3-Pentanone, 1-hydroxy-2-methyl-

Endpoint summary

Description of key information

Skin irritation test (SIT, EpiDerm): irritating

Skin corrosion test (SCT, EpiDerm): inconclusive

Membrane Barrier Test (Corrositex): non-corrosive

Eye irritation test (EpiOcular): irritating

Test for serious eye damage (BCOP): inconclusive

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test-substance No.: 15/0439-1
Batch identification: J 1812
Purity: 90.6 area-%
Homogeneity: The test substance was homogeneous by visual inspection.
Storage stability: The stability under storage conditions over the study period was guaranteed by the sponsor.
pH-value: Ca. 5 (undiluted test substance; determined in the lab prior to start of the GLP study)
Physical state / color: Liquid / yellowish, clear
Storage conditions: Room temperature, protect against humidity ; no direct sunlight

Test system:

human skin model

Source species:

human

Vehicle:

unchanged (no vehicle)

Details on test system:

The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm diameter) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.

Tissue model: EPI-200
Tissue Lot Number: 23309
Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Fifty microliter (50 μL) of the undiluted liquid test substance was applied using a pipette.
Control tissues were concurrently treated with 50 μL of de-ionized water (NC) or with 50 μL of 8 N potassium hydroxide (PC).

Duration of treatment / exposure:

3 minutes at room temperature (2 tissues) and 1 hour in the incubator (2 tissues)

Number of replicates:

Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator, as a rule) and test group (test material, negative control and positive control; 12 tissues per test)
were used.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure mean

Value:

88.4

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure mean

Value:

13.7

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

See "attached background material"

Conclusions:

The borderline result of the skin corrosion test (viability of 13.7% after an exposure period of 1 hour) do not clearly allow the evaluation of the skin corrosion potential. On basis of the results of this study the corrosive potential of the test substance cannot be excluded.

Executive summary:

The potential of Hydroxymethylpentanon to cause dermal corrosion/irritation was assessed by a single topical application of 50 μL of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™).

Two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test-substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 88.4%, and it was 13.7% after an exposure period of 1 hour.

Based on the observed results it has to be concluded, that the results of the test substance Hydroxymethylpentanon should be considered “inconclusive” in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Dec 2015 - Feb 2016

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name of test substance: Hydroxymethylpentanon
Batch identification: J 1812
CAS No.: 27970-79-2
Purity: 90.6 area-%
Homogeneity: The test substance was homogeneous by visual inspection.
pH-value: Ca. 5 (undiluted test substance; determined in the lab prior to start of the GLP study)
Physical state / color: Liquid / yellowish, clear
Storage conditions: Room temperature, protect against humidity ; no direct sunlight

Test system:

human skin model

Source species:

human

Cell type:

other: 3D human skin model

Vehicle:

unchanged (no vehicle)

Details on test system:

On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours.
Three tissues were treated with the test substance, the PC and NC, respectively. Thirty microliter (30 μL) of the undiluted liquid test substance was applied using a pipette. Control tissues were concurrently treated with 30 μL of sterile PBS (NC) or with 30 μL of 5% SDS (PC). A nylon mesh was placed carefully onto the tissue surface of the NC and PC afterwards. The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator. The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were placed into the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period.
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 uL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 uL sterile PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 uL
- Concentration (if solution): 5% SDS

Duration of treatment / exposure:

1 hour (25 minutes at room temperature and 35 minutes in 37°C incubator)

Duration of post-treatment incubation (if applicable):

24 ± 2 hours and another 18 ± 2 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean values

Run / experiment:

negative control

Value:

100

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value

Run / experiment:

positive control

Value:

3.1

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value

Run / experiment:

Hydroxymethylpentanon

Value:

3.3

Negative controls validity:

valid

Positive controls validity:

valid

Executive summary:

The potential of Hydroxymethylpentanon to cause dermal irritation was assessed by a single topical application of 30 μL of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). The irritation test was performed with three EpiDerm™ tissues, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test-substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 3.3%.

