2,6-dimethyloct-7-en-2-ol

Endpoint summary

Description of key information

Skin irritation

In an in vitro skin irritation study according to OECD guideline 439 and GLP, the test item is irritant to skin and under the experimental conditions reported. In an in vitro skin corrosion study according to OECD guideline 431 and GLP, the test item was non-corrosive to skin under the reported experimental conditions.

 

The analogue chemical, dimyrcetol, is a skin irritant in rabbits when tested undiluted.

 

Overall conclusion: In conclusion, the test item is considered to be irritating to skin.

 

Eye irritation

In an in vivo eye irritation study in rabbits, the test article was classified as moderately irritating to the eye of the albino rabbit under the conditions of the study.

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

Study period:

2021-08-03 to 2021-12-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2012-07-06

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2021-06-14

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: Adult human donors

Details on animal used as source of test system:

Not applicable

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international prevalidation study performed by ECVAM. The in vitro skin irritation test using the human skin model EpiDerm™ and EpiSkin™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential. (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm SIT (MatTek Corporation, 82105 Bratislava, Slovakia)
- Tissue batch number: 34190
- Date of analysis of lot tissue functionality and quality: 2021-09-01

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: At 37°C ± 1.5°C and 5 ± 0.5% CO2 for 35 minutes and afterwards at room temperature for 25 minutes

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The units were gently rinsed with PBS several times. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL of 1 mg/mL MTT per well
- Incubation time: 3 hours (± 5 min)
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability, barrier function and reproducibility: The quality of the final product is assessed via:
1) MTT cell viability test (n=3), acceptance criteria: OD (540-570) [1.0-3.0) (for tissue viability)
2) ET-50 assay using 1% Trition X-100 (n=3) and MTT assay, acceptance criteria: ET50 [4.77-8.72 hours) (barrier function)
3) Long-term antibiotic and antimycotic free culture, acceptance criteria: no contamination (sterility)
- Contamination: The absence of HIV1 and 2 antibodies, Hepatitis C antibodies and Hepatitis B antigens HBs as well as funghi, bacteria and mycroplasma were verified beforehand.

NUMBER OF REPLICATE TISSUES: 3 replicate tissues per test item, negative and positive control. Duplicate measurement per replicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues: Since no non-specific colouring potential of the test item was detected in a pre-experiment, no fresh tissue controls were used.
- Killed tissues: The test item did not show direct interaction with MTT. The use of additional killed tissue controls was not necessary.
- N. of replicates: 3 replicates per test item, 3 replicates per controls (negative, positive controls) were used. No colour or MTT interference controls were used.
- Method of calculation used: See "Any other information on materials and methods, incl. tables"

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant or corrosive to skin (category 2 or 1) if the viability after 60 min exposure is equal or less than 50%.
- The test substance is considered to be non-irritant to skin if the viability after 60 min exposure is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 μL

NEGATIVE CONTROL
- Amount applied: 30 μL

POSITIVE CONTROL
- Amount applied: 30 μL
- Concentration: 5% SDS in deionized water

Duration of treatment / exposure:

60 minutes (35 minutes at standard incubation conditions and 25 minutes at room temperature).

Duration of post-treatment incubation (if applicable):

42 ± 4 hours (in 0.9 mL of assay medium for 24 ± 2 hours and, after a medium change, another 18 ± 2 hours) at standard incubation conditions.

Number of replicates:

3 replicate tissues per test item, negative and positive control were tested in a single independent experiment.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 (mean of 3 tissue replicates)

Value:

3.14

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of EpiDermTM SIT for regulatory purposes, technical proficiency was demonstrated by correctly predicting the proficiency chemicals listed in OECD TG 439.

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
- Range of historical values: See "Attached background material"

Interpretation of results:

study cannot be used for classification

Remarks:

This test method cannot resolve between UN GHS Categories 1 and 2, thus classification is only possible in combination with OECD guideline 431.

Conclusions:

In an in vitro skin irritation study according to OECD guideline 439, the test item is irritant to skin according to UN GHS and EU CLP regulation and under the experimental conditions reported.

Executive summary:

This in vitro study according to OECD guideline 439 was performed to assess the skin irritation potential of the test item by means of the Human Skin Model Test. The test item did not prove to be a MTT reducer in the MTT interference pre-experiment. Also, its intrinsic color was not intensive and the OD of the test item in deionised water or isopropanol at 570 nm after blank correction was < 0.08. Therefore, additional tests with freeze-killed tissues or viable tissues (without MTT addition) did not have to be performed. Three tissues each of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes. After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the viability as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system. After treatment with the test item, the mean relative viability value was 3.14% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is irritant to skin. The test item is identified as requiring classification and labelling according to UN GHS/ EU CLP “Category 2” or “Category 1”, since the tissue viability after exposure and post-incubation is less or equal to 50%. Further testing is required to resolve between UN GHS Categories 1 and 2 and decide on the final classification of the test item.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2022-03-22 to 2022-08-25

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:

2019-06-18

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek test protocol “In vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT)”

Version / remarks:

2014-11-07

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcularTM Eye Irritation Test (OCL-200-EIT) For the prediction of acute ocular irritation of chemicals: Identification of chemicals not requiring classification and labelling for eye irritation or serious eye damage

Version / remarks:

2021-02-02

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Justification for test system used:

The Reconstructed Human Epidermis test method is approved by international regulatory agencies as a replacement for the identification of irritants / corrosives in the in vivo Rabbit skin assay (OECD 404).

