4,4'-methylenebis[N-sec-butylaniline]

Endpoint summary

Description of key information

A DEREK assessment, DPRA assay, KeratinoSens^TM assay and a U-Sens^TM assay were performed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a.

DEREK NEXUS did not yield any alerts for skin sensitization of the test item. This is in line with the negative outcome of a DPRA assay, which showed that 4,4’-Methylenebis-N-sec-butylaniline is not expected to bind to protein moieties in vivo. The positive outcome of the KeratinoSens^TM assay indicated that 4,4’-methylenebis-N-sec-butylaniline is able to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes. The outcome of a U-sens™ assay was classified as positive, but this conclusion is based on interference (cytoxicity and colour) in the two experiments and it is not clear if the data indicate activation of dendritic cells. As these data preclude a final conclusion on skin sensitization potential and potency, it was considered scientifically justified to proceed with an in vivo study. A local lymph node assay was performed according to OECD 429 and following GLP principles. Based on the available data, 4,4’-Methylenebis-N-sec-butylaniline is classified as skin sensitizer (Category 1B) according to CLP Regulation (EC) No. 1272/2008.

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:

4 Aug 2018 - 8 Sept 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The CBA/J mouse was chosen as the animal model for this study as recognized by international guidelines as a recommended test system (e.g. OECD, FDA, MHW) to assess skin sensitization.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Remarks:

Inbred, SPF-Quality

Sex:

female

Details on test animals and environmental conditions:

- Source:Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Young adult animals (approx. 9 weeks old)
- Weight at study initiation of dosing: 19.2 to 23.0 g.
- Housing: Animals were group housed in labeled Makrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 44 - 75
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12/12
- Psychological/environmental enrichment: paper and shelters except when interrupted by study procedures/activities.

IN-LIFE DATES: From: 24 Aug 2018 - 17 Sept 2018

Vehicle:

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

No. of animals per dose:

5

Details on study design:

RANGE FINDING TEST
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. Two test item concentrations were tested; a 50% and 100% concentration. Based on the results of the initially treated animals, two additional animals were treated in a similar manner with two lower concentrations (10% and 25%) at a later stage.
The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 10-11 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed.
Two young adult animals (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 prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- 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
- Criteria used to consider a positive response: 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 SI is the ratio of the DPM/animal compared to DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION
Test substance preparation:
Test item dosing formulations (w/w) were homogenized 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 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.
Rationale for vehicle: The vehicle was selected based on trial formulations performed at Charles River and on test substance data supplied by the sponsor. The vehicle was chosen from the vehicles specified in the test guideline.

ADMINISTRATION
Induction- days 1, 2 and 3:The dorsal surface of both ears was topically treated (25 μL/ear) with the test item
Excision of nodes - 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 were euthanized and the draining limph node was excised.
Tissue processing for radioacitivity - day 6: A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze, washed and frozen until the next day.
Radioactivity measurements - day 7: Radioactivity measurements were performed using a Packard scintillation counter (2910TR)

OBSERVATIONS
Mortality/Viability: Twice daily.
Body weights: On day 1 (pre-dose) and day 6 (prior to necropsy).
Clinical signs: Once daily on days 1-6 (on days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on days 1-6 (on days 1 - 3 within 1 hour after dosing) according to a numerical scoring system (see table 1 below). Furthermore, a description of all other (local) effects was recorded according to guidelines.

TERMINAL PROCEDURES
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)

Positive control results:

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.

Key result

Parameter:

SI

Value:

1.5

Test group / Remarks:

5%

Key result

Parameter:

SI

Value:

1.8

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

3.4

Test group / Remarks:

25%

Cellular proliferation data / Observations:

- Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 437, 530 and 1010 DPM, respectively.
- The mean DPM/animal value for the vehicle control group was 299 DPM.
- The SI values calculated for the test item concentrations 5, 10 and 25% were 1.5, 1.8 and 3.4, respectively.

Results  Pre-screen Test

At a 100% and 50% 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 25% and 10% test item concentration, no signs of systemic toxicity were noted and no to very slight irritation was observed and therefore the 25% concentration was selected as highest concentration for the main study.

Skin Reactions / Irritation

The very slight erythema of the ears as shown by three animals treated at 25% on Days 2 or 3 was considered not to have a toxicologically significant effect on the activity of the nodes. 

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 of the auricular lymph nodes were considered normal in size, except for the nodes in one animal treated at 25%, which were considered to be enlarged. 

No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

A LLNA test was performed for 4,4’-methylenebis-N-sec-butylaniline. The test substance could elicit a SI ≥ 3. The data showed a dose response and an EC3 value of 21% was calculated. The test substance has been found to be skin sensitizer and has to be classified as skin sensitizer category 1B according to the GHS, and according to the Regulation (EC) No 1272/2008 should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.

Executive summary:

An LLNA skin sensitization study was performed according to OECD/EC test guidelines and GLP principles. Based on the results of a pre-screen test, the test concentrations were selected at 5, 10 or 25% w/w. The very slight erythema of the ears as shown by three animals treated at 25% on Days 2 or 3 was considered not to have a toxicologically significant effect on the activity of the nodes. All of the auricular lymph nodes were considered normal in size, except for the nodes in one animal treated at 25%, which were considered to be 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 25% were 437, 530 and 1010 DPM, respectively. The mean DPM/animal value for the vehicle control group was 299 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 1.5, 1.8 and 3.4, respectively. Reliable negative controls included, and the six-month reliability check with a positive control  demonstrate the reliability of the model for contact hypersensitivity.

