4-methylpent-3-en-2-one

Endpoint summary

Description of key information

Mesityl oxide has an irritant effect on the skin of the rabbit (OECD 404) following a 4 hour dermal exposure period, even though with a high individual variability. In a bovine corneal opacity and permeability (BCOP, OECD 437), mesityl oxide was not defined as corrosive or severely irritant to the eye. However, since the in vitro irritancy score (41.2)  value is near to the threshold of severe irritancy/corrosion, mesityl oxide might have an irritating potential in vivo. Mesityl oxide was irritant for the respiratory tract in human and animal studies.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in 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 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan England, Hillcrest, Dodgeford Lane, Belton, Loughborough, Leicestershire, LE12 9TE, England.
- Age at study initiation: approximately 15 to 17 weeks
- Weight at study initiation: 3.06 to 3.33 kg
- Housing: 1/cage; grid-bottomed metal cages suspended over trays measuring 48x41x63 cm
- Diet (e.g. ad libitum): ad libitum - STANRAB (P) SQC, Special Diets Services, Witham, Essex CM8 3AD, UK
- Water (e.g. ad libitum): ad libitum - Tap water
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): aproximately 15 to 25
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 6 to 22 June 2010

Type of coverage:

semiocclusive

Preparation of test site:

other: clipping

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 ml/site; 1 site/animal
- Concentration (if solution): as supplied

Duration of treatment / exposure:

4 hours

Observation period:

14 days

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 2.5 x 2.5 cm
- Type of wrap if used: the test item was spread evenly over a square gauze measuring 2.5x2.5 cm.
The square gauze was then placed onto the animal’s skin with the test item in direct contact with the skin.
A strip of synthetic film was placed over the treated site and the whole assembly held in place by encircling the trunk of the animal with a length of elastic adhesive bandage, this forming a semi-occlusive barrier.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): swabbing of the skin with cotton wool soaked with lukewarm water.
- Time after start of exposure: 4 hours

SCORING SYSTEM: Draize scale
Erythema and eschar formation
0 - No erythema
1 -Slight erythema
2 - Well defined erythema
3 - Moderate to severe erythema
4 - Severe erythema (beef redness) to eschar formation preventing grading of erythema

Oedema formation
0 -No oedema
1 - Very slight oedema (barely perceptible)
2 - Slight oedema (edges of area well defined by definite raising)
3 - Moderate oedema (raised approximately 1mm)
4 - Severe oedema (raised more than 1 mm and extending beyond the area of exposure

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

other: 24 to 72 hours

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: Score of 1 recorded also 7 days after treatment; see histopathology

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

other: 24 to 72 hours

Score:

0

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Remarks on result:

other: Presence of desquamation 7 and 14 days after treatment; see histopathology

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

other: 24 to 72 hours

Score:

1.3

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: Score of 1 or 3 recorded respectively 7or 14 days after treatment; see histopathology

Irritation parameter:

edema score

Basis:

animal: # 1, 2 and 3

Time point:

other: 24 h to 14 days

Score:

0

Max. score:

4

Irritant / corrosive response data:

Reaction at the treatment site was recorded in the following three animals:

Rabbit no. 80720001: slight erythema (score of 1) was recorded from 24 hours to 7 days after dosing. No reaction was observed on Day 15. No oedema was observed.
The calculated mean erythema and oedema scores over 24, 48 and 72 hours were 1.0 and 0.0, respectively.

Rabbit no. 80720003: No erythema or oedema (score 0) occurred at the treatment site up to 72 hours after dosing. Desquamation of the treatment site was recorded on Days 8 and 15. No oedema was observed.
The calculated mean erythema and oedema scores over 24, 48 and 72 hours were 0.0.

Rabbit no. 80720005: Well defined erythema (score of 2) was recorded 1 and 24 hours after dosing. Slight erythema (score of 1) at 48 hours, 72 hours and 7 days. Moderate to severe erythema (score of 3) was finally recorded on Day 15. No oedema was observed.
The calculated mean erythema and oedema scores over 24, 48 and 72 hours were 1.3 and 0.0, respectively.

Other effects:

Macroscopic observations
The treatment site showed multiple scabs (up to 1x1 mm) in the female no. 3 and multiple red areas in the cutis (up to 25x8 mm) were noted in the female no. 5, while no macroscopic change at the treatment site was noted in female no. 1.

