cis-2-methyl-4-propyl-1,3-oxathiane

Endpoint summary

Description of key information

Skin irritation (Read-Across: 2-Ethyl-4,4-dimethyl-1,3-oxathiane): not-irritating, OECD 404, 2003

Eye irritation: eye irritating, in vitro EpiOcular EIT, 2014

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:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

30-10-2002 to 06-11-2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study performed under GLP. The source and target substances must have an ether group at the 1-position and a thioether at the 3-position of the cyclic six membered ring and thereby fulfil the 1,3-oxathiane definition. Single alkyl group (preferably smaller than an n-butyl group; should be present at the 2-position. Either a single or dual alkyl groups (preferable smaller than or equal to an n-butyl group) should be present at the 4-position. The target and source share common structural elements in the same relative positions. The source and target have very similar physico-chemical properties and thus have similar expected toxicokinetic behaviour. The substances have similar in silico chemical reactivity predictions. This is observed within available in vivo toxicology testing where low level local and systemic toxicity is demonstrated and comparable between target and source. The substances therefore demonstrate chemical similarity.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in attachment to IUCLID section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across is based on the hypothesis that the source and target substances have common structural features in the same relative positions. The source and target have similar physico-chemical, toxicological properties and because of common metabolism they share common or have similar breakdown products and therefore potential mechanisms of action. Further information is included in attachment to IUCLID section 13.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source and target chemicals have comparable chemical similarity. Further information is included in attachment to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
The source substance is a chemically similar substance with common metabolism and common or similar degradants of the target substance. Further information is included in attachment to IUCLID section 13

4. DATA MATRIX
Further information is included in attachment to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: February 2000; signature: April 2000

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: recognised animal supplier
- Age at study initiation: 12 - 20 weeks
- Weight at study initiation: 2.0 - 3.5 kg
- Housing: Individually housed in suspended metal cages.
- Diet: certified rabbit diet ad libitum
- Water: mains drinking water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod: 12 hours light / 12 hours dark

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5mL
- Concentration (if solution): Test material was used as supplied.

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: dorsal
- Type of wrap if used: semi-occlusive

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, Four hours after application the corset and patches were removed from each animal and any residual test material removed by gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure: 4 hours

SCORING SYSTEM:
Erythema and Eschar Formation
No erythema ................. ....... ... ...... ... ........ .... .. ... .... ..... ..... ...... ...... ... .......... ........... .. .. .0
Very slight erythema (barely perceptible) ... .................... .. .... ....... .......... .. .................... .1
Well-defined erythema .............. ....... .. .................. ....... ... .. ... .... .... ........... ... ............ .... ..2
Moderate to severe erythema ............... ... .. .................. .... .... ...... .... ........... ... ............ .....3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) .. .. ........4

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

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 1h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 24h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 48h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 72h

Score:

0.25

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 7 day

Score:

0

Max. score:

4

Remarks on result:

other: Slight desquamation in one organism observed

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 1h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 24h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 72h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 7 day

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.7

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.7

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.7

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.7

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

Very slight erythema was noted at all treated skin sites one hour after patch removal with very slight to well-defined erythema at the 24 and 48-hour observations and very slight erythema at one treated skin site at the 72-hour observation. Very slight oedema was noted at all treated skin sites at the 24-hour observation and at two treated skin sites at the 48-hour observation. Slight desquamation was noted at one treated skin site at the 7 -day observation. Two treated skin sites appeared normal at the 72-hour observation.

Other effects:

- Other adverse local effects: Sliqht desquamation at day 7 in #2. Applicant assessment indicates that this would have resolved by day 14 by expert judgement.
- Other adverse systemic effects: None reported.