Since 4,4’-methylenebis-N-sec-butylaniline could elicit a SI ≥ 3 and according to the recommendations made in the test guidelines (including all amendments), 4,4’-methylenebis-N-sec-butylaniline would be regarded as skin sensitizer and because the EC3 value is > 2%, it has to be classified as skin sensitizer category 1B according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments). Moreover, according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), 4,4’-methylenebis-N-sec-butylaniline should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.

Endpoint conclusion

Additional information:

No data were available that would preclude performance of the studies to determine the potential for skin sensitization. Therefore, STEP 1 studies were performed, i.e. DEREK assessment; DPRA assay and KeratinoSens^TM assay. As no conclusion on skin sensitising properties could be derived based on this data-set, a second in vitro study, the U-Sens^TM assay was performed.

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for the test item. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. 4,4'-Methylenebis-N-sec-butylaniline is predicted to be not sensitizing to the skin.

A valid DPRA test was performed according to OECD 442C and GLP. For the DPRA assay 4,4'-methylenebis-N-sec-butylaniline was dissolved in isopropanol at 100 mM and dissolved completely. Upon preparation as well as after incubation a precipitate was observed in the co-elution control (CC) and test item samples. No co-elution of the test item with synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was observed. In the cysteine reactivity assay the test item showed 0.4% SPCC depletion while in the lysine reactivity assay the test item showed 0.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 0.2% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed upon addition of the test item to the SPCC and SPCL peptide solutions, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with caution.

A valid KeratinoSens^TM assay was performed according to OECD 442D and GLP. For the KeratinoSens^TM assay 4,4’-methylenebis-N-sec-butylaniline was dissolved in DMSO to a final concentration of 200 mM. The final test concentration ranged from 0.98 – 2000 μM (2-fold dilution series). Two independent experiments were performed. No precipitate was observed at any dose level tested in the first experiment. The test item precipitated at dose levels of 500 mM and upwards in the second experiment. 4,4’-Methylenebis-N-sec-butylaniline showed toxicity (IC30 values of 24 μM and 28 μM and IC50 values of 27 μM and 30 μM in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 4.5 μM and 2.7 μM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 2.71-fold and 4.96-fold in experiment 1 and 2 respectively. The test item is classified as positive in the KeratinoSens^TM assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 μM with a cell viability of >70% compared to the vehicle control.

Subsequently a U-Sens™ assay was performed according to OECD 442E. For this assay, 4,4’-methylenebis-N-sec-butylaniline was dissolved in dimethyl sulfoxide at 50 mg/mL. Two independent experiments were performed. Final test concentrations were between 1- 200 μg/mL in the first experiment and in the second experiment, a narrower dose-response analysis up to 30 µg/mL was performed to investigate the increase in expression of experiment 1 in more detail. No precipitate was observed at any dose level tested. 4,4’-Methylenebis-N-sec-butylaniline showed toxicity (CV₇₀ values of 13 µg/mL in both experiment 1 and 2). In the first and second experiment, induction of the CD86 activity (EC₁₅₀ values of 11 µg/mL and 19 µg/mL in experiment 1 and 2, respectively) was only measured at test concentration with a cell viability < 70%. However, both cytotoxicity and colour interference were observed in both experiments, leading to an individual run conclusion of positive of each experiment. Therefore, 4,4’-methylenebis-N-sec-butylaniline is classified as positive in the U-Sens™ assay since interference (cytoxicity and colour) is observed in both experiments.

In summary, DEREK NEXUS did not yield any alerts for skin sensitization of the test item. This is in line with the negative outcome of a DPRA assay, which showed that 4,4’-Methylenebis-N-sec-butylaniline is not expected to bind to protein moieties in vivo. This implies that key event 1 of the skin sensitizing pathway is not expected to occur. However, the positive outcome of a KeratinoSens^TM assay indicates that 4,4’-methylenebis-N-sec-butylaniline is able to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes (key event 2). As a second in vitro test a U-Sens™ assay is available. The outcome was classified as positive, but this conclusion is based on interference (cytoxicity and colour) in the two experiments and it is not clear if the data indicate activation of dendritic cells (key event 3) as induction was only visible at or near a cytotoxic concentration. Based on these data no conclusion can be drawn on skin sensitizing properties of 4,4’-methylenebis-N-sec-butylaniline. As the current data-set precludes final conclusion on the potency and consequently sub-classification of the substance (skin sensitising 1A or 1B), it was considered scientifically justified to proceed with in vivo testing.

An LLNA skin sensitization study was performed according to OECD/EC test guidelines and GLP principles. Based on the results of a pre-screen test, the test concentrations were selected at 5, 10 or 25% w/w. The very slight erythema of the ears as shown by three animals treated at 25% on Days 2 or 3 was considered not to have a toxicologically significant effect on the activity of the nodes. All of the auricular lymph nodes were considered normal in size, except for the nodes in one animal treated at 25%, which were considered to be 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 25% were 437, 530 and 1010 DPM, respectively. The mean DPM/animal value for the vehicle control group was 299 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 1.5, 1.8 and 3.4, respectively. Reliable negative controls included, and the six-month reliability check with a positive control demonstrate the reliability of the model for contact hypersensitivity. Since 4,4’-methylenebis-N-sec-butylaniline could elicit a SI ≥ 3 and according to the recommendations made in the test guidelines (including all amendments), 4,4’-methylenebis-N-sec-butylaniline would be regarded as skin sensitizer and because the EC3 value is > 2%, it has to be classified as skin sensitizer category 1B according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments). Moreover, according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), 4,4’-methylenebis-N-sec-butylaniline should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, 4,4’-Methylenebis-N-sec-butylaniline is classified as skin sensitizer (Category 1B) according to CLP Regulation (EC) No. 1272/2008.