2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-09-14 to 2016-09-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22th July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

06 July 2012

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:

2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol

EC Number:

230-237-7

EC Name:

2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol

Cas Number:

6976-37-0

Molecular formula:

C8H19NO5

IUPAC Name:

2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/Ca Ola Hsd mice

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: TOXI COOP ZRT. Cserkesz u. 90., 1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals: SPF at arrival, Good conventional during test
- Age at study initiation: 9-11 weeks
- Weight at study initiation: 18.2 - 21.0 g
- Fasting period before study: no
- Housing: group caging (4 mice/cage), Type II. Polypropylene / polycarbonate cages with laboratory bedding
- Diet: ad libitum, ssniff® Rat/Souris-Elevage E complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany
- Water: ad libitum, tap water
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 - 70
- Air changes (per hr): not available
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

other: Pluronic

Remarks:

aqueous 1 % (w/v) Pluronic PE9200

Concentration:

5, 10 and 25 % (max. concentration determined by solubility)

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The substance's solubility in seven recommended solvents was tested. The best solubility was achieved in Plu and EtOH. The maximum feasible concentration in both vehicles was 25 % (w/v). No or not adequate solubility was observed with the other vehicles at this maximum concentration. As Plu is more preferred in the LLNA (as standard vehicle) than EtOH (non-standard vehicle), the test item was formulated with Plu.
- Irritation: No irritation was observed for the three concentration 5, 10 and 25 % in a preliminary test.
- Systemic toxicity: No toxicity was observed for the three concentration 5, 10 and 25 % in a preliminary test.
- Ear thickness measurements: No changes in ear thickness were observed for the three concentration 5, 10 and 25 % in a preliminary test.
- Erythema scores: All scores were 0 for all concentrations, mice and time points (days 1, 2, 3, 4, 5, 6).

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Animals were assigned randomly achieving body weight homogeneity between groups.
- Criteria used to consider a positive response: stimulation index of one concentration > 3

TREATMENT PREPARATION AND ADMINISTRATION: Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, the positive control substance or the vehicles using a pipette, on the dorsal surface of each ear. After the treatments 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:

Significance of the dose-response was evaluated by linear regression using the SI values.

Results and discussion

Positive control results:

The positive control group animals were treated with 25 % (w/v) HCA solution (formulated in AOO) concurrent to the test item groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group.
Significant lymphoproliferative response (SI ≥ 3) was noted for HCA (SI = 6.7). The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed validity of the assay.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.6

Test group / Remarks:

5 %

Results 2

Parameter:

SI

Value:

1

Test group / Remarks:

10 %

Results 3

Key result

Parameter:

SI

Value:

1.7

Test group / Remarks:

25 %

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Disintegration per minute (per mouse (average)):
vehicle control Plu: 772.8
5.0 %: 478.3
10 %: 764.0
25 %: 1290.3

DETAILS ON STIMULATION INDEX CALCULATION: SI = the DPM/mouse of a treated (positive control or test item) group divided by the DPM/mouse of the respective negative control group

EC3 CALCULATION: EC3 value was not calculated as no the SI value observed at the highest test concentration (25 %, w/v) was well below the threshold value of 3.

CLINICAL OBSERVATIONS: No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score ≥ 3) or any other local effect were observed in any treatment group.

BODY WEIGHTS: No significant, treatment related effect on the body weights was observed during the test.

Any other information on results incl. tables

DPM and Stimulation Index Values for all Groups in the Main Test

Dose Group	Measured DPM/group	Group* DPM	DPM/Mouse#	Stimulation Index Values
Vehicle control for the positive control: AOO	9567	9534	2383.5	1
Positive control: 25 % HCA in AOO	64108	64075	16018.8	6.7
Vehicle control for the test item: Plu	3124	3091	772.8	1
Test item: 25 % in Plu	5194	5161	1290.3	1.7
Test item: 10 % in Plu	3089	3056	764	1
Test item: 5 % in Plu	1946	1913	478.3	0.6

HCA = α-Hexylcinnamaldehyde

AOO = Acetone: Olive oil 4:1 (v/v) mixture

Plu = aqueous 1 % (w/v) Pluronic®PE 9200

*Group DPM = measured DPMgroup- average DPMbackground

Average DPMbackground = 33

# Number of animals/group = 4

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In a GLP-Study according to OECD TG 429 (LLNA), the substance, which was tested up to 25 % in aqueous 1% Pluronic (limited due to solubility), did not induce any skin sensitisation.

Executive summary:

The aim of the study according to OECD 429 was to evaluate the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay. A formulation evaluation and a Dose Range Finding test (DRF) were performed to find an appropriate vehicle and the maximum applicable concentration according to the relevant guidelines. Solubility of the test item in vehicles preferred in the LLNA was evaluated and concentration series of 100 %, 50 %, 25 %, 10 % etc. were used. The maximum attainable concentration (based on solubility) was 25 % (w/v) in aqueous 1 % (w/v) Pluronic®PE 9200 (Plu). According to results of the DRF, where no adverse effect was observed up to this maximum concentration, the test item was examined in the main test as 25 %, 10 % or 5 % (w/v) formulations in the selected vehicle of Plu. Appropriate positive control (α-Hexylcinnamaldehyde, HCA), furthermore two negative control groups dosed with the vehicles of the test and positive control groups, respectively, were employed. The positive control item (25 % (w/v) HCA in Acetone:Olive oil 4:1 (v/v) mixture, AOO) induced significant stimulation over the relevant control (SI = 6.7) thus confirming the validity of the assay. No mortality was observed during the main test. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any treatment group. No signs of irritation (monitored by erythema scoring) or any other local effect were observed at the treatment site (ears) in any treatment group. No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (Plu) was noted for the test item at the applied test concentrations. The observed stimulation index values were 1.7, 1.0 and 0.6 at test item concentrations of 25 %, 10 % and 5 % (w/v), respectively. The response was dose-related, however no statistical significance was observed. Additionally the SI value observed at the highest test concentration (25 %, w/v) was well below the threshold value of 3. As 25 % (w/v) was the maximum soluble concentrations achieved in a range of vehicles no significantly higher absorption of the test item is presumable hence the dose-response relationship was considered not biologically significant. Accordingly, the test item was considered to be not a skin sensitizer. In conclusion, under the conditions of the present assay, the test item tested at the maximum feasible concentration of 25 % (w/v, based on solubility) and also at concentrations of 10 % or 5 % (w/v) as formulations (apparently solutions) in a suitable vehicle (aqueous 1 % (w/v) Pluronic®PE 9200) was shown to have no skin sensitization potential in the Local Lymph Node Assay.