Cyclohexanamine, 4,4’-methylenebis[N-(2,2-dimethyl-3-(4-morpholinyl)propylidene)]-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-04-20 to 2021-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

amended 2012-07-20

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 22 2010-07-22

Deviations:

no

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:

(1E)-N-[4-({4-[(E)-[2,2-dimethyl-3-(morpholin-4-yl)propylidene]amino]cyclohexyl}methyl)cyclohexyl]-2,2-dimethyl-3-(morpholin-4-yl)propan-1-imine

EC Number:

954-590-8

Cas Number:

2522560-40-1

Molecular formula:

C31H56N4O2

IUPAC Name:

(1E)-N-[4-({4-[(E)-[2,2-dimethyl-3-(morpholin-4-yl)propylidene]amino]cyclohexyl}methyl)cyclohexyl]-2,2-dimethyl-3-(morpholin-4-yl)propan-1-imine

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: TOXI-COOP ZRT., H-1103, Budapest, Cserkesz u. 90. Hungary
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: 12 weeks
- Weight at study initiation: 19.6 – 22.7 g
- Housing: group-housed to allow social interaction, and with deep wood sawdust bedding, cage: Type II. polypropylene/polycarbonate
- Diet (ad libitum): ssniff® Rat/Souris-Elevage E complete diet (ssniff Spezialdiäten GmbH, D-59494 Soest Germany)
- Water (ad libitum): tap water from watering bottles
- Acclimation period: 21 days
- Indication of any skin lesions: no
- Randomisation: yes, checked by computer software [SPSS/PC+ (4.0.1)] according to the actual body weights verifying the homogeneity and deviations between the groups

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Relative Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

- IN-LIFE DATES: From: 2021-03-31 To: 26-04-2021

Study design: in vivo (LLNA)

Vehicle:

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

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS
- Compound solubility: achieved in AOO at concentration of 75 % (w/v) and below
- Irritation: observed, criteria not met ( which lead to dose reduction)
- Systemic toxicity:
No mortality was observed. Significant loss of body weights was observed in several cases but without dose-relevance. No other symptoms of a significant systemic effect were observed during the test. Loss of hair (on the head and/or base of ears) was observed in the 100 %, 75 % and 50 % (w/v) dose groups. No similar effect (hair loss) was observed in the 25 % and 10 % (w/v) dose groups during the whole test.
A second DRF was performed in which DMF was additionaly used as a vehicle: No mortality was observed. No significant loss of body weights or other symptoms of systemic toxicity were observed in any dose group. Loss of hair and lesion (on the head) were observed in the 100 % (w/v) dose group and in the 50 % (w/v) dose group where the test item was formulated in AOO. Similarly, loss of hair and lesion/scar (on the head) were observed in the 75% and 50 % (w/v) dose groups where formulations were prepared in DMF.

- Ear thickness measurements:
No significantly increased ear thickness values (i. e. an increase of ≥ 25 % compared to the initial value) were observed in any dose group

- Erythema scores:
No significant erythema (sored as ≥ 3) was observed in any test group


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
Exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result is declared positive.

TREATMENT PREPARATION AND ADMINISTRATION
Each mouse was topically treated with 25 µL of the appropriate formulations of the test item, the positive control substance, the physiological saline solution or the vehicle (see Table 3) using a pipette, on the dorsal surface of each ear. After the treatment animals were returned to their cages. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Positive control substance(s):

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

Statistics:

Linear regression was used to evaluate the significance of of the dose-response relationship

Results and discussion

Positive control results:

The positive control item (25 % (w/v) HCA in AOO) induced significant stimulation over the control (SI ≥ 3; actually SI = 17.3), confirming the sensitivity and validity of the assay.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Test group / Remarks:

test item

Remarks on result:

not determinable

Results 2

Key result

Parameter:

SI

Value:

4.6

Test group / Remarks:

10 % (w/v) test item

Results 3

Key result

Parameter:

SI

Value:

35.7

Test group / Remarks:

25 % (w/v) test item

Results 4

Key result

Parameter:

SI

Value:

67.7

Test group / Remarks:

50 % (w/v) test item

Results 5

Key result

Parameter:

SI

Value:

92.2

Test group / Remarks:

100 % (w/v) test item

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Visually larger lymph nodes compared to the relevant negative control (AOO or naive) were observed in the positive control group and in the 100 %, 50 % or 25 % (w/v) dose groups. Appearance of the lymph nodes was normal in both negative control groups and in the 10 % (w/v) dose group.

DETAILS ON STIMULATION INDEX CALCULATION
Significant lymphoproliferative response (SI ≥ 3) was observed for the test item at all tested concentrations. The observed stimulation index values were 92.9, 67.7, 35.7 and 4.6 at test item concentrations of 100 %, 50 %, 25 % and 10 % (w/v), respectively. Significance of the dose-response was evaluated by linear regression using the SI values obtained. Statistical significance for monotonic dose-response correlation was observed (p = 0.049, r 2 = 0.91).

EC3 CALCULATION
Chemicals can be classified according to their relative skin-sensitization potency using EC3 value (dose calculated to induce a stimulation index of 3) calculated by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve according to published method.
EC3 value could not be calculated In this LLNA since all SI-values were above the threshold of 3.

