Cyclohexanone

Endpoint summary

Description of key information

In an OECD guideline study (404) slight to moderate erythema as well as slight edema were observed after an exposure period of 3 hours. After an exposure period of 1 h our slight to marked erythema and after 4 hours slight to moderate erythema were observed. Additionally, in an EpiDerm test in vitro a corrosive potential of the test substance was seen (BASF, 2003). In a transcutaneous electrical resistance assay according to OECD guideline 430, Cyclohexanone did not show a skin corrosion potential (Envigo 2018). Taken together all the information available, it seems likely that the result obtained in the EpiDerm test represents a false positive response; classification is based on the in vivo test, supported by the TER assay.

An in vitro eye irritation test (HET-CAM) showed serious eye damage (BASF, 2006). This was in concordance with an older BASF in vivo study (1966), where severe irritating eye effects were found.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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)

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

ldentification of the animals: Ear tattoo. Age at the beginning of the study: About 7 - 8 months. Body weight range at day 0: 3.82 kg - 4.00 kg
The animals were housed in fully air-conditioned rooms, in which a central air-conditioning system ensured a temperature in the range of 20 - 24°C and a relative humidity in the range of 30 - 70%. Illumination period: 12 h light (6.00 a.m. - 6.00 p.m.) / 12 h darkness (6.00 p.m. - 6.00 a.m.). No. of animals per cage: single housing. Type of diet: Kliba-Labordiät, Provimi Kliba SA, Kaiseraugst, Switzerland (about 130 glanimal per day). Watering: Tap water ad libitum. Acclimatization period: At least 5 days before the beginning of the study.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated skin sites of the same animal

Amount / concentration applied:

0.5 ml

Duration of treatment / exposure:

4 h

Observation period:

14 days

Number of animals:

4

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 3 min

Score:

1.7

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 3 min

Score:

0

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 1 h

Score:

2.7

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 1 h

Score:

0.3

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 4 h

Score:

1.7

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 4 h

Score:

0

After the exposure period of 3 minutes slight to moderate erythema, partly extending beyond the area of exposure and slight edema were observed during the course of the study. Additionally dryness of the skin was noted. The average score (24 to 72 hours) for irritation was calculated to be 1.7 for erythema and 0.0 for edema. The cutaneous reactions (with the exception of dryness of the skin) were reversible in the animal within 7 days after removal of the patch. The exposure period of 1 hour caused slight to marked erythema, extending beyond the area of exposure and slight edema, partly extending beyond the area of exposure in addition to dryness of the skin during the observation period. The average score (24 to 72 hours) for irritation was calculated to be 2.7 for erythema and 0.3 for edema. The cutaneous reactions were reversible in the animal within 14 days after removal of the patch. After the exposure period of 4 hours slight to moderate erythema was noted in addition to dryness of the skin during the course of the study. The average score (24 to 72 hours) for irritation was calculated to be 1.7 for erythema and 0.0 for edema. The cutaneous reactions (with the exception of dryness of the skin) were reversible in all animals within 7 days after removal of the patch.

Interpretation of results:

irritating

Conclusions:

Taking into account the described cutaneous reactions and the average scores for irritation under the test conditions chosen as well as the results of the EpiDermTM Skin Cyclohexanone is considered to be a skin irritant.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(Draft) 2002 and 2000/33/EC

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Test system:

human skin model

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Duration of treatment / exposure:

3 minutes and 1 hour

Details on study design:

As test system EpiDerm 200 skin tissue available as kits containing 24 tissues on shipping agarose was used.
Controls included (1) a negative one using bidistilled water, (2) a positive one using potassium hydroxyde purchased from Sigma-Aldrich (Germany), and a tetrazolium salt (MTT) -reduction control using either bidistilled water or test substance.
Incubation time was 3 minute and 1 hour. The estimation of the corrosive potency of the test substance was based on the metabolic activity within the treated tissue measured by means of a colorimetric assay.
In fact, the reduction of the mitochondrial deshydrogenase activity was used as criterion for determining the cytotoxicity of the test substance.
The estimation of the deshydrogenase activity was based on its property to reduce the yellow-colored and water-soluble MTT into the insoluble, blue-colored formazan. The produced formazan was measured spectrophotometrically (measurement of the optical density of the extract) after having been extracted from the tissue samples with isopropanol.
The formazan production of the test-substance treated samples and that of the negative controls were compared and the quotient of both values was used for estimation of the relative viability of the tissue.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure

Value:

85

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure

Value:

7

The test substance is not able to directly reduce MTT.

