1,3-Cyclohexanedimethanamine, N1,N3-bis(2-methylpropylidene)-

Endpoint summary

Description of key information

Incorez 397 tested in a suitable vehicle (AOO) in concentrations of 10 %, 5 % and 2.5 % was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay. No EC3 value was determined as all tested concentrations revealed SI values above 3.

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:

2011-02-23 to 2011-05-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is conducted in accordance with GLP and OECD/EU guidelines.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90.
- Age at study initiation: young adult mice, 9 weeks (at start of the experiment)
- Weight at study initiation: 16.3 - 19.6 g
- Housing: grouped caging (5 animals/cage)in type II. polypropylene/polycarbonate cages
- Diet: Animals received ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany, ad libitum
- Water: tap water for human supply, ad libitum
- Acclimation period: 7 and 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: :22 ± 3 °C
- Humidity: 30 – 70 %
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Vehicle:

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

Concentration:

10, 5, 2.5 % (v/v)

No. of animals per dose:

5 animals per dose

Details on study design:

Dose selection:
The preliminary test was performed in two steps: in the first round the vehicle control (AOO) and test item concentrations of 100 % (the undiluted test item itself) and 50 % in the vehicle (AOO) was tested. In the second round 50 % and 25 % concentrations were examined. The applicability of the undiluted test item was acceptable. The 50 % and 25 % concentrations of test item in AOO were real solutions and applicability of these formulations on the ears of animals was also acceptable.
In the first step groups of 2 CRL: NMRI BR mice were treated with the vehicle, the test item (100 %) and the 50 % test item formulation. All animals were observed for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded prior to the first treatment (Day 1) and prior to termination (Day 6). Both ears of each mouse were observed for erythema and scored. Ear thickness measurement was not performed in the first round. In the second step groups of 2 CRL: NMRI BR mice were treated with the concentrations of 50 % and 25 %. Beside body weight measurement and observation of signs of toxicity and irritation, measurement of ear thickness was taken using digital micrometer on
Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6.
Mortality was not observed either in the first or the second test. In the first test signs of systemic toxicity were observed in the 100 % dose group (the undiluted test item). The symptoms were hunched back posture (2/2 animals), narrow eyes (1/2 animals), decreased activity (2/2 animals). No significant treatment related effect on body weights was observed. Excessive local irritation was observed at concentration of 100 % indicated by erythema score 3 observed on each treatment days (Day 1: 1/2 animals, Day 2 and 3: 2/2 animals) and also on the first non treatment day (Day 4: 1/2 animals). Additionally, local tissue effect (necrosis) was observed on each animal at both concentrations tested (observed from Day 3 and from Day 5 at concentrations of 100 % and 50 %, respectively). Discoloured (yellowish) hair (on the crown of animals) was observed from Day 5 at concentration of 100 % and on Day 6 at concentration of 50 %.
Loss of hair was also observed on 1 animal in the 100 % treatment group. In the second test no significant treatment related effect on body weights was observed. No other sign of systemic toxicity was observed in this test. Excessive local irritation, indicated by increase of ear thickness > 25 % was observed at both tested concentrations (2/2 animals in the 50 % treatment group and 1/2 animals in the 25 % treatment group) on Day 6 (at termination of the test). Additionally, local tissue effect (necrosis) and/or scar were observed on each animal at both concentrations tested (observed from Day 4 and from Day 5 at concentrations of 50 % and 25 %, respectively).
Based on the observed effects (irritation and/or toxicity) concentrations of 100 % and 50 % were declared to be unacceptable for further testing. Although in the 25 % treatment group the test item did not cause redness or obvious irritation, the local tissue effect (necrosis) observed was considered to be also unacceptable. According to this, test item concentration of 10 % (the next from the concentration series recommended by the relevant guideline) was selected to be the highest test concentration in the main test.

In the main assay thirty female CRL: NMRI BR mice were allocated to six groups of five animals each:
- Three groups received the appropriate formulation of Incorez 397 at concentrations of 10 %, 5 % or 2.5 %,
- two groups received the positive control substance at concentrations of 50 and 25 %,
- the negative control group received the vehicle (AOO).
Each substance was applied on the external surface of each ear (25 µL/ear) of the animals for three consecutive days (Day 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the obtained values were used to calculate stimulation indices (SI).

