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Endpoint summary

Description of key information

In a first instance, in vitro testing was considered to address the potential of the Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid to be sensitising to the skin.

Considering that the substance is an UVCB, it is not technically feasible to conduct a study in accordance with the OECD Testing Guideline 442C.

Testing conducted in accordance with the OECD Testing Guideline 442D returned a negative result.

Although the study performed in accordance with the OECD Testing Guideline 442E provided a negative result, it was not technically feasible to conduct the test above 100 µg/mL. This study shall therefore be considered as inconclusive.

Consequently, only one in vitro study returned a conclusive result, which is not sufficient to reach a conclusion on the skin sensitisation potential of the registered substance, and in vivo testing had to be performed.

The guinea pig Maximization test (GPMT) was selected since the Local Lymph Node Assay (LLNA) has shown to provide false positive results for fatty acids, such as 12-hydroxystearic acid. This testing returned a positive result.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Remarks:

Guinea-Pig Maximization Test (GPMT)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

26 Apr 2021 - 09 Jul 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: JMAFF Guidelines (2000), including the most recent revisions.

Version / remarks:

2000

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The Dunkin Hartley guinea pig was chosen as the animal model for this study as recognized by international guidelines as a recommended test system (e.g. OECD, FDA, MHLW). The test method and number of animals are based on the test guidelines. The guinea pig Maximization test was selected since the Local Lymph Node Assay has shown to provide false positive results for fatty acids, such as 12-HYDROXYSTEARIC ACID (12-HAS ).

Specific details on test material used for the study:

Physical Description: White powder
Purity/Composition: UVCB, no correction factor required
Storage Conditions: Keep tightly closed, in a dry and cool place and protected from heat and ignition sources

Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.

The dosing formulations were kept at room temperature until dosing. The dosing formulations and vehicle were stirred until and during dosing.

No adjustment was made for specific gravity of the vehicle.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’Arbresle, France
- Females: nulliparous and non-pregnant: yes
- Age at study initiation: Young adult animals (approximately 4-5 weeks old)
- Weight at study initiation: 275 to 303g
- Housing: Group housed (up to 5 animals of the same sex and same dosing group together) in Noryl cages (Tecniplast; 74 cm x 54 cm x 25 cm height) containing sterilized wooden fibers as bedding material equipped with water bottles. For psychological/environmental enrichment, animals were provided with shelters.
- Diet: Complete maintenance diet for guinea pigs (MS-H, SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum, except during designated procedures. In addition, hay was provided daily
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 23
- Humidity (%): 44 to 75*
- Air changes (per hr): Ten or greater with 100% fresh air (no air recirculation).
- Photoperiod (hrs dark / hrs light): 12/12

- IN-LIFE DATES: From: 29 April 2021 To: 09 July 2021

*The values that were outside the targeted mean humidity range (40-70%) occurred for three days and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study.

Route:

intradermal and epicutaneous

Vehicle:

corn oil

Concentration / amount:

20% for the intradermal induction and 40% for the epidermal induction.

Day(s)/duration:

Intradermal induction: 7 days. Epidermal induction: 48 hours.

Adequacy of induction:

highest technically applicable concentration used

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Concentration / amount:

5% for the challenge phase.

Day(s)/duration:

24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Test animals: 10
Control animals: 5

Details on study design:

RANGE FINDING TESTS:
Series of test item concentrations were tested. Practical feasibility of administration determined the highest starting concentration for each route. The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 20%, 10%, 5%, 2%, 1%.
The test system and procedures were identical to those used during the main study, unless otherwise specified. The four animals selected were between 4 and 9 weeks old. No body weights were determined.

Intradermal injections:
A series of four test item concentrations was tested, the highest concentration being the maximum concentration that could technically be injected. Two animals received two different concentrations in duplicate (0.1 mL/site) in the clipped scapular region. The injection sites were assessed for irritation 24 and 48 hours after treatment.

Epidermal application:
A series of four test item concentrations was tested, the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each or an equivalent amount when dosed with a spatula) per animal to the clipped flank, using Metalline patches (2x3 cm) mounted on Medical tape which were held in place with Micropore tape and subsequently Coban elastic bandage.
The animals receiving intradermal injections were treated with the lowest concentrations and two other animals with the highest concentrations.
After 24 hours, the dressing was removed and the skin cleaned of residual test item using water.
The treated skin areas were assessed for irritation 24 and 48 hours after removal of the dressings.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1
1) Intradermal injections on day 1:
- Site: scapular region. One of each pair was on each side of the midline and from cranial to caudal:
Three pairs of intradermal injections:
1) 0.1 mL: FCA (50% in water for injection)
2) 0.1 mL: test substance at a 20% concentration (control animals: 0.1 mL corn oil)
3) 0.1 mL: 1:1 mixture of the test substance at a 40% concentration + FCA (undiluted)
- Readings: on day 3 (48 hrs after the injections)