Viability of the test substance treated tissues determined after an exposure period of 3 minutes was 85%. Viability of the test substance treated tissues determined after an exposure period of 1 hour was 7%. These results indicate a corrosive potential of the test substance.

Interpretation of results:

corrosive

Reference 3

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 430 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER))

Version / remarks:

2015

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Sponsor's identification: cyclohexanone
Batch number: STBF4198V
Product Code: W390909
CAS number: 108-94-1
Purity: 99.8%
Appearance: clear colorless liquid
Recertification date: 30 November 2021
Storage conditions: room temperature in the dark (<30 °C)

Test system:

isolated skin discs

Source species:

rat

Source strain:

Wistar

Details on animal used as source of test system:

One female Wistar (RccHan™:WIST) strain rat was supplied by Envigo RMS (UK) Limited, Oxon, UK. At the start of pelt preparation the rat was 21 to 23 days old.

Vehicle:

unchanged (no vehicle)

Details on test system:

Skin disc preparation:
Following an acclimatization period of 2 days the animal was shaved to remove hair from the dorsal surface. The shaved area was washed using an antibiotic wash. After 3 days a second antibiotic wash was performed. Two days later the animal was killed using ascending concentrations of carbon dioxide followed by cervical dislocation. The animal was in the telogen phase of hair growth and little or no hair growth was visible. When the animal had been humanely killed the dorsal skin was removed from the rat as a single pelt. Care was taken during the procedure to avoid unnecessary damage to the pelt. Excess fat was removed and the pelt mounted, epidermal side uppermost, onto a polytetrafluoroethylene (PTFE) tube. The tissue was secured in place using a rubber “O” ring. Excess tissue was trimmed away and the “O” ring/PTFE interface sealed with soft paraffin wax. The tube was supported by a clamp inside a labelled 30 mL glass receptacle containing 10 mL electrolyte solution (154 mM MgSO4).
Skin disc quality control:
Two skin discs of approximately 0.79 cm2 were taken from the pelt and the TER measured as a quality control procedure. Each disc had to give a resistance value of greater than 10 kΩ in order for the remainder of the pelt to be used in the assay. If either disc fell below the 10 kΩ threshold, the pelt was discarded. The quality control discs were then discarded and new discs from the acceptable pelt were mounted on the PTFE tubes.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test item was used as supplied. A volume of 150 μL was applied to the inner epidermal surface.

Duration of treatment / exposure:

The test item, positive and negative control were applied to the epidermal surface of three skin discs per test group for a contact period of 24 hours.

Number of replicates:

3 per test group

Irritation / corrosion parameter:

transcutaneous electrical resistance (in kΩ)

Run / experiment:

mean value test item

Value:

7.2

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

transcutaneous electrical resistance (in kΩ)

Run / experiment:

mean positive control

Value:

880.3

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

transcutaneous electrical resistance (in kΩ)

Run / experiment:

mean negative control

Value:

18.7

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, under the conditions of this transcutaneous electrical resistance assay and following assessment of the data, cyclohexanone was non-corrosive and is considered unlikely to have the potential to cause corrosion in vivo.

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:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Principles of method if other than guideline:

The study was performed according to the methods described in the following publications:
• Lüpke N.P. (1985): Hen’s Egg Chorio allantoic Membrane Test for Irritation Potential. Fd. Chem. Toxic. 23, pp. 287 – 291.
• Spielmann, H. (1995): HET-CAM Test. In: Methods in Molecular Biology, 43 (eds.: O’Hare, S. and Atterwill, C. K.) pp. 199 – 204.
• Spielmann, H. et al. (1996): Results of a Validation Study in Germany on Two In Vitro Alternatives to the Draize Eye Irritation Test the HET-CAM Test and the 3T3 NRU Cytotoxicity Test. ATLA 24, pp. 741 – 858.
In addition the study follows the testing strategy for determination of eye irritation/corrosion as given in the following guideline: Organization for Economic Co-operation and Development (OECD), OECD Guidelines for testing of chemicals, Guideline No. 405: "Acute Eye Irritation / Corrosion", adopted April 24, 2002.