Reference 3

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name of test substance: Hydroxymethylpentanon
Batch identification: J 1812
CAS No.: 27970-79-2
Purity: 90.6 area-%
Homogeneity: The test substance was homogeneous by visual inspection.
pH-value: Ca. 5 (undiluted test substance; determined in the lab prior to start of the GLP study)
Physical state / color: Liquid / yellowish, clear
Storage conditions: Room temperature, protect against humidity ; no direct sunlight

Test system:

artificial membrane barrier model

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Duration of treatment / exposure:

A membrane disc coated with the biobarrier matrix was placed into one vial containing the CDS and 500 μL of the undiluted test substance was added onto the membrane disc. An electronic time clock was started with the application. The vial was observed for three minutes for any change in the CDS.
If no color change was observed within three minutes, the remaining membranes were treated with the test substance. An electronic time clock was started with each application. The vials were observed continuously for the first ten minutes. Thereafter the vials were observed for approximately ten minutes around the time points relevant for evaluation (see table in section 3.8) or until breakthrough of the test substance occurred.

Number of replicates:

Four vials containing the CDS were used for the test substance. In addition, one vial was used for the PC, NC and for the color (blank) control, each.

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

mean

Value:

> 60

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

see attached background material for detailed results.

Conclusions:

According to the findings in the Corrositex assay under the conditions tested, the test substance was not corrosive.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

weight of evidence

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
Test-substance No.: 15/0439-1
Batch identification: J 1812

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protect against humidity ; no direct
sunlight

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: freshly slaughtered cattle; Schlachthof Mannheim, Schlachthofstr. 21, 68165
Mannheim, Germany
- Age of the animals: minimum 12 months, maximum 60
months
- indication of any existing defects or lesions in ocular tissue samples: no

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL of the undiluted liquid test substance

Duration of treatment / exposure:

approximately 10 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to
3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour.
After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an
opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 544 opacity units1 were discarded. The remaining corneas were then distributed into negative control, positive control and treatment groups.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
de-ionized water

POSITIVE CONTROL USED
100% ethanol, 100% dimethylformamide

APPLICATION DOSE AND EXPOSURE TIME
750 μL of the undiluted liquid test substance was applied into the anterior chamber
Exposure time: approximately 10 minutes

POST-INCUBATION PERIOD: yes. 2 hours

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red).
- POST-EXPOSURE INCUBATION:
The corneas were incubated for further 2 hours at about 32 °C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.


SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
A study is considered acceptable if the PC gives an IVIS that falls within two standard
deviations of the current historic mean.
The NC responses should result in opacity and permeability values that are not higher than the established upper limits.
Since the IVIS per treatment group is determined from the mean of three single corneas, the
variability between the corneas treated per test substance should be acceptably low.
In cases of borderline results in the first testing run, a second testing run should be considered (but not necessarily required), as well as a third one in case of discordant mean IVIS results between the first two testing runs. In this context, a result in the first testing run is considered borderline if the predictions from the 3 corneas were non-concordant, such that:
- 2 of the 3 corneas gave discordant predictions from the mean of all 3 corneas, OR,
- 1 of the 3 corneas gave a discordant prediction from the mean of all 3 corneas, AND the
discordant result was >10 IVIS units from the cut-off threshold of 55.

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

39.7

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: permeability score

Run / experiment:

mean

Value:

0.514

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

47.4

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

See "attached background material"

Irritant / corrosive response data:

See "attached background material"

See "attached background material"

Conclusions:

The borderline result of the BCOP Test (mean IVIS 47.4) does not clearly allow for the evaluation of risk of serious eye damage. On basis of the results of this study a potential of the
test substance to bear a risk of serious eye damage cannot be excluded.

Executive summary:

The potential of Hydroxymethylpentanon to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 μL of the undiluted test substance to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test substance for 10 minutes followed by a 2-hours postincubation period. In addition to the test substance a negative control (NC; de-ionized water) and two positive controls (PC1: 100% ethanol / PC2: 100% dimethylformamide) were applied to three corneas each.

Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance.

Hydroxymethylpentanon showed an IVIS of 47.4 in the BCOP Test. Therefore, a potential of the test substance to bear a risk of serious eye damage cannot be excluded, the study has to be considered inconclusive.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test-substance No.: 15/0439-1
Batch identification: J 1812

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protect against humidity ; no direct
sunlight

Species:

human

Details on test animals or tissues and environmental conditions:

The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinocytes used to model the human corneal epithelium. The EpiOcularTM tissues (surface 0.6 cm²) are cultured on cell culture inserts (MILLICELLs, 10 mm diameter) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.