Table 1. Individual skin reactions

Skin reaction	Observation time	Individual score - Rabbit number
		1	2	3
Erythema	1 hour	1	1	1
	24 hours	2	2	1
	48 hours	1	2	1
	72 hours	0	1	0
	7 Days	0	0 D	0
Oedema	1 hour	0	0	0
	24 hours	1	1	1
	48 hours	1	1	0
	72 hours	0	0	0
	7 Days	0	0	0

D - desquamation

Mean scores per organism at 24, 48 and 72h:

Erythemea/Escar Formation:

1: total = 3; mean score = 1.0

2: total = 5; mean score = 1.7

3: total = 2; mean score = 0.7

Oedema Formation:

1: total = 2; mean score = 0.7

2. total = 2; mean score = 0.7

3. total = 1; mean score = 0.3

Interpretation of results:

not irritating

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The target substance: is not considered to be irritating to the skin.

Executive summary:

The study was performed on a source substance according to EU Method B.4 and OECD 404 to assess the primary skin irritancy potential of the test item in accordance with GLP in New Zealand White rabbits. 0.5 ml of the test item was introduced under a 2.5 cm x 2.5 cm cotton gauze patch and placed in position on the shorn skin. The patch was secured in position with a strip of surgical adhesive tape. To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset and the animals were returned to their cages for the duration of the exposure period. After 4 hours of exposure to the test item, the pads and collars were removed and the test sites were gently cleaned of any residual test item with distilled water. Individual dose sites were scored according to the Draize scoring system at approximately 1, 24, 48, and 72 hours and at 7 days after patch removal. Very slight erythema was noted at all treated skin sites one hour after patch removal with very slight to well-defined erythema at the 24 and 48-hour observations and very slight erythema at one treated skin site at the 72-hour observation. Very slight oedema was noted at all treated skin sites at the 24-hour observation and at two treated skin sites at the 48-hour observation. Slight desquamation was noted at one treated skin site at the 7 -day observation. Two treated skin sites appeared normal at the 72-hour observation. Under the conditions of this study the source substance was not classified as irritating to the skin.

The target substance: is not expected to produce positive local irritation responses adequate for classification under Regulation (EC) 1272/2008.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30-10-2002 to 06-11-2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: February 2000; signature: April 2000

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: recognised animal supplier
- Age at study initiation: 12 - 20 weeks
- Weight at study initiation: 2.0 - 3.5 kg
- Housing: Individually housed in suspended metal cages.
- Diet: certified rabbit diet ad libitum
- Water: mains drinking water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod: 12 hours light / 12 hours dark

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5mL
- Concentration (if solution): Test material was used as supplied.

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: dorsal
- Type of wrap if used: semi-occlusive

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, Four hours after application the corset and patches were removed from each animal and any residual test material removed by gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure: 4 hours

SCORING SYSTEM:
Erythema and Eschar Formation
No erythema ................. ....... ... ...... ... ........ .... .. ... .... ..... ..... ...... ...... ... .......... ........... .. .. .0
Very slight erythema (barely perceptible) ... .................... .. .... ....... .......... .. .................... .1
Well-defined erythema .............. ....... .. .................. ....... ... .. ... .... .... ........... ... ............ .... ..2
Moderate to severe erythema ............... ... .. .................. .... .... ...... .... ........... ... ............ .....3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) .. .. ........4

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

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 1h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 24h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 48h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 72h

Score:

0.25

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 7 day

Score:

0

Max. score:

4

Remarks on result:

other: Slight desquamation in one organism observed

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 1h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 24h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 72h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 7 day

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.7

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.7

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.7

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.7

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

Very slight erythema was noted at all treated skin sites one hour after patch removal with very slight to well-defined erythema at the 24 and 48-hour observations and very slight erythema at one treated skin site at the 72-hour observation. Very slight oedema was noted at all treated skin sites at the 24-hour observation and at two treated skin sites at the 48-hour observation. Slight desquamation was noted at one treated skin site at the 7 -day observation. Two treated skin sites appeared normal at the 72-hour observation.

Other effects:

- Other adverse local effects: Sliqht desquamation at day 7 in #2. Applicant assessment indicates that this would have resolved by day 14 by expert judgement.
- Other adverse systemic effects: None reported.