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Species:

other: in vitro test

Strain:

other: in vitro test

Vehicle:

other: Pluronic PE 6200

Controls:

no

Amount / concentration applied:

10, 20, 40%

Duration of treatment / exposure:

up to a maximum time period of 3.5 minutes

Observation period (in vivo):

210 seconds

Number of animals or in vitro replicates:

3 eggs for 10% concentration, 2 eggs for 20% concentration and 1 egg for 40% concentration

Details on study design:

Type of eggs: Fresh, fertilized hen eggs produced under controlled SPF conditions.
Strain/quality: White Leghorn, SPAFAS Inc., USA, SPF Premium.
Origin: Charles River Deutschland GmbH, Extertal.
Identification: At start of incubation period continuous numbering of the eggs with a felt pen.
Weight range at start of incubation period: 48.5 g – 61.1 g
Randomization: A randomization was not performed. For each solvent eggs of comparable weight (± 10 g) were used.
Reasons for the selection: Hen eggs are recommended as preferred type of eggs in the quoted references. Analogous to test animals, controlled SPF housing guarantees a defined microbiological status of the eggs.
Climate: Breeding in an incubator at constant temperature of 37.5°C (± 0.5°C) and a relative humidity of 62.5% (± 7.5%).
Automatic rotating device: Until including incubation day 8 and/or day 9, the eggs were rotated automatically 5 times a day. On the day before application the eggs were placed with the blunt end upward and were not rotated until preparation. The incubation conditions were checked daily. Deviations were recorded.
Candling of the eggs: The eggs were candled before the start of incubation and on the 9th and/or 10th day. Any defective or unfertilized eggs were discarded.

Irritation parameter:

in vitro irritation score

Run / experiment:

10 % in Pluronix PE 6200, 210 sec

Value:

0

Irritation parameter:

in vitro irritation score

Run / experiment:

20 % in Pluronic PE 6200, 210 sec

Value:

1 - 2

Irritation parameter:

in vitro irritation score

Run / experiment:

40 % in Pluronic PE 6200, 210 sec

Value:

2

Concentration	Egg No.	Time (seconds) until appearance of		Grading of effects		Additional findings
		Haemorrhagia	Coagulation	Haemorrhagia	Coagulation
10% in Pluronic PE 6200	1	210	210	0	0	none
	2	210	210	0	0	none
	3	210	210	0	0	none
Mean:	n = 3	210	210	0	0
20% in Pluronic PE 6200	1	60	60	1	2	intravascular coagulation
	2	76	76	1	2	intravascular coagulation
Mean:	n = 2	68	68	1	2
40% in Pluronic PE 6200	1	20	20	2	2	intravascular coagulation
Mean:	n = 1	20	20	2	2

Grading: 0 = no reaction; 1 = slight; 2 = moderate; 3 = severe

Interpretation of results:

highly irritating

Conclusions:

Based on the results of this study and applying the evaluation criteria it is concluded, that the threshold concentration for effects indicating serious eye damage was > 10% < 20% for Cyclohexanone.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

BASF-internal standard

GLP compliance:

no

Species:

rabbit

Strain:

Vienna White

Details on test animals or tissues and environmental conditions:

Male rabbit weighing 2.84 kg, femal rabbit weighing 2.7 kg.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.05 ml

Duration of treatment / exposure:

24 h

Observation period (in vivo):

8 days

Number of animals or in vitro replicates:

2

Irritation parameter:

conjunctivae score

Time point:

other: 1h

Score:

1

Max. score:

3

Irritation parameter:

conjunctivae score

Time point:

24 h

Score:

1

Max. score:

3

Irritation parameter:

conjunctivae score

Time point:

other: 8d

Score:

1

Max. score:

3

Irritation parameter:

chemosis score

Time point:

other: 1h

Score:

3

Max. score:

3

Irritation parameter:

chemosis score

Time point:

24 h

Score:

1

Max. score:

3

Irritation parameter:

cornea opacity score

Time point:

24 h

Score:

2

Max. score:

3

Irritation parameter:

cornea opacity score

Time point:

other: 8d

Score:

2

Max. score:

3

Irritant / corrosive response data:

Results after 1 hour: Slight reddening, severe edema, slight cloudiness.
Results after 24 hours: Slight reddening and edema, moderately cloudiness.
Results after 8 days: slight reddening, moderately cloudiness, scar (eye lid).
Results with common salt: no irritation

Interpretation of results:

other: serious damage for eyes

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the data available, cyclohexanone is classified for its irritating properties according to Regulation (EC) No. 1272/2008 (H315, H318 and H 335).