2) Topical application on day 8:
- Amount: 0.5 mL 40% test substance (control animals: 0.5 mL corn oil)
- Area: approximately 6 cm^2
- Exposure period: 48 hours (occlusive)
- Readings: scores were rated directly after patch removal

B. CHALLENGE EXPOSURE (all animals, with the 5% test substance and the vehicle)
- Day of challenge: day 22
- Exposure period: 24 hours (occlusive)
- Site: flank
- Amount: 0.1 mL
- Readings: scores were rated 24 and 48 hours after patch removal

OBSERVATIONS
Mortality/Moribundity Checks: 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.
Toxicity: At least once daily.
Body weights: Animals were weighed individually on Day 1 (pre-dose) and after termination of the study
Necropsy: No necropsy was performed.
Irritation: "please refer to section "Any other information on materials and methods incl. tables" for details on scoring system."

Challenge controls:

Not applicable.

Positive control substance(s):

yes

Remarks:

The results of the latest reliability check, performed in March/April 2021 with Alpha-Hexylcinnamaldehyde, are reported. It was concluded that the female guinea pig of the Dunkin Hartley strain is an appropriate animal model for the performance of studies

Positive control results:

The latest reliability check shows a sensitisation rate of 80%.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

5%

No. with + reactions:

3

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

vehicle

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

5%

No. with + reactions:

3

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

vehicle

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

50% Alpha- Hexylcinnamaldehyde, technical grade

No. with + reactions:

8

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

50% Alpha- Hexylcinnamaldehyde, technical grade

No. with + reactions:

7

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

- Signs of irritation during induction:

Slight to moderate erythema was observed during the intradermal induction (conc. 20%) at the injection sites in control and test animals. Scabbing was observed in animals which received an intra-dermal injection of the test item.

Moderate erythema was observed following the epidermal induction (conc. 40%) in all 10 test animals. After epidermal exposure no skin reactions were evident in the control animals.

- Evidence of sensitisation of the challenge concentration:

Skin reactions (erythema) of grade 1, were observed in three experimental animals in response to the 5% test item concentration. No skin reactions were evident in the control animals.

- Toxicity / Mortality:

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

- Body Weights:

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

Category 1B according to Regulation (EC) No 1272/2008 (CLP).

Conclusions:

In a guinea pig maximisation test method, the potential of the substance for skin sensitisation was tested according to OECD 406 guideline and in accordance with GLP principles, showing a sensitization rate of 30 percent.

Executive summary:

In a guinea pig maximisation test method, the potential of the substance for skin sensitisation was tested according to OECD 406 guideline and in accordance with GLP principles. The capability of the test item to induce contact hypersensitivity was evaluated in females of guinea pig, Dunkin Hartley strain. Ten animals were exposed to the test item, at concentration of 20% for the intradermal induction and 40% for the epidermal induction. The control group consisted of 5 animals exposed to the vehicle, corn oil, during the induction phase. At challenge, 5% of the test item in corn oil was applied on the clipped skin of all the animals. Skin reactions were evaluated 24 and 48 hours after treatment. Slight to moderate erythema was observed during the intradermal induction (conc. 20%) at the injection sites in control and test animals. Scabbing was observed in animals exposed to the test item during the induction phase. Moderate erythema was observed following the epidermal induction (conc. 40%) in all 10 test animals. After epidermal exposure no skin reactions were evident in the control animals. No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

In the challenge phase, skin reactions of grade 1 were observed in experimental animals in response to the 5% test item concentration. No skin reactions were evident in the control animals.

The skin reactions observed in response to a 5% test item concentration in three out of ten experimental animals in the challenge phase were considered indicative of sensitization, based on the absence of any response in the control animals.

These results indicate a sensitization rate of 30 per cent.

Based on these results:

According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) the substance should be classified as skin sensitizer (Category 1).

According to Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), the substance should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

1B

Respiratory sensitisation

Endpoint conclusion

Additional information:

