thermomycolin

Endpoint summary

Description of key information

Skin corrosion (OECD 431): not corrosive

Skin irritation (OECD 404): not irritating

Eye irritation (OECD 405): not irritating

Eye corrosion (OECD 438): not corrosive

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Sept. 23-24, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test procedure was a GLP study performed similar to the principles of OECD guideline 431 (2010)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

29 July 2016

Deviations:

yes

Remarks:

Fresh MTT solution was prepared - no negative influence on the study outcome.

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Vehicle:

unchanged (no vehicle)

Remarks:

Direct application to the EpiDermTM, but wetted with water

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg was applied to the individual reconstituted skin membranes and wetted with 50 µL milliQ water

Duration of treatment / exposure:

3 min and 1 hour

Vehicle:

water

Details on study design:

The test compound Subtilisin and two other enzyme samples were assessed by being applied topically to the EpiDermTM Skin Model for 3 minutes and 60 minutes. Subsequently, the effect on the tissue viability was determined, based on the reduction of MTT to a purple formazan precipitate by mitochondrial dehydrogenase activity. The test compound was tested as a neat product. Positive control substance was 8M KOH.
The study was conducted to comply with the following guidelines:
- Organisation for Economic Co-operation and Development (OECD). New test guideline 431. OECD Guideline for the testing of chemicals. Draft proposal for a new guideline 431. In vitro skin corrosion: human skin model test (2002).
- Commission directive 2000/33/EC. Official Journal of the European Communities L136/90 – 107. Annex I, B.40. Skin Corrosion.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

79.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure

Value:

80.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The test compound Subtilisin batch PPA 1619, was examined for in vitro skin corrosive potency. The EpiDermTM Skin Model (EPI-200) was exposed to the test compound for 3 minutes and one hour, after which the viability of the EpiDermTM Skin Model was assessed by measuring the conversion of MTT. Based on this assay, the test compound Subtilisin was predicted as non-corrosive.

Formazan production in EpiDerm^TMskin membranes and negative and positive controls for 3 minutes and 1 hour:

                                                                

3-minute exposure
Test group	Test compound	A540(% of control)
NC	Negative control (demineralised water)	100.0
A	Subtilisin (PPA 1619)	79.1
PC	Positive control (8 M KOH)	10.2
1-hour exposure
Test group	Test compound	A540(% of control)
NC	Negative control (demineralised water)	100.0
A	Subtilisin (PPA 1619)	80.8
PC	Positive control (8 M KOH)	8.2

 

 

Interpretation of results:

GHS criteria not met

Conclusions:

There is regulatory acceptance in the EU that a substance can be considered corrosive (Skin Corrosive Cat.1A) based on a positive result in the human epidermis model test. Negative in vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant and shall therefore be subject to further evaluation.

Executive summary:

An EpiDerm^TMSkin irritation test was conducted to assess the potential of the test substance, Subtilisin (batch PPA 1619), to cause skin corrosion, by means of an in vitro test. The EpiDerm^TMtest has been accepted as a replacement to the in vivo rabbit skin irritation test, by the European Centre for the Validation of Alternative Methods (ECVAM), and a draft proposal for a new OECD test guideline (TG 431) was followed. The study was conducted in accordance with GLP. It was concluded that the test substance, subtilisin, was predicted as a non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Oct. 12-27, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test procedure was according to the OECD 404 and according to GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Bred by Charles River, UK
- Weight at study initiation: between 2.45 - 2.65 kg
- Age at study initiation: Young adults, approx. 3 months
- Housing: Individually, in animal room with control of temperature (15-23°C) and humidity (40-70%RH)
- Diet (e.g. ad libitum): Standard diet ad libitum
- Water (e.g. ad libitum): Automatic, ad libitum
- Acclimation period: 7 days
- Photoperiod (hrs dark / hrs light): 12 hrs/12hrs

IN-LIFE DATES: From: 1994-10-12 To: 1994-10-27

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

not required

Amount / concentration applied:

0.5 mL of the test material diluted to retail activity (445 mg test material made up to 1000 mg with water), corresponding to a solution with 4.5% active enzyme protein

Duration of treatment / exposure:

4 hours

Observation period:

16 days

Number of animals:

3

Details on study design:

The test compound Subtilisin was assessed by semiocclusive application of 0.5 mL of the test material formulation to the closely-clipped dorsa of three New Zealand White rabbits for 4 hrs.
The study was conducted to comply with the following guidelines:
- Organisation for Economic Co-operation and Development (OECD) test guideline 404.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: Very slight exfoliation was evident until termination of the study (day16) in the present animal.

