Betaine hydrochloride

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23/01/2018-01/02/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2015

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2009

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

batch number: 20161110
solid white powder

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human epidermis (SkinEthic RHE® model, Episkin SA, RHE/S/17)
- Tissue batch number(s): 18-RHE-006
- Production date: not reported
- Shipping date: not reported
- Delivery date: 30 January 2018
- Date of initiation of testing: 30 January 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: not reported
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 42 minutes after the test item application, the human epidermis were washed with 25 x 1 mL of DPBS (Dutscher - Batch No. 7530417).
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: ELx800 absorbance microplate reader
- Wavelength: 570 nm
- Filter: not reported
- Filter bandwidth: not reported
- Linear OD range of spectrophotometer: not reported

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
There is no direct interaction between the test item and MTT

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test item is considered as non-irritant to skin in accordance with UN GHS No Category: if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is > 50%.
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2): if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is “non corrosive”.
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1): if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and in absence of information on a skin corrosion test.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

16 mg/0.5 cm2

Duration of treatment / exposure:

42min

Duration of post-treatment incubation (if applicable):

42h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

95.4

Negative controls validity:

valid

Remarks:

mean tissue viability 100%

Positive controls validity:

valid

Remarks:

mean tissue viability 1.6%

Interpretation of results:

GHS criteria not met

Conclusions:

The test item betaine hydrochloride has to be considered as Non-irritant to skin. It corresponds to UN GHS No Category.
No hazard statement or signal word is required.

Executive summary:

The aim was to evaluate the possible irritating effects of the test item betaine hydrochloride after topical application on in vitro human reconstructed epidermis (SkinEthic RHE model).

The test item was applied as supplied, during 42 minutes, at the dose of 16 mg to 3 kuvubg reconstructed human epidermis of 0.50 cm2 previously moistened with 10 microliters of distilled water. The application was followed by a rinse with 25 ml of DPBS and a 42 hours post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

The mean percent viability of the treated tissues was 95.4%, versus 1.6% in the positive control (5% dodecyl sulfate)

In accordance with the regulation EC No.1272/2008, the test item betaine hydrochloride has to be considered as non-irritant to skin. It corresponds to UN GHS No Category. No hazard statement or signal word is required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg per eye

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption have been used for this assay. The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 - 2.5 kg).
Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 12 February 2018 at 8:20 am.
Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline.
The eyes were enucleated at Phycher on 12 February 2018 at 9:50 am.
The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.
The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 32.5°C and 33.9°C.

EQUILIBRATION AND BASELINE RECORDINGS
After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.
Once all eyes had been examined and approved, the eyes were incubated between 45 and 54 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED
physiological saline – Dutscher Batch No. 3012808; expiry date of batch: June 2019

SOLVENT CONTROL USED
not applicable

POSITIVE CONTROL USED
sodium hydroxide CAS No.:1310-73-2 – Fisher Scientific, Batch No. 1550248, expiry date: 21 August 2020

APPLICATION DOSE AND EXPOSURE TIME
Immediately following the zero reference measurements, three eyes (in their holder) were removed from the superfusion apparatus, placed in a horizontal position, and 30 mg of the test item was applied, as supplied, during 10 seconds to the cornea of each eye such that the entire surface of the cornea was evenly covered with the test item.
Then the eyes were rinsed twice with 10 mL of physiological saline at ambient temperature. The eye (in its holder) was subsequently returned to the superfusion apparatus in the original upright position.

OBSERVATION PERIOD
Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: the eyes were rinsed twice with 10 mL of physiological saline at ambient temperature.
- Indicate any deviation from test procedure in the Guideline: none

METHODS FOR MEASURED ENDPOINTS:
All observations of the cornea and measurement of corneal thickness were performed using a Haag- Streit BP900 slit-lamp microscope with depth-measuring device no. I. For the measurement of corneal thickness, the slit-width was set at 91⁄2, equalling 0.095 mm. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which was determined only at pretreatment and 30 minutes after test item exposure) were determined at each of the above time points.

SCORING SYSTEM:
- Mean corneal swelling (%): Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope. It was expressed as a percentage and was calculated from corneal thickness measurements. The mean percentage of corneal swelling for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for each test item.
- Mean maximum opacity score: Corneal opacity was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score was then given for each test or control item.
- Mean fluorescein retention score at 30 minutes post-treatment: The mean fluorescein retention value for all test eyes was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item.

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. Yes

Irritation parameter:

cornea opacity score

Remarks:

maximal mean

Value:

0.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: corresponding to ICE class II

Irritation parameter:

fluorescein retention score

Remarks:

mean

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: corresponding to ICE class IV

Irritation parameter:

percent corneal swelling

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: corresponding to ICE class I

Interpretation of results:

study cannot be used for classification

Remarks:

inconclusive for classification

Conclusions:

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category “no prediction can be made”, as defined by the OECD guideline No.438. Therefore, the test item betaine hydrochloride is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method. Additional testing (in vitro and/or in vivo) are required to establish a definitive classification.

Reference 2

Endpoint:

eye irritation: in vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study need not be conducted because the substance is a strong acid (pH <= 2.0) or base (pH => 11.5)

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation/corrosion:

The substance is not classified a skin irritant because in the selected test it doesn't meet the classification criteria of the CLP regulation n. 1272/2008.

Eye irritation:

The substance is classified a severe eye irritant based on ICE in vitro test (inconclusive) and its low pH (<2).