Microscopic observations
The histopathological evaluation of the above-mentioned treatment sites revealed in female no. 1 focal slight acute inflammation in the dermis and in the female no. 80720003 focal scabs and chronic inflammatory reaction in the dermis.
Female no. 80720005 showed a most relevant pathological change in the treatment site such as multifocal mild haemorrhage associated to hyperplasia of follicular basal cell (hair follicles) and squamous cell layer with acanthosis of the epidermis.
As reported in the literature, the follicular basal cell hyperplasia may be considered to contribute to repair the epidermal wounding.

Interpretation of results:

Category 2 (irritant)

Remarks:

Migrated information based on the histopathological examinations Criteria used for interpretation of results: expert judgment

Conclusions:

These results indicate that the test item, Mesityl oxide, has an irritant effect on the skin of the rabbit following a 4 hour dermal exposure period, even though with a high individual variability. Reaction was moderately stronger in one animal and recovery was not completed in two animals at the end of the 14-day observation period.

Executive summary:

The acute dermal irritation of Mesityl oxide (purity 99.87%) was investigated in the rabbit in an OECD 404 study. A 0.5 mlaliquot of the substance was applied to the clipped dorsal skin of 3 animals for a period of 4 hours. The resulting reaction to treatment was assessed approximately 1, 24, 48, 72 hours, 7 and 14 days after the end of the exposure period. In the first rabbit slight erythema (score of 1) was recorded from 24 hours to 7 days after dosing. No reaction was observed on Day 15 (calculated mean erythema score over 24, 48 and 72 hours equal to 1.0). No oedema was observed. In the second rabbit, no erythema or oedema (scores of 0) occurred at the treatment site up to 14 days after dosing. Desquamation of the treatment site was recorded on Days 8 and 15. In the third rabbit, well defined erythema (score of 2) was recorded 1 and 24 hours after dosing, slight erythema (score of 1) at 48 hours, 72 hours and 7 days. Moderate to severe erythema (score of 3) was finally recorded on Day 15 (calculated mean erythema score over 24, 48 and 72 hours equal to 1.3). No oedema was observed. There was no indication of a systemic effect of treatment. Body weight changes were not remarkable. The histopathological evaluation of the treatment sites revealed, a focal and slight acute inflammation in the dermis in the 1^stfemale, focal scabs and chronic inflammatory reaction in the dermis in the 2^ndfemale and a multifocal and mild haemorrhage associated to hyperplasia of follicular basal cell (hair follicles) and squamous cell layer with acanthosis of the epidermis in the 3^rdfemale. As reported in the literature, the follicular basal cell hyperplasia may be considered to contribute to repair the epidermal wounding. These results indicate that the test item, Mesityl oxide, has an irritant effect on the skin of the rabbit following a 4-hour dermal exposure period, even though with a high individual variability. Reaction was moderately stronger in one animal and recovery was not completed in two animals at the end of the 14-day observation period.           

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

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:

other: Fully GLP Compliant

Qualifier:

according to guideline

Guideline:

other: OECD 437 "Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants (adopted 7 September 2009)

Deviations:

no

GLP compliance:

yes

Species:

other: Bovine

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
Eye supply: Slaughter house
Age of animals : 6-12 months
Killing time: Approximately 8:30 to 11:00 in the morning (used in early afternoon).
Transport condition: HBSS 1x plus Penicillin/streptomicin maintained at approximately 4°C.
Approximate test starting time: 13:30

PREARATION OF CORNEAS

- Eyes were examined for the presence of any defects
- Each cornea with 2-3 mm of surrounding sclera was dissected from the eye using a scalpel, scissors and forceps and placed into a Petri dish containing HBSS.
- Each cornea was mounted into a pre-warmed testing chamber with the endothelial surface of the cornea placed in contact with the
O-ring of the posterior part of the chamber.
- The chamber was then filled with EMEM without phenol red maintained at 32 ± 1°C (posterior part of the chamber first to maintain convexity).

EQUILIBRATION OF CORNEAS
- incubation in a liquid bath at 32 ± 1°C at least 1 hour; the two chambers were drained (anterior first) and re-filled with EMEM without phenol maintained at 32 ± 1°C (posterior first).
Selection : Basal opacity of corneas was determined by means of an opacitometer
Corneas were distributed in treatment groups starting from those with minor opacity (maximum basal opacity score: 1).