Clinical observations:
During the study (from Day 1 to Day 6) each animal was observed at least once daily for any clinical signs, including local irritation and systemic toxicity. Individual records were maintained.
Measurement of body weight:
Individual body weights were recorded on Day 1 (beginning of the test) and at Day 6 (prior to ³HTdR injection) with a precision of +/- 0.1 g.
Evaluation of the results:
DPM was measured for each group. The results were expressed as DPN (DPM divided by the number of lymph nodes) following the industry standard for data presentation. Stimulation index (SI = DPN of a treated group divided by DPN of the negative control group) for each treatment group was also calculated.
Interpretation of results:
The test item is regarded as a sensitiser if both of the following criteria are fulfilled:
- Exposure to at least one concentration of the test item result in an incorporation (tritiated methyl thymidine) of at least 3-fold or greater than recorded in control mice
- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either
local toxicity or immunological suppression.

Positive control substance(s):

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

Statistics:

EC 3 calculation was not possible, as all SI values were above 3.

Positive control results:

The positive control group animals were treated with 50 % or 25 % (w/v) HCA solution (dissolved in AOO) concurrent to the test item groups.
No mortality, significant cutaneous reactions or signs of toxicity were observed. Significant lymphoproliferative response (SI above 3) was noted for HCA with a stimulation index value of 3.9 at concentration of 50 %, while an SI value of 2.4 was observed at concentration of 25 %. The response observed for positive control was dose-related. The results of the positive control demonstrated the appropriate performance of the assay in accordance with the OECD Guideline 429 and confirmed the validity of the assay.

Key result

Parameter:

SI

Value:

5.5

Test group / Remarks:

2.5 % Incorez 397 in AOO

Key result

Parameter:

SI

Value:

9

Test group / Remarks:

5 % Incorez 397 in AOO

Key result

Parameter:

SI

Value:

10.5

Test group / Remarks:

10 % Incorez 397 in AOO

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Group DPN (measured DPN group values – average DPN background values) obtained for the negative control group, Incorez 397 10 %, 5 %, 2.5 % groups were 14214, 149878, 127451, 78728 respectively. Or expressed as DPN/node with the following results 1421.4, 14987.8, 12745.1, 7872.8, obtained in the negative control group, Incorez 397 10 %, 5 %, 2.5 % groups respectively.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In conclusion, under the conditions of the present assay Incorez 397, tested as formulations in a suitable vehicle (AOO) in concentrations of 10 %, 5 % and 2.5 % was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.

Executive summary:

The aim of the study was to determine the skin sensitisation potential of Incorez 397 following dermal exposure in the Local Lymph Node Assay. The test item was a liquid, hence the maximum concentration of 100% (the undiluted test item) according to the OECD guideline 429 could be tested. A preliminary solubility test was performed to select suitable vehicle for the further test concentrations. Solubility of the test item in Acetone: Olive oil 4:1 (v/v) mixture (AOO) was examined and found to be acceptable. Since AOO is the most preferred solvent by the OECD 429 guideline, no other solvents were examined. The preliminary irritation/toxicity test was performed. Based on the observed effects (irritation and/or toxicity) concentrations of 100 % and 50 % were declared to be unacceptable for further testing. Although in the 25 % treatment group the test item did not cause redness or obvious irritation, the local tissue effect (necrosis) observed was considered to be also unacceptable. According to this, test item concentration of 10 % was selected to be the highest test concentration in the main test.

In the main assay 30 female CBA/Ca mice were allocated to six groups of five animals each:

- three groups received Incorez 397 at concentrations of 10 %, 5 % or 2.5 %,

- the negative control group received the vehicle (AOO),

- the positive control groups received alpha-Hexylcinnamaldehyde (HCA) at concentrations of 50 % or 25 %.

Each substance was applied on the external surface of each ear (25 µL/ear) of the animals for three consecutive days (Day 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the obtained values were used to calculate stimulation indices (SI).