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: Exfoliation was seen until day 13 in this animal, all other effects were cleared at day 10.

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.3

Max. score:

4

Reversibility:

fully reversible

Irritant / corrosive response data:

Very slight or well defined erythema and very slight to moderate oedema were observed at the test sites of two animals during the first 72 hrs after bandage removal. One of these animals also showed a dry abrasion over the majority of the test site during this time. One week after treatment, very slight erythema and exfoliation were evident in both animals. The exfoliation persisted until day 13 in one animal and to termination of the study in the other. No dermal response was observed at the tests site of the third animal at any time during the observation period.
The mean values for erythema and oedema recorded 24, 48 and 72 hours after treatment did not exceed the limit values considered to indicate a significant inflammatory response.

Interpretation of results:

GHS criteria not met

Conclusions:

According to EC-standards (as published in the Official Journal of the European Communities, L 110 A, 4 May 1993), the test material is not irritating to skin. Concentration of solution applied was 4.5% expressed as active enzyme protein.

CLP: not classified

Executive summary:

An in vivo skin irritation test was conducted to assess the potential of the test substance, Subtilisin, batch PPA 4852, to cause skin irritation. The OECD test guideline 404 was followed. 0.5 mL of the test material formulation was applied under semiocclusive conditions to the closely-clipped dorsa of three New Zealand White rabbits for 4 hrs. The study was conducted in accordance with GLP. The study concluded that the test substance, Subtilisin, was non-irritant to the skin.

However due to the very slight exfoliation still seen in one animal at the termination of the study, Subtilisin should be regarded as slightly irritant to skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

June 24-27 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test procedure was a GLP study performed according to OECD guideline 405

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

02 Oct 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Centre Lagro, France
- Age at study initiation: young adults
- Weight at study initiation: 2008-2210 g
- Housing: induvidually in stainless steel cages with perforated floor
- Diet (e.g. ad libitum): Standard diet ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23.9 C (target 17 - 23 C)
- Humidity (%): 30-70 %
- Air changes (per hr): ca 10 air changes per hr
- Photoperiod (hrs dark / hrs light): 12 hrs light/ 12 hrs dark cycle

IN-LIFE DATES: From: 24 June 2002 To: 27 June 2002

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): Undiluted test material

Observation period (in vivo):

The reactions were judged 1, 24, 48 and 72 hours after treatment

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No washing

SCORING SYSTEM: According to OECD 405

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 0.7

Max. score:

3

Reversibility:

fully reversible within: 72 hrs

Remarks on result:

other: Small haemorrhage on the nictitating membrane at 1h reading, cleared at 24h

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.7

Max. score:

3

Reversibility:

fully reversible within: 72 hrs

Remarks on result:

other: Small haemorrhage on the nictitating membrane at 1h reading, cleared at 24h

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 hrs

Remarks on result:

other: Small haemorrhage on the nictitating membrane at 1h reading, cleared at 24h

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 hrs

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 hrs

Remarks on result:

other: Discharge score 1 at 1 h, fully cleared at 24h

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 hrs

Remarks on result:

other: Discharge score 2 at 1 h, fully cleared at 24h

Irritation parameter:

cornea opacity score

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritation parameter:

iris score

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritant / corrosive response data:

At the 1 hour reading, Subtilisin generally caused slight or moderate redness and slight or moderate swelling of the conjunctivae and slight or moderate ocular discharge in the rabbits. In addition, all rabbits showed a small haemorrhage on the nictitating membrane. After 24 hours, slight to moderate redness and swelling was observed in all treated rabbits. After 48 hours only slight redness was observed and 72 hours after treatment, all eye effects had cleared completely.

Interpretation of results:

other: CLP/ EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

ClP: not classified

Executive summary:

The study was carried out according to OECD guideline no. 405. Three rabbits were treated as follows: One rabbit was treated first and two more the following day. An amount of 0.1 ml of the undiluted test substance was instilled in the conjunctival cul-de-sac of one of the eyes of the rabbit. After administration, the upper and the lower eye lid were carefully closed and subsequently held together for at least one second before releasing to prevent loss of material. The other eye remained untreated and served as a control. The reactions of the eyes were judged at 1 h, 24 h, 48 h and 72 h after treatment.