CLINICAL OBSERVATIONS:
No mortality or symptoms of systemic toxicity were observed during the test. Loss of hair and/or lesions (on the top of the head) was observed in the 100 % and 50 % (w/v) dose groups. The symptoms were not excessive in any of the affected groups. No similar symptoms were observed in the 25 % or 10 % (w/v) dose groups.

BODY WEIGHTS
Body weight decrease by > 5 % was observed in the following groups: AOO control group (1 of the 4 animals, 7 % decrease); 100 % (w/v) dose group (1 of the 4 animals, 7 % decrease); 50 % (w/v) dose group (1 of the 4 animals, 9 % decrease); 25 % (w/v) dose group (1 of the 4 animals, 8 % decrease); 10 % (w/v) dose group (1 of the 4 animals, 11 % decrease). The group mean body weights did not decrease significantly in these test groups. No body weight decrease by > 5 % was observed in the naive or positive control groups.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness).
No significant occurrence of erythema was observed in any treatment group. Ear thicknesses were measured in all test groups. No significant increase (compared to the initial values) was observed in the negative control groups (naive or AOO) and in the 25 % and 10 % (w/v) dose groups. Significantly increased ear thicknesses (i.e. ≥ 25 % increase compared to the initial value) were observed in the 100 % (w/v) dose group (2 of the 4 animals) and in the 50 % (w/v) dose group (3 of the 4 animals) as well as in the positive control group (2 of the 4 animals): the maximum increase was 31.6 %, 31.6 % or 26.3 %, respectively.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Under the conditions of the present assay, the test item tested at the maximum concentration of 100 % (w/v, as the undiluted form) and also at concentrations of 50 %, 25 % or 10 % (w/v) as formulations in a suitable vehicle (Acetone : Olive oil 4:1 (v/v) mixture; AOO) was shown to have skin sensitization potential in the Local Lymph Node Assay in mice.

Executive summary:

The aim of this study was to determine the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay in mice. The pooled treatment group approach was used in this test. The maximum dose selection was performed according to the relevant guidelines and based on results of a formulation evaluation and two consecutive Dose Range Finding tests (DRFs). The liquid test item was suitable for application on the ears of animals at 100 % (w/v) concentration (i.e. as the undiluted form). Lower test concentrations were achieved by dilution with Acetone : Olive oil 4:1 (v/v) mixture (AOO, the most preferred standard vehicle in the LLNA. Based on results of the DRFs no obvious significant adverse effects (either systemic or local) of the test item were expected. According to this, the test item was examined in the main test as the undiluted form (100 % concentration) and as 50 %, 25 % and 10 % (w/v) formulations in AOO with the aim of testing at highest possible concentrations. In addition, an appropriate positive control (α-Hexylcinnamaldehyde, HCA), as well as a negative control groups dosed with physiological saline solution (as naive control for the undiluted test item) and the vehicle (AOO) of the test and positive control groups were employed. The positive control item (25 % (w/v) HCA in AOO) induced significant stimulation over the control (SI ≥ 3; actually SI = 17.3), confirming the sensitivity and validity of the assay. No mortality or signs of systemic toxicity were observed during the main test. No significant, treatment related effect on the body weights was observed in any dose group (although body weight decrease by > 5 % were observed in some cases). Irritation was monitored by observation of erythema and also by ear thickness measurements. No significant erythema was observed in any test group during the test. Significantly increased ear thicknesses (i.e. ≥ 25 % increase compared to the initial value) were observed in the 100 % (w/v) dose group (2 of the 4 animals) and in the 50 % (w/v) dose group (3 of the 4 animals) as well as in the positive control group (2 of the 4 animals): the maximum increase was 31.6 %, 31.6 % or 26.3 %, respectively. In addition, hair loss and/or lesions (on the top of the head) were observed in these dose groups, but this symptom was not considered excessive. Significant lymphoproliferative response (SI ≥ 3) was observed for the test item at all tested concentrations. The observed stimulation index values 92.9, 67.7, 35.7 and 4.6 at test item concentrations of 100 %, 50 %, 25 % and 10 % (w/v), respectively. Statistically significant correlation for monotonic dose-response was observed (p = 0.049, r2 = 0.91; evaluated by linear regression using the SI values obtained). Ear thickness measurement indicated that the test item may cause irritation which could have contributed to the increased lymphoproliferation and may cause over estimation of the skin sensitization at high test concentrations. On the other hand, the effect was not excessive and comparable with the effect observed for the positive control (which was considered as normal). The loss of hair, observed in the 100 % and 50 % (w/v) dose groups may also indicate an adverse effect although it was also not considered excessive. Additionally at 25 % and 10 % (w/v) test concentrations (where no similar effects were observed) the SI values were also clearly above the threshold value of 3. Based on all these and in accordance with the relevant guidelines, it is concluded that the test item is a skin sensitizer. Chemicals can be classified according to their relative skin-sensitization potency using EC3 value (dose calculated to induce a stimulation index of 3) calculated by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve according to published method. In this LLNA no EC3 could be calculated by using this method hence the test item cannot be classified according to the published data for classification of contact allergens. Nevertheless, it can be stated that the test item is at least a moderate skin sensitizer as the concentration of 10 % (w/v) induced a response above the threshold value of 3.