Table 1. Individual skin reactions

Skin reaction	Observation time	Individual score - Rabbit number
		1	2	3
Erythema	1 hour	1	1	1
	24 hours	2	2	1
	48 hours	1	2	1
	72 hours	0	1	0
	7 Days	0	0 D	0
Oedema	1 hour	0	0	0
	24 hours	1	1	1
	48 hours	1	1	0
	72 hours	0	0	0
	7 Days	0	0	0

D - desquamation

Mean scores per organism at 24, 48 and 72h:

Erythemea/Escar Formation:

1: total = 3; mean score = 1.0

2: total = 5; mean score = 1.7

3: total = 2; mean score = 0.7

Oedema Formation:

1: total = 2; mean score = 0.7

2. total = 2; mean score = 0.7

3. total = 1; mean score = 0.3

Interpretation of results:

not irritating

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test item is not considered to be irritating to the skin.

Executive summary:

The study was performed to EU Method B.4 and OECD 404 to assess the primary skin irritancy potential of the test item in accordance with GLP in New Zealand White rabbits. 0.5 ml of the test item was introduced under a 2.5 cm x 2.5 cm cotton gauze patch and placed in position on the shorn skin. The patch was secured in position with a strip of surgical adhesive tape. To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset and the animals were returned to their cages for the duration of the exposure period. After 4 hours of exposure to the test item, the pads and collars were removed and the test sites were gently cleaned of any residual test item with distilled water. Individual dose sites were scored according to the Draize scoring system at approximately 1, 24, 48, and 72 hours and at 7 days after patch removal. Very slight erythema was noted at all treated skin sites one hour after patch removal with very slight to well-defined erythema at the 24 and 48-hour observations and very slight erythema at one treated skin site at the 72-hour observation. Very slight oedema was noted at all treated skin sites at the 24-hour observation and at two treated skin sites at the 48-hour observation. Slight desquamation was noted at one treated skin site at the 7-day observation. Two treated skin sites appeared normal at the 72-hour observation. Under the conditions of this study the substance is not classified as irritating to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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

Study period:

24-09-2014 to 26-09-2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well reported study performed under GLP; using a commercially available human corneal epithelium model to manufacturer guideline with no significant deviations. Guideline currently under validation by the ECVAM.

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation EpiOcular™ Eye Irritation Test (OCL-200-EIT)

Deviations:

yes

Remarks:

Tissue viability in meeting the acceptance criterion: the mean OD of three tissues is used at OD >= 1.0 instead of mean of two tissues according to guideline; no significant effect on the reliability of the study

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2012; signature: April 2013

Species:

other: human-derived epidermal keratinocyte tissues

Strain:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

other: Three tissues were used for the positive and negative control groups.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 uL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

30 minutes

Number of animals or in vitro replicates:

Three tissues were used for each treatment group (test item, negative control and positive control)

Details on study design:

