reaction mass of N1,N3-bis(3-aminopropyl)-2-methyl-cyclohexane-1,3-diamine and N1,N3-bis(3-aminopropyl)-4-methyl-cyclohexane-1,3-diamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

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:

Reaction products of acrylonitrile and 2-methylcyclohexane-1,3-diamine and 4-methylcyclohexane-1,3-diamine, hydrogenated

EC Number:

948-816-4

Molecular formula:

C13H30N4

IUPAC Name:

Reaction products of acrylonitrile and 2-methylcyclohexane-1,3-diamine and 4-methylcyclohexane-1,3-diamine, hydrogenated

Specific details on test material used for the study:

Batch identification Bähr63/Dest/F4-5

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Supplier: Envigo RMS GmbH,
C/O Postfach 553
NL-5800 AN Venray

Age on day 0: 8 – 12 weeks
Identification: The single housed animals were identified by cage cards
Body weight on day 0: 17.0 g – 20.0 g (pretest)
17.0 g – 23.1 g (main test)

The animals were housed in fully air-conditioned rooms. Central air-conditioning guaranteed a temperature range of 20 – 24°C and a relative humidity range of 45 – 65%. There were no deviations from these ranges.

Illumination period: 12 hours light from 06:00 h to 18:00 h; 12 hours darkness from 18:00 h to 06:00 h
Type of cage: Polycarbonate cages type MII with mesh wire tops supplied by BECKER & Co., Castrop-Rauxel, Germany
No. of animals per cage: 1 animal
Watering: Drinking water ad libitum

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

1%, 5%, 10%

No. of animals per dose:

5

Details on study design:

Form of application: Epicutaneous application is simulating dermal contact with the compound, which can occur under practical use conditions.
Application volume: 25 µL per ear
Site of application: Dorsal surface of both ears
Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site

On study day five, 20 µCi 3H-thymidine in 250 µL sterile saline were injected into the tail vein of the mice.

Positive control substance(s):

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

other:

Results and discussion

Positive control results:

The sensitivity of mice (CBA/CaOlaHsd) and the reliability of experimental techniques were assessed by regularly using a known sensitizer as recommended by the test guideline.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1

Test group / Remarks:

vehicle

Results 2

Parameter:

SI

Value:

1.31

Test group / Remarks:

1%

Results 3

Parameter:

SI

Value:

1.97

Test group / Remarks:

5%

Results 4

Key result

Parameter:

SI

Value:

4.11

Test group / Remarks:

10%

Any other information on results incl. tables

test group	treatment	H-thymidine incorporation stimulation index (SI)	cell count	lymph node weight stimulation	ear weight stimulation index
1	vehicle propylene glycol	1.00	1.00	1.00	1.00
2	1% in propylene glycol	1.31	1.24	1.12	1.01
3	5% in propylene glycol	1.97	1.43	1.29	1.04
4	10% in propylene glycol	4.11	1.69	1.54	1.17
5	vehicle propylene glycol (exp. 2)	1.00	1.00	1.00	1.00
6	10% in propylene glycol (exp. 2)	3.82	1.93	1.72	1.24

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The skin sensitizing potential of Reaction mass of N1,N3-bis(3-aminopropyl)-2-methyl-cyclohexane-1,3-diamine and N1,N3-bis(3-aminopropyl)-4-methyl-cyclohexane-1,3-diamine was assessed using the radioactive Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of cells in the auricular lymph nodes to repeated application of the test substance to the dorsal skin of the ears.
Groups of 5 female CBA/CaOlaHsd mice each were treated with 1%, 5% and 10% (w/w) preparations of the test substance in propylene glycol (PG) or with the vehicle alone. A second experiment was performed as within the main test two out of five animals treated with the 10% concentration showed excessive ear skin irritation. These findings were not observed within the pretest hence, the 10% concentration was chosen for the main experiment.
To increase the data base for evaluation, a second experiment was conducted with groups of 5 female CBA/CaOlaHsd mice each, being treated with the 10% concentration or with the vehicle alone.

No signs of systemic toxicity were noticed in all animals during general observation.

When applied as 10% preparation in PG, the test substance induced a biologically relevant (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3) and statistically significant increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes. These findings were observed for the first and the second experiment. The increases at 5% and 1% test-substance preparation failed to reach the cutoff and thus lie below the threshold of immunological relevance.

Concomitantly, the 10% test-substance preparation induced a biologically relevant and statistically significant response (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. This effect was also observed for the second experiment. The SI of the 5% and 1% test-substance preparation was below the border of biological relevance.

Statistically significant increases for the 3H-thymidine incorporation and lymph node cell counts were noted at the 5% concentration.

In addition, statistically significant increases in lymph node weights were noted at the 10% (first and second experiment) and 5% concentration.

The 10% concentrations caused increases in ear weights (first and second experiment: mean SI 1.17 and 1.24, respectively) at the border of biological relevance. However, within the first experiment two out of five animals of the 10% concentration revealed excessive ear skin irritation (SI >1.25). After the second experiment again two animals of the 10% concentration test group revealed SI values above the cutoff for excessive ear skin irritation and two further animals showed SI values close to the cutoff. Besides, the increases in ear weights were statistically significant at this concentration in both experiments.

While the lymph node reactions were biologically relevant in one animals in the absence of excessive irriation, ear weight increases were observed for all other animals with lymph node reaction after treatment with the 10% concentration.

Hence in summary, the lymph node reactions observed are not considered to indicate a skin sensitization reaction but to be caused by excessive irritation of the ear skin.

Executive summary:

It is concluded that Reaction mass of N1,N3-bis(3-aminopropyl)-2-methyl-cyclohexane-1,3-diamine and N1,N3-bis(3-aminopropyl)-4-methyl-cyclohexane-1,3-diamine does not exhibit a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.