At the 1 hour reading, Subtilisin generally caused slight or moderate redness and slight or moderate swelling of the conjunctivae and slight or moderate ocular discharge in the rabbits. In addition, all rabbits showed a small haemorrhage on the nictitating membrane. After 24 hours, slight to moderate redness and swelling was observed in all treated rabbits, while the haemorrhage on the nictitating membrane was no longer present. After 48 hours only slight redness was observed and 72 hours after treatment, all eye effects had cleared completely. According to EC-standards (as published in the Official Journal of the European Communities, L 110 A, volume 36, 4 May 1993), Subtilisin batch PPA 6865 is not irritating to eyes.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

May 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to GLP and the procedures were according to the later OECD guideline 438 (adopted 2009).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

6 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Vehicle:

unchanged (no vehicle)

Controls:

not specified

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The chicken eye cornea was treated with 0.03 mL
- Concentration (if solution): undiluted test sample

Duration of treatment / exposure:

The exposure period was 10 seconds

Observation period (in vivo):

The eyes were examined at ca 0, 30, 75, 120, 180 and 240 minutes after treatment.

Details on study design:

Subtilisin was examined for potential irritation/corrosive properties in an ex vivo bioassay, the Chicken Enucleated Eye Test (CEET). Three main parameters were measured to disclose possible adverse effects, the corneal swelling, corneal opacity and fluorescein retention of damaged epithelial cells.
The study was carried out at the contract lab. TNO. Three enucleated chicken eyes per sample and one control per test run were selected for testing. At time t = 0, immediately after a zero reference measurement, the following procedure was applied for each test eye: the eye cornea was treated with 0.03 ml of the undiluted test sample. After an exposure period of 10 seconds, the corneal surface was rinsed thoroughly with 20 ml isotonic saline of ambient temperature. The control eye was treated with saline only. The eyes were examined at ca 0, 30, 75, 120, 180 and 240 minutes after treatment. Fluorescein retention was only evaluated at ca 30 minutes after treatment. All examinations were carried out with the slit-lamp microscope.
In addition, the eyes were collected for histopathological examination of the cornea.

Irritation parameter:

fluorescein retention score

Value:

0

Irritation parameter:

cornea opacity score

Value:

0

Other effects / acceptance of results:

After treatment, the corneal thickness of the test eyes generally remained unchanged or only showed a very slight increase. None of the three eyes showed any corneal opacity or fluorescein retention. Microscopic examinations of the treated corneas did not reveal any additional information on possible adverse effects.

Interpretation of results:

other: non-corrosive (Eye Irrit. 2 or not classified according to Regulation (EC) No. 1272/2008)

Conclusions:

Under the conditions of the test, the test substance was shown to have no corrosive potential in the Isolated chicken eye (ICE) test prediction model. The result does not allow for the non-classification or classification as irritant of the test substance and therefore further evaluation and/or data generation is required.

Executive summary:

Subtilisin was examined for potential irritation/corrosive properties in an ex vivo bioassay, the Chicken Enucleated Eye Test.

Three main parameters were measured to disclose possible adverse effects, the corneal swelling, corneal opacity and fluorescein retention of damaged epithelial cells.

Three enucleated chicken eyes per sample and one control were selected for testing. The individual eye cornea was treated with 0.03 ml of the undiluted test sample. After an exposure period of 10 seconds, the corneal surface was rinsed with 20 ml isotonic saline. The control eye was treated with saline only. The eyes were examined at ca 0, 30, 75, 120, 180 and 240 minutes after treatment. All examinations were carried out with the slit-lamp microscope.

In addition, the eyes were collected for histopathological examination of the cornea.

The results showed that after treatment, the corneal thickness of the test eyes generally remained unchanged or only showed a very slight increase. None of the three eyes showed any corneal opacity or fluorescein retention. Microscopic examinations of the treated corneas did not reveal any additional information on possible adverse effects.

According to the EC classification system of the Chicken Enucleated Eye Test, it was concluded that the tested Subtilisin batch was not irritating to eyes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for read-across

There are no data for skin and eye irriation / corrosion available for thermomycolin (CAS 52233-31-5). To fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VII, 8.1 and 8.2, read-across from appropriate substances is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. According to Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met”. In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across) “to avoid the need to test every substance for every endpoint”.

For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substances are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

As no experimental/ measured data are available on skin and eye irriation / corrosion of thermomycolin (CAS 52233-31-5), read-across to reliable data on the analogue substance subtilisin (CAS 9014-01-1) was applied.

