By-product from Guanidinoacetic acid manufacturing

Endpoint summary

Description of key information

A GLP Study according to OECD guideline 439 as beeen performed to to determine the skin irritation potential of "By-product from Guanindinoacetic acid manufacturing". As no irritating effects were observed a study to determine the corrosivity of the test item did not have to be conducted.

A GLP Study according to OECD guideline 437 has beeen performed to to determine the eye irritation potential of "By-product from Guanindinoacetic acid manufacturing". No stand-alone prediction can be made regarding the classification of the test substance By-product from Guanidinoacetic acid manufacturing according to the evaluation criteria.

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:

2020-11-24 - 2021-03-01

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

201009MA2 solid

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of
normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of
the species of interest and closely mimics the biochemical and physiological properties of the upper
parts of the human skin, i.e. the epidermis.

Vehicle:

other: Dulbecco’s phosphate buffered saline (DPBS)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg + 25 µL DPBS

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 59.2

Vehicle controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The relative mean
tissue viability after 60 min of exposure and 42 h post-incubation was > 50%. The test item is therefore
classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

In the present study the skin irritant potential of By-product from Guanidinoacetic acid manufacturing
was analysed. The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human
epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404, [7]) to
distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test
substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was
applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity
measured by formazan production from MTT after a 60 min exposure and 42 h post-incubation period
and compared to those of the concurrent negative controls.
To check the non-specific MTT-reducing capability of the test item 25 mg of the test item was mixed
per 1 mL MTT medium and incubated for 60 min at 37  1 °C in the incubator. The mixture did not turn
blue/purple. Thus, the additional test with freeze-killed tissues and the quantitative corrections were
not necessary.
To check the colouring potential of the test item 25 mg of the test item was mixed per 300 μL aqua
dest. and/or per 300 μL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for
60 min. The mixture showed no relevant colouring detected by unaided eye-assessment. Thus, the
additional test with viable tissues and the quantitative corrections were not necessary.
The test item showed no non-specific reduction of MTT and no relevant colouring potential after
mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of
possible false-negative results were necessary.
The test item showed no irritant effects. The mean relative tissue viability (% negative control) was
> 50% (59.2%) after 60 min treatment and 42 h post-incubation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28.01.2021 - 2021-03-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

201009MA2 solid

Species:

cattle

Details on test animals or tissues and environmental conditions:

The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany.
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

The test item was suspended with physiological saline 0.9% NaCl to give a 20% concentration

Duration of treatment / exposure:

4 h ± 5 min

Number of animals or in vitro replicates:

3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive control treated with imidazole 20% in physiological saline 0.9% NaCl

Irritation parameter:

in vitro irritation score

Value:

ca. 4.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Remarks:

With an IVIS > 3

Conclusions:

No stand-alone prediction can be made regarding the classification of the test substance By-product from Guanidinoacetic acid manufacturing according to the evaluation criteria. Further testing in another suitable method is required.

Executive summary:

The eye irritancy potential of By-product from Guanidinoacetic acid manufacturing was investigated in the bovine corneal opacity and permeability assay.

Preparation of the test item: The test item was suspended with physiological saline 0.9% NaCl (see 10.3) to give a 20% concentration.
Visual observation after treatment: None of the corneas treated with By-product from Guanidinoacetic acid manufacturing showed any opacity of the tissue.

Mean in vitro irritation score: 4.10
Classification:

☐ UN GHS No Category
☒ no stand-alone prediction can be made
☐ UN GHS Category 1

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.
The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

No skin irritating effects could be observed in the performed shin irritation study. Therefore "By-product from Guanidinoacetic acid manufacturing" does not have to be classified as irritant or corrosive to skin.

For eye irritation no stand-alone prediction can be made regarding the classification of the test substance By-product from Guanidinoacetic acid manufacturing according to the evaluation criteria.