- Details of the test procedure used: EpiOcular™ tissues were shipped at 2- 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt, EpiOcular™ tissues were kept in the refrigerator. After 24 hours and at least 1 hour prior to start of the assay, tissues were transferred to 6-well plates with 1 mL assay medium, pre-incubated, and transferred again to 6-well plates with assay medium. Prewarming: All incubations were done in the incubator at 37 ± 1.5 °C, 5 ± 0.5% CO2. Treatment: 37 ± 1.5 °C, 5 ± 0.5% CO2. MTT Assay: 37 ± 1.5 °C, 5 ± 0.5% CO2. After pre-incubation of EpiOcular™ tissues and after completion of the treatment with 20 uL PBS, the negative and positive control and the test item was added into the insert atop the concerning EpiOcular triplicate tissues. The plates were placed into the incubator for 30 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2. After the end of the treatment period the inserts were removed immediately from the cell culture plates. The tissues were rinsed with PBS for 15 times and submerged 3 times in PBS to remove any residual test material. The surface of the skin equivalents was carefully tried using cotton tips as well as by plotting the inserts with blotting paper. The inserts were then placed in 12-well plates supplemented with 5 mL medium per well. After 12 minutes incubation the inserts were transferred to 6-well plates containing 1 mL fresh medium per well. Following, the cell culture plates were placed in the incubator for about 120 minutes. Afterwards the cell viability was determined with the MTT assay.
- RhCE tissue construct used, including batch number: EpiOcular-Kit LotNo.: 19187, KitA
- Doses of test chemical and control substances used: 50 uL (neat test item)
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable):
Exposure was for 30 minutes at room temperature.
Post exposure immersion: After the end of the treatment intervals the inserts were removed immediately from the cell culture plates. The tissues were gently rinsed with 15 times with each 1 mL PBS in order to completely remove any residual test material. The surface of the skin equivalents were carefully dried using cotton tips as well as by plotting the inserts with blotting paper. The inserts will then be placed in 12-well plates supplemented with 5 mL medium per well. After 12 ± 0.5 minutes incubation the inserts will be transferred to 6-well plates containing 1 mL fresh medium per well.
Post-exposure incubation: The cell culture plates will be placed in the incubator for 120 ± 15 minutes. Afterwards the cell viability will be determined with the MTT assay.
- Justification for the use of a different negative control than ultrapure H2O (if applicable): Not applicable.
- Justification for the use of a different positive control than neat methyl acetate (if applicable): Not applicable.
- Description of any modifications to the test procedure: Not applicable.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): Yes. Optical evaluation of the MTT -reducing capacity of the test item after 1 hour incubation with MIT-reagent did not show blue colour. The substance was considered to not directly reduce MTT.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): Triplicate.
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm microplate reader.
- Description of the method used to quantify MTT formazan
- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable): Not applicable.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: Per SOP (and ECVAM validation).
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Yes (see full study report).
- Complete supporting information for the specific RhCE tissue construct used: Yes (see full study report).
- Reference to historical data of the RhCE tissue construct: Not available.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: Not available.
- Positive and negative control means and acceptance ranges based on historical data: Yes (per reference literature).
- Acceptable variability between tissue replicates for positive and negative controls: Yes. Standard deviation was < 6.0%.
- Acceptable variability between tissue replicates for the test chemical: Yes. Standard deviation was < 2.0%.

SCORING SYSTEM:
A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of formazan formed, as monitored by the absorbance. The results for the time-dependant cytotoxicity curves can be presented as the arithmetic mean ± standard deviation. By setting the absorption of the negative control to 100% cell survival the relative absorption calculated for the test item and the positive control can also be described as tissue viability.

Tissue Viability = 100 x (absorption-testitem/positive control –Absorption-blank) / (Absoption-neg.control – Absoption-blank)

Irritation parameter:

other: TEST ITEM: Mean Relative Absorbance (% of negative control)

Remarks:

30 minutes (exposure)

Run / experiment:

mean

Value:

45.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

S.D. 10.5% (n=3)

Irritation parameter:

other: POSITIVE CONTROL: Mean Relative Absorbance (% of negative control)

Remarks:

30 minutes (exposure)

Run / experiment:

mean

Value:

34.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

S.D. = 0.9 % (n=3)

Irritation parameter:

other: NEGATIVE CONTROL: Mean Relative Absorbance (% of negative control)

Remarks:

30 minutes (exposure)

Run / experiment:

mean

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

S.D. = 5% (n=3)

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Concurrent positive control was within the expected range.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. Standard deviation was < 6.0%.
- Acceptance criteria met for positive control: Yes. Standard deviation was < 6.0%.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

Table 1 Results after treatment

Group	Mean Absorbance* Tissue 1, 2, 3 Minus Mean Blank	Mean Absorbance of 3 tissues	Relative Absorbance (%) Tissue	SD (%)	Mean Relative Absorbance (% of negative control)
Negative control	1.540	1.460	102.7	5.9	100
Negative control	1.559		104.0
Negative control	1.401		93.2
Positive control	0.546	0.498	34.6	2.2	34.1
Positive control	0.501		31.6
Positive control	0.501		35.9
Test item	0.729	0.668	47.2	1.3	45.8
Test item	0.702		45.3
Test item	0.692		44.7

* mean of three replicate wells after blank correction

** relative absorbance (rounded) : 100 x (absorbance-testitem/positivecontrol) / (absorbance-neg.control)

 

Optical evaluation of the MTT -reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour. The test item was considered to not directly reduce MTT.