Vehicle:

unchanged (no vehicle)

Controls:

other: 3 corneas for each treatment with the positive or negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 ml/cornea for 3 corneas
- Concentration (if solution): as supplied

POSITIVE CONTROL
Identity: Sodium hydroxide (NaOH)
Sigma Cat. No. : S5881
CAS : 1310-73-2
Batch no. : 00014227117
Purity: 98.7 %
- Amount(s) applied (volume or weight with unit): 0.75 ml/cornea for 3 corneas
- Concentration: 10% (w/v) solution in water (sterile water Bieffe Medital, batch 08K2901).

NEGATIVE CONTROL
Identity: Physiological saline (0.9% NaCl; Bieffemedital batch no. 07K1701)
- Amount(s) applied (volume or weight with unit): 0.75 ml/cornea for 3 corneas
- Concentration: As supplied

Duration of treatment / exposure:

Corneas were exposed in horizontal position for 10 minutes incubated in a liquid bath at 32 ± 1°C.

Number of animals or in vitro replicates:

3 corneas for treatment with the test item

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: After exposure, corneas (epythelial side) were rinsed thoroughly with EMEM with phenol red.
Finally, the anterior chamber was re-filled with EMEM without phenol red maintained at 32 ± 1°C.
- Time after start of exposure: 2 hours in a liquid bath at 32 ± 1°C.

TOOL USED TO ASSESS OPACITY: Opacitometer

PERMEABILITY
- On completion of the opacity measurements, EMEM was removed from both chambers (anterior first).
- The posterior chamber was re-filled with fresh EMEM without phenol red maintained at 32 ± 1°C.
- The anterior chamber (epythelial surface) was treated with 1 ml aliquot of sodium fluorescein solution 0.4% in EMEM without phenol red maintained at 32°C ± 1°C. The fluorescein solution used for treatment was checked for concentration (3.864 mg/ml, using a reference curve 0-20 µg/ml - 96.6% of the theoretical concentration).
- The corneas were horizontally incubated in a liquid bath at 32 ± 1°C for approximately 90 minutes.
- The medium was removed from the posterior chamber (endothelial surface) and the optical density measure.

TOOL USED TO ASSESS OPACITY: Spectrophotometer set at 490 nm

Irritation parameter:

other: in vitro irritancy score (IVIS)

Basis:

other: Mesityl oxide

Time point:

other: 10 min

Score:

41.2

Irritation parameter:

other: in vitro irritancy score (IVIS)

Basis:

other: positive control

Time point:

other: 10 min

Score:

165.4

Irritation parameter:

other: in vitro irritancy score (IVIS)

Basis:

other: negative control

Time point:

other: 10 min

Score:

0.35

Irritant / corrosive response data:

The test item induced a slight increase of the corneal opacity with a mean value equal to 11.0.
At the macroscopic observation the three corneas showed slight opacity.

With reference to the permeability, the permeability of the cornea after treatment with fluorescein was similar to that of positive control (slightly minor) with a mean observed OD490 value equal to 2.0114, thus indicating a significant alteration of corneal barrier.

Negative control gave the expected results.
The positive control induced opacity of the whole cornea surface with a mean increase of the opacity value equal to 133.0. The corneal permeability was also increased with a mean observed OD490 value equal to 2.1587. One of the three values was excluded since the OD490 value, 2.384, was over the upper limit of detection of this experiment (OD490 = 2.360 for a 20 µg/ml fluorescein solution).

Mesityl oxide: ASSESSMENT OF OCULAR SEVERE IRRITATION/CORROSION BOVINE CORNEAL OPACITY/PERMEABILITY (BCOP) ASSAY

  

Treatment code		Opacity Value		Macroscopic observation		OD490
N1		0*		No macroscopic changes		0,002
N2		1		No macroscopic changes		0,002
N3		0		No macroscopic changes		0,006
	Mean	0,3			Mean	0,0033
	SD	0,6			SD	0,0023
	CV %	200,0			CV %	69,70
P1		140		Cornea heavily opaque		1,974
P2		126		Cornea heavily opaque		2,384°
P3		134		Cornea heavily opaque		2,350
	Mean	133,3			Mean	2,1620
	SD	7,0			SD	0,2659
	CV %	5,3			CV %	12,30
Corrected Mean		133,0		Corrected Mean		2,1587
(negative control)				(negative control)
A1		13		Cornea slightly opaque		2,275
A2		9		Cornea slightly opaque		1,909
A3		12		Cornea slightly opaque		1,860
	Mean	11,3			Mean	2,0147
	SD	2,1			SD	0,2268
	CV %	18,6			CV %	11,26
Corrected Mean		11,0		Corrected Mean		2,0114
(negative control)				(negative control)