The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epiderm™ Skin Corrosion Test Kit (Epi-200 SCT), MatTek Corporation, 82105 Bratislava, Slovakia
- Tissue batch number: 36141
- Quality control date: 2022-05-18
- Date of initiation of testing: 2022-03-25

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (3 minutes exposure) 37 ± 1.5°C and 5 ± 0.5% CO2 (60 minutes exposure)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Several washing steps with PBS
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Tissue viability was determined for each tissue batch in the course of the supplier's qualitiy control and was within the acceptance criteria. The laboratory's historical control data can be found in the attachments.
- Barrier function: Barrier function was determined for each tissue batch in the course of the supplier's qualitiy control and was within the acceptance criteria.
- Morphology: Barrier function was determined for each tissue batch in the course of the supplier's qualitiy control and was within the acceptance criteria. Thus, cells were considered to have a functional morphology.
- Contamination: Contamination (HIV-1 virus, hepatitis B virus, hepatitis C virus and bacterial, yeast or other funghi) was tested for every tissue batch in the course of the supplier's qualitiy control. No contamination was detected.
- Reproducibility: The test results were within the laboratory's historical control data which can be found in the attachments.

NUMBER OF REPLICATE TISSUES: Duplicate tissues for test item, negative and positive control treatment for each timepoint.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: Not needed as no MTT reduction or interference was detected in the pre-experiments.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: A single experiment using 2 tissue replicates per test item/positive control/ negative control and time point was conducted.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 μL (79.4 μL/cm^2 according to guideline)

NEGATIVE CONTROL
- Amount applied: 50 μL (79.4 μL/cm^2 according to guideline)

POSITIVE CONTROL
- Amount applied: 50 μL (79.4 μL/cm^2 according to guideline)
- Concentration: 8.0 N

Duration of treatment / exposure:

3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

No post-treatment incubation was conducted

Number of replicates:

Duplicate tissues for test item, negative and positive control treatment for each timepoint.

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean of 2 tissue replicates (60 min exposure)

Run / experiment:

1

Value:

49.69

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean of 2 tissue replicates (3 min exposure)

Run / experiment:

1

Value:

95.97

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory has demonstrated proficiency in the EpiDerm Skin Corrosion Test as specified by the Certificate of Proficiency.

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

Interpretation of results:

study cannot be used for classification

Remarks:

This test method cannot resolve between UN GHS Categories 1 and 2, thus classification is only possible in combination with OECD guideline 439.

Conclusions:

In an in vitro skin corrosion study according to OECD guideline 431 and GLP, the test item was non-corrosive to skin under the reported experimental conditions.

Executive summary:

This in vitro study according to OECD guideline 431 and GLP was performed to assess the corrosive potential of the test item by means of the Human Skin Model Test with EpiDerm™tissues models. The test item did not reduce MTT (pre-test for direct MTT reduction), and it did not change colour when mixed with deionised water and isopropanol (pre-test for colour interference). Consequently, additional tests with freeze-killed and viable tissues and non-specific killed tissues to determine correction factors for calculating the true viability in the main experiment were not necessary. Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (Dulbecco’s Phosphate-Buffered Saline (DPBS)) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively. After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period. The 1 hour exposure caused a decrease of the cell viability < 15% of the negative control. The CV in the range 20 – 100% viability between the tissue replicates is ≤ 30%, thus the validity of the test system and the specific batch of the tissue models is confirmed. After exposure of the tissues to the test item the relative absorbance value was 95.97% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was 49.69%. Both values did not reach the threshold for corrosivity which is defined to be < 50% after the 3 minutes exposure or ≥ 50% after 3 minutes exposure and < 15% after the 1 hour exposure. Therefore, the test item is considered to be non-corrosive. In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item is non-corrosive to skin according to EU CLP and UN GHS.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

other: Read-across, see cross-reference

Justification for type of information:

Please refer to IUCLID section 13 for Read Across Justification.

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: 100% dimyrcetol

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: 100% dimyrcetol

Irritation parameter:

erythema score

Basis:

animal #4

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: 100% dimyrcetol

Irritation parameter:

edema score

Basis:

animal #4

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: 100% dimyrcetol

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: 100% dimyrcetol

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: 100% Dimyrcetol

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: 100% dimyrcetol

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.6

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: 100% dimyrcetol

Reference 4

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

Test substance identified by name only. The observation period was only 7 days.