In vitro skin sensitisation
A KeratinoSens™ assay was performed with Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid (BA-1801) according to OECD guideline 442D and GLP principles. The test item was suspended in dimethyl sulfoxide (DMSO) at 10 mg/mL. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.5 – 100 µg/mL (2-fold dilution series). The highest test concentration was considered to be the limit of solubility. The test item precipitated at dose levels of 25 µg/mL and upwards. Three independent experiments were performed. Overall it is concluded that the test conditions were adequate and that the test system functioned properly.
In the first experiment, the test item showed toxicity (IC30 value of 0.10 µg/mL ). However, no IC50 value could be calculated due to the no clear dose-response observed with viability values between 62-77% for concentrations between 0.05 and 13 µg/mL. No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (Imax) was 1.20-fold. The test item is classified as inconclusive in the individual run since negative results (<1.5-fold induction) were observed at test concentrations up to 100 µg/mL with a cell viability of mostly >70% compared to the vehicle control.
In the second experiment, the test item showed toxicity (IC30 and IC50 values of < 0.05 µg/mL). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (Imax) was 1.11-fold. The test item is classified as negative in the individual run since negative results (<1.5-fold induction) were observed at test concentrations up to 100 µg/mL with a cell viability of < 70% compared to the vehicle control.
Since the first two experiments were not concordant, a third experiment was performed to provide a final conclusion.
In the third experiment, the test item showed toxicity (IC30 and IC50 values of < 0.05 µg/mL). A fluctuation in toxicity was observed at dose level 25 µg/mL at which the viability showed a fluctuation up to 146%. Since this was a clear outlier compared to the other dose levels in this experiment, it is likely the fluctuation was caused by an incidental fluctuation. No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (Imax) was 1.40-fold.
The test item is classified as negative in the individual run since negative results (<1.5-fold induction) were observed at test concentrations up to 100 µg/mL with a cell viability of < 70% compared to the vehicle control.
Overall, the test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed in two out of three experiments at test concentrations up to 100 µg/mL with cell viability < 70% compared to the vehicle control.
In conclusion, Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid (BA-1801) is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

An in vitro U-SENS assay was performed with BA-1801 according to OECD 442E and in accordance with GLP principles. The test item was suspended in dimethyl sulfoxide at maximum 25 mg/mL. The stock was diluted to five test concentrations (1, 10, 20, 50 and 100 µg/mL). No precipitate was observed at any dose level tested in the first and second experiments. In the third experiment at the dose levels of 20 to 100 µg/mL, either precipitation or dead cell parts were observed at the end of the treatment. Three independent experiments were performed.
In all experiments the positive (TNBS) and negative (LA) controls were considered valid. Overall it is concluded that the test conditions were adequate and that the test system functioned properly.
In the first experiment, the test item showed no toxicity (no CV70 value) and no biologically relevant induction of the CD86 activity (no EC150 value) was measured at any of the test concentrations. The individual run conclusion is considered negative.
In the second experiment, test item showed no toxicity (no CV70 value). An induction of the CD86 activity (CD86-IgG1 S.I. = 150%) was measured at the concentrations of 1 and 100 µg/mL. In absence of dose response, no EC150 value can be determined. Since the responses were not dose related and the first experiment was considered negative, a third experiment was performed to draw a final conclusion.
In the third experiment, the test item showed no toxicity (no CV70 value) and no biologically relevant induction of the CD86 activity (no EC150 value) was measured at any of the test concentrations. The individual run conclusion is considered negative.
Based on the three experiments, the test item is classified as negative in the U-Sens™ assay since negative results (<150% increase) were observed at test concentrations with a cell viability of >70% compared to the vehicle control in two out of three experiments. The negative result is however limited to concentrations up to 100 µg/mL, due to the limited solubility of the test item. Therefore, the maximum concentration was not in compliance with the OECD 442E guideline and the result must therefore be regarded as inconclusive.

in vivo skin sensitisation
In a guinea pig maximisation test method, the potential of the substance for skin sensitisation was tested according to OECD 406 guideline and in accordance with GLP principles. The capability of the test item to induce contact hypersensitivity was evaluated in females of guinea pig, Dunkin Hartley strain. Ten animals were exposed to the test item, at concentration of 20% for the intradermal induction and 40% for the epidermal induction. The control group consisted of 5 animals exposed to the vehicle, corn oil, during the induction phase. At challenge, 5% of the test item in corn oil was applied on the clipped skin of all the animals. Skin reactions were evaluated 24 and 48 hours after treatment. Slight to moderate erythema was observed during the intradermal induction (conc. 20%) at the injection sites in control and test animals. Scabbing was observed in animals exposed to the test item during the induction phase. Moderate erythema was observed following the epidermal induction (conc. 40%) in all 10 test animals. After epidermal exposure no skin reactions were evident in the control animals. No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.
In the challenge phase, skin reactions of grade 1 were observed in experimental animals in response to the 5% test item concentration. No skin reactions were evident in the control animals.
The skin reactions observed in response to a 5% test item concentration in three out of ten experimental animals in the challenge phase were considered indicative of sensitization, based on the absence of any response in the control animals.
These results indicate a sensitization rate of 30 percent.

Justification for classification or non-classification

A Guinea Pig Maximization Test was conducted in accordance with the OECD Testing Guideline 406, which indicated a sensitization rate of 30% with an intradermal induction at 20% test substance. Therefore, reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid (BA-1801) should be classified as skin sensitizer (Category 1B) in accordance with the CLP Regulation.