Registration Dossier

Diss Factsheets

Administrative data

Description of key information

N,N’[(methylimino)bis(trimethylene)]bis(stearamide) was not a skin sensitiser in a local lymph node assay according to OECD guideline 429 (2010) and EU Method B.42 (2012).

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:
2017-11-01 to 2017-11-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Deviations:
yes
Remarks:
Age of animals at study initiation was 13 - 14 weeks, instead of 8 -12 weeks. It was considered that these deviations do not affect the validity of the study.
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc , Postbus 6174, 5960 AD Horst / The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Pre-test: 12 - 13 weeks, main study: 13 - 14 weeks
- Weight at study initiation: main study: 18.9 to 24.7 g
- Housing: Makrolon Type III (main study), with wire mesh top
- Diet: 2018C Teklad Global 18% protein rodent diet ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days prior to the start of dosing
- Indication of any skin lesions: not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 45-65%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.
- IN-LIFE DATES: From: 2017-11-01 To: 2017-11-14
Vehicle:
propylene glycol
Concentration:
0%, 5%, 10% and 25%
No. of animals per dose:
4 females
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 25% suspension in PG.

- Irritation:
- Systemic toxicity:
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals.
Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 25% once daily each on three consecutive days.

- Ear thickness measurements:
In the pre-test, the ear thickness was determined prior to the first application of the test item (day 1), on day 3, and on day 6 prior to sacrifice using a micrometer.

- Erythema scores:
/ = No visible erythema
1 = Very slight erythema
2 = Well defined erythema
3 = Moderate to severe erythema
4 = Severe erythema to formation of eschar which prevents grading of erythema




MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
The animals were distributed into the test groups at random.

- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
• Second, that 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.


TREATMENT PREPARATION AND ADMINISTRATION:

Test Item Administration
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% in PG. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (diameter approx. 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

Administration of 3H-methyl-thymidine
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 20.2 µCi of 3H-methyl thymidine (equivalent to 80.9 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Terminal Procedure
Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.

Preparation of Single Cell Suspensions
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.

Determination of cellular proliferation (incorporation of 3HTdR)
The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a ß-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
Experiment performed in October 2017 (Envigo study number 1868000). Positive control substance: α-Hexylcinnamaldehyde:
10 % test group = SI 2.77; 25 % test group = SI 5.70 (EC3 = 11.2 % (w/v)
Key result
Parameter:
SI
Value:
0.64
Test group / Remarks:
5 % Test group
Key result
Parameter:
SI
Value:
1.12
Test group / Remarks:
10 % Test group
Key result
Parameter:
SI
Value:
1.21
Test group / Remarks:
25 % Test group
Key result
Parameter:
EC3
Remarks on result:
not determinable
Remarks:
The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.).


DETAILS ON STIMULATION INDEX CALCULATION
Refer to table below

EC3 CALCULATION
The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

CLINICAL OBSERVATIONS:
No deaths occurred during the study period. No signs of systemic toxicity were observed during the study period. On day 3, the animals (including the vehicle controls) showed an erythema of the ear skin (Score 1).

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Preliminary test:

At the tested concentrations of 10 and 25 % the animals did not show any signs of systemic toxicity.

On day 1 and 2, the animal treated with 25% test item concentration showed an erythema of the ear skin (Score 1) and on day 6 scaly ears. The animal treated with 10% test item concentration did not show any signs of local skin irritation.

Main study:

Calculation and Results of Individual Data

Test item concentration %

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

21

---

---

---

---

---

BG II

16

---

---

---

---

0

1

7534

7515.5

8

939.4

1.00

5

2

4794

4775.5

8

596.9

0.64

10

3

8449

8430.5

8

1053.8

1.12

25

4

9134

9115.5

8

1139.4

1.21

1    =  Control Group

2-4 =  Test Group

a)  =  The mean value was taken from the figures BG I and BG II

b)  =  Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

Interpretation of results:
GHS criteria not met
Conclusions:
Stimulation Indices (S.I.) of 0.64, 1.12, and 1.21 were calculated for concentrations of 5, 10, and 25% in PG, respectively.
These results indicate that the test substance does not elicit an SI ≥ 3 and thus N,N’[(methylimino)bis(trimethylene)]bis(stearamide) was not a skin sensitiser under the test conditions of this study.
Executive summary:

In a local lymph node assay according to OECD guideline 429 (2010) and EU Method B.42 (2012) N,N’[(methylimino)bis(trimethylene)]bis(stearamide) dissolved in PG was assessed for its possible contact allergenic potential using test item concentrations of 5, 10, and 25%. The highest achievable test item concentration was 25% in PG.

All treated animals survived the scheduled study period and no signs of systemic toxicity were observed. On day 3, the animals (including the vehicle controls) showed an erythema of the ear skin (score1,= Very slight erythema).

Stimulation Indices (S.I.) of 0.64, 1.12, and 1.21 were calculated for concentrations of 5, 10, and 25% in PG, respectively. The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

These results indicate that the test substance does not elicit an SI ≥ 3 and thus N,N’[(methylimino)bis(trimethylene)]bis(stearamide) was not a skin sensitiser under the test conditions of this study.

In conclusion, N,N’[(methylimino)bis(trimethylene)]bis(stearamide) does not need to be classified as skin sensitizer according the criteria of CLP, EU GHS (Regulation (EC) No 1272/2008).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

In a local lymph node assay according to OECD guideline 429 (2010) and EU Method B.42 (2012)N,N’[(methylimino)bis(trimethylene)]bis(stearamide)dissolved in PG was assessed for its possible contact allergenic potential using test item concentrations of 5, 10, and 25%.The highest achievable test item concentration was 25% in PG.

All treated animals survived the scheduled study period and no signs of systemic toxicity were observed. On day 3, the animals (including the vehicle controls) showed an erythema of the ear skin (score1,= Very slight erythema).

Stimulation Indices (S.I.) of 0.64, 1.12, and 1.21 were calculated for concentrations of 5, 10, and 25% in PG, respectively. The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

These results indicate that the test substancedoes notelicit an SI ≥ 3 andthusN,N’[(methylimino)bis(trimethylene)]bis(stearamide) was not a skin sensitiser under the test conditions of this study.

In conclusion, N,N’[(methylimino)bis(trimethylene)]bis(stearamide) does not need to be classified as skin sensitizer according the criteria of CLP, EU GHS (Regulation (EC) No 1272/2008).

Justification for classification or non-classification

N,N’[(methylimino)bis(trimethylene)]bis(stearamide) does not need to be classified as skin sensitizer according the criteria of CLP, EU GHS (Regulation (EC) No 1272/2008).

Categories Display