Tissue model: OCL-200
Tissue Lot Number: 21593
Supplier: MatTek In Vitro

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

fifty microliter (50 μL) of the undiluted liquid test substance was applied covering the whole tissue surface.
Control tissues were concurrently applied with 50 μL of sterile de-ionized water (NC) or with 50 μL of methyl acetate (PC).

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

2

Details on study design:

EXPERIMENTAL PROCEDURE

To assess the ability of the test material to directly reduce MTT a pretest was performed. The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 3 hours. A negative control (de-ionized water) was tested concurrently. If the color of the MTT
solution or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT.
-Basic procedure:
Two tissues were treated with each, the test substance, the PC and the NC.
Pre-incubation of the tissues:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours.
-Pretreatment of the tissues:
After the pre-incubation the tissues were pre-treated with 20 μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.
-Application of the test substance:
Using a pipette, fifty microliter (50 μL) of the undiluted liquid test substance was applied covering the whole tissue surface.
Control tissues were concurrently applied with 50 μL of sterile de-ionized water (NC) or with 50 μL of methyl acetate (PC).
After application, the tissues were placed into the incubator until the total exposure time of 30 minutes was completed.
-Removal of the test substance and postincubation period:
To remove the test substance, the tissues were washed with sterile PBS. For this purpose the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well prewarmed medium (post-soak immersion) in order to remove residual test substance. After 12 minutes of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium. Subsequently, the tissues were incubated at standard culture conditions for 2 hours.
-MTT incubation:
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT-incubation.
The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

- RhCE tissue construct used, including batch number:
Tissue model: OCL-200
Tissue Lot Number: 21593
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

- Evaluation of results:
The irritation potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile water. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 60%.

Irritation parameter:

other: viability value

Run / experiment:

1

Value:

4.3

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: viability value

Run / experiment:

2

Value:

6

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: viability value

Run / experiment:

mean

Value:

5.2

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: no

Conclusions:

Based on the observed result for the EpiOcular Test alone Hydroxymethylpentanon shows an eye irritation potential under the test conditions chosen. However, the test method does not
yet allow for the evaluation of serious eye damage.

Executive summary:

The potential of Hydroxymethylpentanon to cause ocular irritation was assessed by a single topical application of 50 μL of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™). Two EpiOcular™ tissues were incubated with the test substance for 30 minutes followed by a 2-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test-substance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability.

The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues was 5.2%.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion:

The potential of Hydroxymethylpentanon to cause dermal corrosion/irritation was assessed by a single topical application of 50 μL (corrosion test) or 30 μL (irritation test) of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). For the corrosion test two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each. The irritation test was performed with three EpiDerm™ tissues, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test-substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The following results were obtained in the EpiDerm™ skin corrosion/irritation test:

Corrosion test: The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 88.4%, and it was 13.7% after an exposure period of 1 hour.

Irritation test: The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 3.3%.

Additionally, a membrane barrier test (Corrositex) was performed to assess the corrosive potential of the test substance. According to the prediction model, the test substance was not corrosive.

The borderline result of the skin corrosion test do not allow for a clear evaluation of the skin corrosion potential. On basis of the data available the corrosive potential of the test substance cannot be excluded.

Eye irritation:

The potential of Hydroxymethylpentanon to cause ocular irritation was assessed by a single topical application of 50 μL of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™). Two EpiOcular™ tissues were incubated with the test substance for 30 minutes followed by a 2-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test-substance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues was 5.2%, indicating that the substance is at lease irritating to eyes.

To determine whether Hydroxymethylpentanon would cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 μL of the undiluted test substance to the epithelial surface of isolated bovine corneas (BCOP test). Three corneas were treated with the test substance for 10 minutes followed by a 2-hours postincubation period. In addition to the test substance a negative control (NC; de-ionized water) and two positive controls (PC1: 100% ethanol / PC2: 100% dimethylformamide) were applied to three corneas each. Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of 47.4 for the test substance.

The borderline result of the BCOP test do not allow for a clear evaluation of the eye damage potential. On basis of the results of this study potential of the test substance to cause severe eye damage cannot be excluded.

Justification for classification or non-classification

Based on the data available, Hydroxymethylpentanon is irritating to skin and irritating to eyes, however as to whether it is corrosive to skin and has the potential to cause serious eye damage is not clear. In line with the precautionary principle, classification as corrosive to skin category 1C (H314) as well as causes serious eye damage (H318) is assigned.