 

The relative absorbance values of the test item, corresponding to the cell viability, did not decrease below 60% compared with the result of the negative control.

 

The acceptability criteria) of the assay were met.

The corrected mean OD values of the three tissues exposed to the negative control were>= 1.0 (1.540, 1.559, 1.401)

The mean tissue viability of the positive control is 34.1% of the negative control (<= 60%)

The standard deviation of the three identically treated skin equivalents of each dose group was <= 20% (below 6%)

Interpretation of results:

irritating

Remarks:

Criteria used for interpretation of results: expert judgment

Conclusions:

Under the conditions of this study the test substance is an eye irritant.

Executive summary:

The study was performed to assess the irritancy potential of the test item to the eye means of the Human Cornea Model Test using the EpiOcular™ model. The study was performed to GLP. The method was according to manufacturer guideline. Tissues of the human cornea model EpiOcular™ were pre-treated for 30 minutes with PBS. The tissues were exposed to each 50 uL of the test item and of the positive control: methyl acetate (neat) and the negative control (deionised water) for 30 minutes in triplicate. Afterwards the cell viability was determined using the MTT assay. After treatment with the negative control, the mean OD of the three tissues was >= 1.0. Compared with the value of the negative control, treatment with the positive control induced a sufficient decrease in the relative absorbance, and the mean tissue viability was <= 60% of the negative control. The relative standard deviation between each the three identically treated equivalents of each dose group was <= 20%. Consequently, the test met the acceptance criteria. Irritating effects were observed following incubation with test item with a mean tissue viability of 45.8%. The relative absorbance values of the test item, corresponding to the cell viability, decreased below 60% compared with the result of the negative control; consequently, the test item is considered to possess an eye irritating potential.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

18-07-1979 to 08-07-1979

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Pre-GLP study following a method equivalent to a recognised guideline. The study was completed at a 50% v/v dose level within triethyl citrate vehicle and therefore is given limited reliability scoring.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: 16 CFR 1500.42

Deviations:

no

Principles of method if other than guideline:

The method was in accordance with the 16 CFR 1500.42 and US CPSC requirements

GLP compliance:

no

Species:

rabbit

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier.
- Age at study initiation: Young adults.

Vehicle:

other: triethyl citrate

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 grammes (total test item and vehicle)

VEHICLE
- Concentration (if solution): 50% v/v
- Lot/batch no. (if required): not reported.

Duration of treatment / exposure:

3 days

Observation period (in vivo):

4 days (observations weree completed every day from day 1 to day 7 post treatement).

Number of animals or in vitro replicates:

6 (sexes not specified)

Details on study design:

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

SCORING SYSTEM: Draize scoring system. Reference 16 CFR 1500.42.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

24

Remarks on result:

other: n = 6; maximum category score = 4

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: day 7

Score:

0

Max. score:

24

Remarks on result:

other: n = 6; maximum category score = 4

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

12

Remarks on result:

other: n =6; maximum category score = 2

Irritation parameter:

iris score

Basis:

mean

Time point:

other: day 7

Score:

0

Max. score:

12

Remarks on result:

other: n =6 ; maximum category score = 2

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0.277

Max. score:

18

Reversibility:

fully reversible

Remarks on result:

other: n =6 ; maximum category score = 3; conjuctivae redness - maximum score = 2 in number #2 reversed in 72 hours

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: day 7

Score:

0

Max. score:

18

Remarks on result:

other: n =6 ; maximum category score = 3

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0.277

Max. score:

24

Reversibility:

fully reversible

Remarks on result:

other: n =6 ; maximum category score = 4; chemosis - maximum score = 1 reversed in 72 hours

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: day 7

Irritant / corrosive response data:

No positive corneal scores. No positive iridal scores. No positive conjnctivae redness mean scores (following scoring at 24, 48 and 72 h). No positive chemosis mean scores. Any effects fully reversed within 72 hours.