N1-3: Negative control

P1-3 : Positive control

A1-3: Test item

 

*: value read was -1; it was assumed to be 0 for mean calculation.

°: this value was outside the spectrophotometer range, and thus it is excluded from mean calculation

Interpretation of results:

irritating

Remarks:

Migrated information category 2 Criteria used for interpretation of results: other: REGULATION (EC) No 1272/2008

Conclusions:

The treatment with the test item induced a slight effect on corneal opacity and a strong effect on corneal permeability.
According to the criteria stated in the OECD guideline for this test, the test item is not defined as corrosive or severely irritant to the eye.
However, since the calculated IVIS value is near to the threshold of severe irritancy/corrosion, the test item might have an irritating potential in vivo.

Executive summary:

The potential of the test item Mesityl oxide to cause corrosion/severe irritation by using the Bovine corneal opacity and permeability (BCOP) Assay was examined in agreement with OECD guideline no. 437 (adopted 7 September 2009). The test item was tested without any further dilution (being a liquid) on the epithelial surface of three idoneous bovine corneas for an exposure period of 10 minutes and a recovery period of 2 hours. Positive and negative controls [a 10% (w/v) sodium hydroxide (NaOH) solution and physiological saline alone, respectively] were concurrently tested in the same number of replicates at the same treatment volume (0.75 ml). The mean opacity detected at the end of the test item recovery period was 11.0. After the determination of opacity the epithelial surface of every cornea was treated with a 0.4% solution of sodium fluorescein in EMEM for 90 minutes to investigate alteration in permeability. Mean OD₄₉₀value of the medium from the endothelial surface side was 2.0114 (similar to the positive control mean value of 2.1587). Positive and negative controls gave the expected results, indicating the good functioning of the test system. These results indicate that the test item induces slight changes on corneal opacity and has a strong effect on corneal permeability. The calculatedin vitroirritancy score (IVIS) for the test item is 41.2. According to the criteria stated in the OECD guideline for this test, the test item is not defined as corrosive or severely irritant to the eye. However, since the calculated IVIS value is near to the threshold of severe irritancy/corrosion, the test item might have an irritating potential in vivo.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin irritation

The acute dermal irritation of Mesityl oxide (purity 99.87%) was investigated in the rabbit in an OECD 404 study (Corvaro, 2010b). A 0.5 mlaliquot of the substance was applied to the clipped dorsal skin of 3 animals for a period of 4 hours. The resulting reaction to treatment was assessed approximately 1, 24, 48, 72 hours, 7 and 14 days after the end of the exposure period. In the first rabbit slight erythema (score of 1) was recorded from 24 hours to 7 days after dosing. No reaction was observed on Day 15 (calculated mean erythema score over 24, 48 and 72 hours equal to 1.0). No oedema was observed. In the second rabbit, no erythema or oedema (scores of 0) occurred at the treatment site up to 14 days after dosing. Desquamation of the treatment site was recorded on Days 8 and 15. In the third rabbit, well defined erythema (score of 2) was recorded 1 and 24 hours after dosing, slight erythema (score of 1) at 48 hours, 72 hours and 7 days. Moderate to severe erythema (score of 3) was finally recorded on Day 15 (calculated mean erythema score over 24, 48 and 72 hours equal to 1.3). No oedema was observed. There was no indication of a systemic effect of treatment. Body weight changes were not remarkable. The histopathological evaluation of the treatment sites revealed, a focal and slight acute inflammation in the dermis in the 1^stfemale, focal scabs and chronic inflammatory reaction in the dermis in the 2^ndfemale and a multifocal and mild haemorrhage associated to hyperplasia of follicular basal cell (hair follicles) and squamous cell layer with acanthosis of the epidermis in the 3^rdfemale. As reported in the literature, the follicular basal cell hyperplasia may be considered to contribute to repair the epidermal wounding. These results indicate that the test item, Mesityl oxide, has an irritant effect on the skin of the rabbit following a 4-hour dermal exposure period, even though with a high individual variability. Reaction was moderately stronger in one animal and recovery was not completed in two animals at the end of the 14-day observation period.