Skin Irritation / Corrosion

The skin corrosion potential of subtilisin was investigated in a study according to a draft proposal for a new OECD test guideline OECD 431 and in compliance with GLP (Maas, 2004). The EpiDermTM Skin model was exposed to 25 mg of the unchanged test material for 3 minutes and 1 hour. The MTT test revealed mean tissue viabilities of 79.1% and 80.8%. Therefore, the test material is concluded to be non-corrsive to skin.

The skin irritation potential of subtilisin was investigated in a study according to OECD 404 and in compliance with GLP (Rees, 1994). 0.5 mL of the test material formulation was applied under semiocclusive conditions to the closely-clipped dorsa of 3 New Zealand White rabbits for 4 hours. The mean values for erythema and oedema recorded 24, 48 and 72 hours after treatment did not exceed the limit values considered to indicate a positive response. However, very slight exfoliation was observed in one animal at the termination of the study, and therefore it was concluded that subtilisin was a mild irritant to skin. Nevertheless, the effects observed in the study were not severe enough to meet the criteria for classification as a skin irritant.

Furthermore, an additional in vivo skin irritation test based on the procedure of the US Federal Register, § 191.11, 17 Sept. 1964 is available. The study was conducted before GLP was implemented but the principles were the same (Lightowler and Gardner, 1977). 0.5 mL of the test material formulation (3.4% expressed as active enzyme protein) was applied under semiocclusive conditions to the closely-clipped dorsa of 12 New Zealand White rabbits for 24 hours. The mean values for erythema and oedema recorded 24 and 72 hours after treatment did not exceed the limit values considered to indicate a positive response. Thus, the test material is not concluded to be a skin irritant.

Eye Irritation / Corrosion

The eye corrosion potential of subtilisin was investigated tested in a study according to OECD 438 and in compliance with GLP (Prinsen, 2002). Three enucleated chicken eyes were exposed to 30 μL of the neat/ undiluted test material for 10 seconds and was incubated for another 4 hours after rinsing and removal of the test material. Three main parameters were measured to disclose possible adverse effects: the corneal swelling, corneal opacity and fluorescein retention of damaged epithelial cells. The results showed that after treatment the corneal thickness of the test eyes generally remained unchanged or only showed a very slight increase. None of the three eyes showed any corneal opacity or fluorescein retention. Microscopic examinations of the treated corneas did not reveal any additional information on possible adverse effects. Therefore, the test material is not regarded as an eye irritant.

The eye irritation potential of subtilisin was investigated in a study according to OECD 405 and in compliance with GLP (Prinsen, 2002). Three rabbits were treated as follows: One rabbit was treated first and two more the following day. An amount of 0.1 mL of the undiluted test material was instilled in the conjunctival cul-de-sac of one of the eyes of the rabbit. After administration, the upper and the lower eye lid were carefully closed and subsequently held together for at least one second before releasing to prevent loss of material. The other eye remained untreated and served as a control. At the 1 hour reading, the test material generally caused slight or moderate redness and slight or moderate swelling of the conjunctivae and slight or moderate ocular discharge in the rabbits. In addition, all rabbits showed a small haemorrhage on the nictitating membrane. After 24 hours, slight to moderate redness and swelling was observed in all treated rabbits, while the haemorrhage on the nictitating membrane was no longer present. After 48 hours only slight redness was observed and 72 hours after treatment, all eye effects had cleared completely. The mean values for erythema and oedema recorded 24, 48 and 72 hours after treatment did not exceed the limit values considered to indicate a positive response. Therefore, test material is not regarded as an eye irritant.

Despite the outcome of the available skin and eye irritation/corrosion studies, which concluded that subtilisin was neither a skin nor an eye irritant, due to its catalytic activity (protease), subtilisin nevertheless has the potential to cause skin and eye irritation.

The source substance subtilisin has a harmonised classification in Annex VI of the Regulation (EC) 1272/2008. The substance is classified for the following:

- Skin Irritant Category 2 (H315)

- Eye Damage Category 1 (H318)  

  

Conclusion on skin and eye irritation properties

Reliable data available for the read-across analogue substance subtilisin (CAS 9014-01-1) on skin and eye irritation/corrosion indicate that thermomycolin (CAS 52233-31-5) is not a skin or eye irritant.

Justification for classification or non-classification

The source substance subtilisin has a harmonised classification in Annex VI of the Regulation (EC) 1272/2008. Therefore,  as skin and eye irritation data on the source substance subtilisin has been read-across to thermomycolin, the registered substance is also classified for the following:

- Skin Irritant Category 2 (H315)

- Eye Damage Category 1 (H318)