Betaine hydrochloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07/02/2018-20/02/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Version / remarks:

2010

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: test method B.51 of the council regulation No.640/2012 of 06 July 2012

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Test material information

Constituent 1

Reference substance name:

Betaine hydrochloride

EC Number:

209-683-1

EC Name:

Betaine hydrochloride

Cas Number:

590-46-5

Molecular formula:

C5H12NO2.Cl

IUPAC Name:

2-(trimethylazaniumyl)acetate

impurity 1

Reference substance name:

Minor constituents present in very low concentration and not affecting the hazardous properties of the substance

Molecular formula:

Traces of metals such as V, Mo, Zr, Ba, Sn, Ti

IUPAC Name:

Minor constituents present in very low concentration and not affecting the hazardous properties of the substance

impurity 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Water

Test material form:

solid: particulate/powder

Details on test material:

lot number 20161110

Specific details on test material used for the study:

batch number: 20161110
solid white powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Female CBA/J (CBA/JRj) strain mice (SPF caw) were supplied by Elevage Janvier Labs (F-53941 Le Genest Saint Isle). On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant. After an acclimatisation period of at least five days under stabling and nutritional conditions identical to those of the test, the animals were selected at random and given a number unique within the study by indelible ink-marking on the tail and a number written on a cage card. At the start of the main study the animals were 8 weeks old.
The animals were weighed at the beginning and at the end of the study.

Study design: in vivo (LLNA)

Vehicle:

other: Pluronic L92 (1%) (CAS 9003-11-6)

Concentration:

0 - 10 - 25 - 50 %

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
As no information was available regarding irritant potential or systemic toxicity of the test item in the mouse, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the test item diluted at 50% in Pluronic® L92 at 1% (maximun concentration administrable) to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed daily from Day 1. Any signs of toxicity or excessive local irritation noted during this period were recorded. Ear thickness was recorded on Day 1, Day 3 and on Day 6. The bodyweight of the mouse was recorded on Day 1 (prior to dosing) and Day 6.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT

Criteria used to consider a positive response:
Ear thickness measurements and recording of local reactions were performed in order to assess any possible irritant effect of the test item, as possible irritancy may be involved in false positive lymphoproliferative responses.
On day 1 and on day 3 (before application) as well as on day 6 (after sacrifice) of each experiment, the thickness of the right ear of each animal of the vehicle control and treated groups was measured by a micrometer.
Furthermore, on day 6, punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and weighed in order to assess the irritation potential of the test item and the two lymph nodes per mouse were weighed.
Any irritation reaction (erythema and oedema) was recorded in parallel. Any other observation (dryness, presence of residual test item...) was noted.
The irritation level of the test item is determined according to the following table:
% increase in ear thickness between day 1 and day 3 and/or between day 1 and day 6 Interpretation
< 10 % Non irritant
10 – 25 % Slightly irritant*
> 25 % Irritant
The test item should also be considered as an irritant if the score of erythema is higher or equal to 3.

On day 6 (end of the test), the animals were euthanized with sodium pentobarbital (Dolethal®). The draining auricular lymph nodes from the four mice were excised.
A single cell suspension of the lymph node cells of 4 mice of each group was prepared by gentle mechanical tissue disaggregation through a 200-mesh cell strainers in 4 mL of PBS (Ca2+ / Mg2+ - free) containing 0.5% BSA into a well of a multi-well 6.
10 μL of this cell suspension was diluted in 10 mL of physiological saline solution (NaCl 0.9%). The lymphocyte cells were counted using a cell counter (Beckman Coulter Z2).
For the run, the lower size selected was 5 μm and the upper size selected was 15 μm (the average size of a lymphocyte is 8 μm).

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of four mice were treated with the test item diluted at the concentrations of 50%, 25% and 10% in Pluronic® L92 at 1%. The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the test vehicle alone in the same manner.

Positive control substance(s):

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

Results and discussion

Positive control results:

An EC1.6 value of 14.58% was observed for the test item α-Hexylcinnamaldehyde, resulting in a classification as a sensitiser in Category 1, Sub-category 1B, in accordance with the Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.74

Test group / Remarks:

10%

Results 2

Key result

Parameter:

SI

Value:

1.01

Test group / Remarks:

25%

Results 3

Key result

Parameter:

SI

Value:

1.01

Test group / Remarks:

50%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In view of these results, under these experimental conditions, the test item betaine hydrochloride does not have to be classified as a sensitizer, in accordance with the criteria for classification, packaging and labelling of dangerous substances and mixtures of the Regulation No. 1272/2008.
No signal word or hazard statement is required.