No mortality or systemic clinical signs were observed during the study. No treatment related effects were observed on animal body weights in any treated groups. No cutaneous reactions were observed at the site of the treatment in any of the treated groups. Stimulation index values of the test item were 10.5, 9.0 and 5.5 at treatment concentrations of 10 %, 5 % and 2.5 %, respectively. The stimulation index values were compatible with a biological dose-related response.

Normally, the EC3 value (the theoretical concentration of the test item in the test solution, leading a three fold increase of lymph node cell proliferation over the control) is estimated by linear interpolation using the reported SI values and the corresponding concentrations immediately above and below the SI value of 3. Since no SI value below 3 was observed with the test item, no estimation of the EC3 value was performed in this LLNA. α-Hexylcinnamaldehyde, dissolved in AOO at concentrations of 50 % and 25 % (% w/v) was used as positive control.

A significant lymphoproliferative response (SI >= 3) was noted for the positive control chemical at concentration of 50 % with a stimulation index value of 3.9, while an SI value of 2.4 was observed at concentration of 25 %. The response observed for positive control was dose-related. The results of the positive control demonstrated the appropriate performance of the assay in accordance with the OECD Guideline 429 and confirmed the validity of the assay.

Under the conditions of the present assay Incorez 397 tested in a suitable vehicle (AOO) in concentrations of 10 %, 5 % and 2.5 % was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The aim of the study was to determine the skin sensitisation potential of Incorez 397 following dermal exposure in the Local Lymph Node Assay. The test item was a liquid, hence the maximum concentration of 100% (the undiluted test item) according to the OECD guideline 429 could be tested. A preliminary solubility test was performed to select suitable vehicle for the further test concentrations. Solubility of the test item in Acetone: Olive oil 4:1 (v/v) mixture (AOO) was examined and found to be acceptable. Since AOO is the most preferred solvent by the OECD 429 guideline, no other solvents were examined. The preliminary irritation/toxicity test was performed. Based on the observed effects (irritation and/or toxicity) concentrations of 100 % and 50 % were declared to be unacceptable for further testing. Although in the 25 % treatment group the test item did not cause redness or obvious irritation, the local tissue effect (necrosis) observed was considered to be also unacceptable. According to this, test item concentration of 10 % was selected to be the highest test concentration in the main test.

In the main assay 30 female CBA/Ca mice were allocated to six groups of five animals each:

- three groups received Incorez 397 at concentrations of 10 %, 5 % or 2.5 %,

- the negative control group received the vehicle (AOO),

- the positive control groups received alpha-Hexylcinnamaldehyde (HCA) at concentrations of 50 % or 25 %.

Each substance was applied on the external surface of each ear (25 µL/ear) of the animals for three consecutive days (Day 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the obtained values were used to calculate stimulation indices (SI).

No mortality or systemic clinical signs were observed during the study. No treatment related effects were observed on animal body weights in any treated groups. No cutaneous reactions were observed at the site of the treatment in any of the treated groups. Stimulation index values of the test item were 10.5, 9.0 and 5.5 at treatment concentrations of 10 %, 5 % and 2.5 %, respectively. The stimulation index values were compatible with a biological dose-related response.

Normally, the EC3 value (the theoretical concentration of the test item in the test solution, leading a three fold increase of lymph node cell proliferation over the control) is estimated by linear interpolation using the reported SI values and the corresponding concentrations immediately above and below the SI value of 3. Since no SI value below 3 was observed with the test item, no estimation of the EC3 value was performed in this LLNA. α-Hexylcinnamaldehyde, dissolved in AOO at concentrations of 50 % and 25 % (% w/v) was used as positive control.

A significant lymphoproliferative response (SI >= 3) was noted for the positive control chemical at concentration of 50 % with a stimulation index value of 3.9, while an SI value of 2.4 was observed at concentration of 25 %. The response observed for positive control was dose-related. The results of the positive control demonstrated the appropriate performance of the assay in accordance with the OECD Guideline 429 and confirmed the validity of the assay.

Under the conditions of the present assay Incorez 397 tested in a suitable vehicle (AOO) in concentrations of 10 %, 5 % and 2.5 % was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results INCOREZ 397 has to be classified and labelled as skin sens. cat.1, H317 (may cause an allergic skin reaction) according to Regulation 1272/2008/EC (CLP) respectively.