Individual scoring data was presented within the study report. Only #2 had a positive redness score that fully resolved within 72 hours. No other significant effects were noted.

Interpretation of results:

not classified

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study the test substance would not be considered as an eye irritant.

Executive summary:

The study was performed to assess the irritancy potential of the test item to the eye following a single application in the albino rabbit. The non-GLP study was completed under 16 CFR 1500.42 following a guideline similar to OECD 405. A mass of 0.1 grams of the test item at 50% v/v in triethyl citrate vehicle was placed into the conjunctival sac of one eye of 6 animals. The other eye remained untreated and was used for control purposes. Assessment of ocular damage/irritation was made approximately 24, 48 and 72 hours following treatment with observations daily up to 7 days. No corneal opacity or iritis was observed. Conjunctival redness irritation was observed in four out of six test animals on all day one but was fully reversed within 3 days. Chemosis was low and transient within 3 days. Under the conditions of this study the test item is not considered to be an eye irritant. Based on the applicants recalculation of the mean scores following grading at 24, 48 and 72h, since not more than 4 out of 6 rabbit mean scores met the EU classification criteria. Further there was complete absence of Corenal Opacity and Corneal Iritis and full reversibility of effects was shown within 21-days. The substance has the potential to produce mild transient eye irritation but is insufficient for classification.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

Key Study: OECD TG 404, 2003 : Read-Across SOURCE (2-Ethyl-4,4-dimethyl-1,3-oxathiane) : The study was performed to EU Method B.4 and OECD 404 to assess the primary skin irritancy potential of the test item in accordance with GLP in New Zealand White rabbits. 0.5 ml of the test item was introduced under a 2.5 cm x 2.5 cm cotton gauze patch and placed in position on the shorn skin. The patch was secured in position with a strip of surgical adhesive tape. To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset and the animals were returned to their cages for the duration of the exposure period. After 4 hours of exposure to the test item, the pads and collars were removed and the test sites were gently cleaned of any residual test item with distilled water. Individual dose sites were scored according to the Draize scoring system at approximately 1, 24, 48, and 72 hours and at 7 days after patch removal. Very slight erythema was noted at all treated skin sites one hour after patch removal with very slight to well-defined erythema at the 24 and 48-hour observations and very slight erythema at one treated skin site at the 72-hour observation. Very slight oedema was noted at all treated skin sites at the 24-hour observation and at two treated skin sites at the 48-hour observation. Slight desquamation was noted at one treated skin site at the 7 -day observation. Two treated skin sites appeared normal at the 72-hour observation. Under the conditions of this study the substance is not classified as irritating to the skin.

 

Weight of Evidence: Since there was not a clear demonstration of a mean score greater than or equal to 2.3 in the scoring at 24, 48 and 72 hours and/or expected to persist to the end of a 14-day observation period. The substance would not be considered to be irritating. Further in vivo testing for skin irritation is unjustified.

 

Eye irritation:

Key Study: in vitro EpiOcular EIT, 2014 : The study was performed to assess the irritancy potential of the test item to the eye means of the Human Cornea Model Test using the EpiOcular™ model. The study was performed to GLP. The method was according to manufacturer guideline. Tissues of the human cornea model EpiOcular™ were pre-treated for 30 minutes with PBS. The tissues were exposed to each 50 uL of the test item and of the positive control: methyl acetate (neat) and the negative control (deionised water) for 30 minutes in triplicate. Afterwards the cell viability was determined using the MTT assay. After treatment with the negative control, the mean OD of the three tissues was >= 1.0. Compared with the value of the negative control, treatment with the positive control induced a sufficient decrease in the relative absorbance, and the mean tissue viability was <= 60% of the negative control. The relative standard deviation between each the three identically treated equivalents of each dose group was <= 20%. Consequently, the test met the acceptance criteria. Irritating effects were observed following incubation with test item with a mean tissue viability of 45.8%. The relative absorbance values of the test item, corresponding to the cell viability, decreased below 60% compared with the result of the negative control; consequently, the test item is considered to possess an eye irritating potential.