Eye irritation

The potential of the test item Mesityl oxide to cause corrosion/severe irritation by using the Bovine corneal opacity and permeability (BCOP) Assay was examined in agreement with OECD guideline no. 437 (adopted 7 September 2009) (Corvaro, 2010c). The test item was tested without any further dilution (being a liquid) on the epithelial surface of three idoneous bovine corneas for an exposure period of 10 minutes and a recovery period of 2 hours. Positive and negative controls [a 10% (w/v) sodium hydroxide (NaOH) solution and physiological saline alone, respectively] were concurrently tested in the same number of replicates at the same treatment volume (0.75 ml). The mean opacity detected at the end of the test item recovery period was 11.0. After the determination of opacity the epithelial surface of every cornea was treated with a 0.4% solution of sodium fluorescein in EMEM for 90 minutes to investigate alteration in permeability. Mean OD₄₉₀value of the medium from the endothelial surface side was 2.0114 (similar to the positive control mean value of 2.1587). Positive and negative controls gave the expected results, indicating the good functioning of the test system. These results indicate that the test item induces slight changes on corneal opacity and has a strong effect on corneal permeability. The calculated in vitro irritancy score (IVIS) for the test item is 41.2. According to the criteria stated in the OECD guideline for this test, the test item is not defined as corrosive or severely irritant to the eye. However, since the calculated IVIS value is near to the threshold of severe irritancy/corrosion, the test item might have an irritating potential in vivo.

Mesityl oxide scored an injury grade of 5 (i.e., 0.02 mL undiluted test compound gives a score of over 5.0 points and 0.005 mL not over 5.0 points - out of a maximum of 20) on a scale of 1 to 10, 18 to 24 hours after instillation into the eyes of rabbits (Carpenter and Smyth, 1946).

Respiratory tract irritation

With respect to the respiratory tract, the sensory irritation in the upper part of the respiratory tract was studied by determining the concentration associated with a 50% decrease in the respiratory rate (RD50). Using male Swiss OF1 mice, the RD50 for 4-methylpent-3-ene-2-one was 61 ppm (250 mg/m3) (De Ceaurriz et al., 1984).

Brondeau et al. (1990) found a concentration-related decrease in white blood cell counts (leucopenia) without any change in differential or red blood cell counts in rats after 4-hour exposures to 18, 68, 86, and 130 ppm (74, 278, 352, 532 mg/m3), being statistically different (p< 0.05) from controls at the 2 higher concentrations. This effect was adrenal dependent and Brondeau et al. regarded it as an associative response to sensory irritation.

In testing sensory response of exposure to various organic vapors, Silverman et al. (1946) found that a majority of the 24 subjects experienced some degree of eye and nasal irritation 3 to 5 min after the start of a 15 -min exposure at 25 and 50 ppm (ca.100 and 205 mg/m3), respectively. One of the objectionable after-effects of this solvent was the persistent unpleasant taste that remained with many subjects for 3 to 6 hours after the exposure. Based on the unpleasant taste and nasal irritation at 50 ppm (ca. 205 mg/m3), the majority of the subjects suggested 25 ppm (ca. 100 mg/m3) to be the highest concentration that would be satisfactory for an 8-hour day.

Justification for selection of skin irritation / corrosion endpoint:
GLP guideline study

Justification for selection of eye irritation endpoint:
GLP guideline study

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: irritating

Effects on respiratory irritation: irritating

Justification for classification or non-classification

Regulation (EC) No 1272/2008

Not classified.

Proposed self classification

According to Regulation (EC) No 1272/2008

Classification		Labelling
Hazard Class and Category Code(s)	Hazard Statement Code(s)	Pictogram Signal Word Code(s)	Hazard Statement Code(s)	Suppl. Hazard statement code(s)
Skin Irritation 2 Eye Irritation 2 STOT 3	H315 H319 H335	GHS07	H315 H319 H335

According to Directive 67/548/EEC

Classification	Risk phrases	Indication(s) of danger
Xi; R36/37/38	36/37/38	Xi