group / Remarks:

20 % w/w

Remarks on result:

other: Mean SI value

Cellular proliferation data / Observations:

- Skin Reactions / Irritation: The very slight erythema of the ears as shown by one animal treated at 5%, four animals treated at 10% and all animals treated at and 20% throughout the observation period 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 two animals treated at 5% and all animals at 10% and 20% throughout the observation period, which did not hamper scoring of the skin reactions.
- Ear Thickness: Variation in ear thickness during the observation period were less than 25% from Day 1 pre-dose values for the majority of animals. The variation slightly exceeded 25% for all animals treated at 10% and two animals treated at 20% on Day 3 and for one animal treated at 5% and one animal treated at 10% on Day 6. As no clear correlation was seen with the DPM values, these findings were considered not to have affected the outcome for these animals.
- 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 vehicle control group and several animals treated at 10% (2 animals) and 20% (3 animals) were considered normal in size. The auricular lymph nodes of all animals treated at 5% and several animals treated at 10% (3 animals) and 20% (2 animals) were considered slightly enlarged. 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 5%, 10% and 20% were 1245, 938 and 853 DPM, respectively. The mean DPM/animal value for the vehicle control group was 644 DPM. The SI values calculated for the test item concentrations 5, 10 and 20% were 1.9, 1.5 and 1.3, respectively.
- Vehicle control: The mean DPM/animal value for the vehicle control group was 644 DPM.

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

Group	TSI (%)^1	Animal	Size nodes^2		DPM^3	Mean DPM ± SEM^4	mean SI ± SEM
			Left	Right
1	0	1 2 3 4 5	n n n n n	n n n n n	890 819 589 302 618	644 ±103	1.0 ±0.2
2	5	6 7 8 9 10	+ + + + +	+ + + + +	745 1038 2234 1115 1094	1245 ±256	1.9 ±0.4
3	10	11 12 13 14 15	+ + n + n	+ + n + n	880 729 1513 428 1139	938 ±184	1.5 ±0.3
4	20	16 17 18 19 20	n + + n n	n + + n n	830 1003 795 928 711	853 ±51	1.3 ±0.1

¹TS = test item (% w/w).

²Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +, ++ or +++: degree of enlargement, n:

considered to be normal).

³DPM= Disintegrations per minute.

⁴SEM = Standard Error of the Mean.

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 20%, the test item was not considered to be a skin sensitizer. It was established that the EC3 value (if any) exceeds 20%. Therefore, the substance is not considered to be a skin sensitizer.

Executive summary:

The skin sensitization of the test item was investigated in a Local Lymph Node Assay following OECD Guideline 429, EPA OPPTS 870.2600 and EC No. 640/2012 Part B. The test concentrations of 5, 10 and 20 % w/w were administered to three groups of five female mice on three consecutive days by topical application to the dorsal surface of each ear. Five vehicle control animals were similarly treated with N,N-dimethylformamide. Three days after test item application 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 and the draining (auricular) lymph node of each ear was excised. 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.

 

The very slight erythema of the ears as shown by one animal treated at 5%, four animals treated at 10% and all animals treated at and 20% throughout the observation period 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 two animals treated at 5% and all animals at 10% and 20% throughout the observation period, which did not hamper scoring of the skin reactions.

 

Variation in ear thickness during the observation period were less than 25% from Day 1 pre-dose values for most animals. The variation slightly exceeded 25% for all animals treated at 10% and two animals treated at 20% on Day 3 and for one animal treated at 5% and one animal treated at 10% on Day 6. As no clear correlation was seen with the DPM values, these findings were considered not to have affected the outcome for these animals.

 

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.

The auricular lymph nodes of all animals treated at 5% and several animals treated at 10% (3 animals) and 20% (2 animals) were considered 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 5%, 10% and 20% were 1245, 938 and 853 DPM, respectively. The mean DPM/animal value for the vehicle control group was 644 DPM. The SI values calculated for the test item concentrations 5, 10 and 20% were 1.9, 1.5 and 1.3, respectively.

There was no indication that the test item elicits a SI ≥3 when tested up to the test item concentration of 20%. It was established that the EC3 value (if any) exceeds 20%. Therefore the substance is not considered to be a skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In the KeratinoSens^TMassay no biologically relevant induction was measured as the test item was cytotoxic. As the test substance could not be tested at concentrations up to 1000 µM due to precipitation and, more important, cytotoxicity, the KeratinoSens^TMassay was determined to be inconclusive according to OECD 442D. Therefore, no conclusion on skin sensitisation properties and potency can be drawn from the in vitro test performed and an LLNA assay, according to the OECD 429 guideline, was commissioned.

Mean DPM/animal values for the experimental groups treated with test item concentrations of 5, 10 and 20%, in the LLNA, were 1245, 938 and 853 DPM, respectively. The mean DPM/animal value for the vehicle control group was 644 DPM. The SI values calculated for the test item concentrations 5, 10 and 20% were 1.9, 1.5 and 1.3, respectively. Since there was no indication that the test item elicits a SI ≥3 when tested up to 20%, Reaction product of 1,5-naphthylene diisocyanate and cyclohexylamine was not considered to be a skin sensitizer.

Justification for classification or non-classification

Based on the currently available in vitro data, no assessment of classification can be made, as the in vitro results were inconclusive. Therefore, an LLNA was performed to assess the classification requirement of the NDI category substances; and this concluded that Reaction product of 1,5-naphthylene diisocyanate and cyclohexylamine is not a skin sensitiser and therefore does not require classification for skin sensitisation. This conclusion is considered to be appropriate for all NDI category substances.