 

Supporting Study: eq. to OECD 405, 1979: The non-GLP study was completed under 16 CFR 1500.42 following a guideline similar to OECD 405. A mass of 0.1 grams of the test item at 50% v/v in triethyl citrate vehicle was placed into the conjunctival sac of one eye of 6 albino rabbits. The other eye remained untreated and was used for control purposes. Assessment of ocular damage/irritation was made approximately 24, 48 and 72 hours following treatment with observations daily up to 7 days. No corneal opacity or iritis was observed. Conjunctival redness irritation was observed in four out of six test animals on all day one but was fully reversed within 3 days. Chemosis was low and transient within 3 days. Under the conditions of this study the test item is not considered to be an eye irritant. Based on the applicants recalculation of the mean scores following grading at 24, 48 and 72h, since not more than 4 out of 6 rabbit mean scores met the EU classification criteria. Further there was complete absence of Corenal Opacity and Corneal Iritis and full reversibility of effects was shown within 21-days. The substance has the potential to produce mild transient eye irritation but is insufficient for classification.

 

Weight of Evidence: Determination is made based on in vitro data supported by in vivo data at a 50% v/v dose (equiv. or similar to OECD 405, 1979) demonstrating absence of significant effects. The substance based can be considered to be irritating to the eye. There was a complete absence of corneal opacity and iritis reported and expert judgement would indicate that irreversible damage to the eye is not expected or supported based on the available data.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for dermal irritation.

 

The substance meets classification criteria under Regulation (EC) No 1272/2008 for eye irritation category 2: H319

For skin irritation, the weight of evidence indicates that there was not a clear demonstration of a mean score greater than or equal to 2.3 in the scoring at 24, 48 and 72 hours and/or expected to persist to the end of a 14-day observation period. The substance would not be considered to be irritating. Further in vivo testing for skin irritation is unjustified.

For eye irritation, the weight of evidence indicates that the substance has the potential to cause transient irritating effects to the eye sufficient for classification. An available in vitro EpiOcular EIT study indicates that the substance has a potential to be eye irritating. The magnitude of effect for the substance was less than the positive control (methyl acetate; eye irritant category 2: H319) which had an effect on the RHE tissues within the range of the literature (Y. Kaluzhny et al. 2011) thereby validating the assay. It was just below the alternative 50% prediction cut-off (S. Kolle et al. 2011) and suggestive of the substance being in the lowest order of effect within the CLP Regulation (EC) 1272/2008 classification criteria. Furthermore, an available in vivo study at a 50% v/v dose (equiv. or similar to OECD 405, 1979) demonstrated no effects in vivo on corneal opacity and iritis and with mild effects primarily associated with conjunctivae redness but which were fully reversible within 7 days. The substance is of demonstrated low order of acute toxicity (insufficient for classification) and based on absence of skin irritation in structural analogue(s) studies – the overall evidence is indicative of reversible irritating effects on the eye which meet the EU classification criteria category 2. The substance does not meet the EU Directive 67/548/EEC criteria for eye irritation based on the main effect being conjunctival redness and due to a differences within the mean redness scoring criteria to that of Regulation (EC) 1272/2008; classification in the directive criteria was not supported.

Other data available to the applicant supports the above conclusion.

 

References:

1. Guidance on Application of the CLP Criteria, ECHA, version 5.0, July 2017

2. ECHA Guidance on Information Requirements R7.a: section 7.2; 166-200, version 2.4, 2014

3. L. Scott et al., Toxicology in Vitro 24, 1–9, 2010

4. Y. Kaluzhny et al., ATLA 39, 339-364, 2011

5. S. Kolle et al., ATLA 39, 365–387, 2011