Qualifier:

according to guideline

Guideline:

other: Annex V, EEC Directive 79/831

Version / remarks:

The system described in the Official Journal of the European Communities No. L257, 16th September 1983 was used to classify materials (Commission Directive of 29th July 1983 adapting technical progress for the 5th time, Council Directive 67/548/EEC).

Deviations:

yes

Remarks:

The observation period was only 7 days.

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Housing: Animals were individually housed in anodised aluminum cages with grid floors and no bedding materials were provided in the cages.
- Diet: Ad libitum (commercially available pelleted rabbit diet (Product Ref. 680, Dalgety-Spillers Ltd.))
- Water: Ad libitum (tap water)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-67
- Air changes (per hr): Not indicated, but room was air-conditioned
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

other: Tested either undiluted or as 50% solution in DEP

Controls:

no

Amount / concentration applied:

A 0.5 mL aliquot of the test material was placed evenly over a 2.5 cm square of surgical lint B.P. The lint square was then placed onto the animals skin, on the left flank and immediately caudal to the last rib, with the test material in contact with the skin. In the test, a second test material was applied in the same manner to the right flank. Lint patches were held in place with Elastoplast bandages (10 cm wide). The test material was applied a group of four animals either neat (undiluted) or as a 50% solution.

Duration of treatment / exposure:

4 hours. After the treatment period, the adhesive tapes was removed and the treated sites cleansed by gently swabbing with cotton wool soaked in warm water.

Observation period:

One hour after removeal and at 4, 24, 48, 72 and 168 hours after patch removal, animals were assessed for signs of reaction to treatment. Animals were examined under a standard light source designed to comply with the requirements of B.S. 950 Part 1 (Artificial Daylight for the Assessment of Colour). Irritation was assessed numerically as defined in Table 1.

Number of animals:

4

Details on study design:

The day before treatment, rabbits were restrained and the dorsal surfaces of the trunk clipped free of hair using a Oster Model A2 clipper with Angra blade. Groups of 4 healthy animals with intact skin were selected for the study.

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: 100% dimyrcetol

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: 100% dimyrcetol

Irritation parameter:

erythema score

Basis:

animal #4

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: 100% dimyrcetol

Irritation parameter:

edema score

Basis:

animal #4

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: 100% dimyrcetol

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: 100% dimyrcetol

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: 100% Dimyrcetol

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: 100% dimyrcetol

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.6

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: 100% dimyrcetol

Irritant / corrosive response data:

Sample 8A (50% Dimyrcetol, See Table 2)

Well defined erythema and very slight oedema of the skin were apparent in 3/4 rabbits and very slight erythema in 1/4 rabbits 1 hour after dosing. Some increase in severity of oedematous response was exhibited by 2/4 animals at 24 hours, with slight reaction observed. Further increases in response were apparent at the 48 hour examination with well defined erythema noted in all 4 rabbits. Oedema of the skin ranged from very slight in one rabbit, slight in two rabbits, and moderate in one rabbit. Slight desquamation of the surface of treated skin was also observed in one rabbit. The irritant response seen in one rabbit had declined by the 72 hour observation, with very slight erythema remaining. There were no remaining reactions at treated sites on any rabbit by day 7 after dosing.

Sample 8B (100% Dimyrcetol, See Table 3)

Well defined erythema was observed in all 4 rabbits and very slight oedema of the treated skin in three rabbits was apparent at the one hour observation. Oedematous responses increased and at 24 hours after dosing slight reaction was observed in three rabbits and very slight reaction was noted in the remaining rabbit of the group. Well defined erythema and slight oedema were present at the treated site of all four rabbits 48 and 72 hours after dosing. This response had declined within 7 days with very slight erythema remaining in 3 rabbits and very slight oedema in all 4 rabbits. Slight desquamation from the treated skin was also observed in one rabbit.

Table 2:  Sample 8A Results

 

Rabbit No. /Observation Time	Erythema Score	Oedema Score	Comments
909
1 hour	2	1
24 hour	2	2
48 hour	2	3
72 hour	2	3
168 hour	0	0
914
1 hour	1	0
24 hour	1	0
48 hour	2	1	Slight desquamation
72 hour	1	0	Very slight desquamation
168 hour	0	0
915
1 hour	2	1
24 hour	2	1
48 hour	2	2
72 hour	2	2
168 hour	0	0
917
1 hour	2	1
24 hour	2	2
48 hour	2	2
72 hour	2	2
168 hour	0	0

 

Table 3:  Sample 8B Results

 

Rabbit No. /Observation Time	Erythema Score	Oedema Score	Comments
909
1 hour	2	0
24 hour	2	2
48 hour	2	2
72 hour	2	2
168 hour	0	1
914
1 hour	2	1
24 hour	2	1
48 hour	2	2
72 hour	2	2
168 hour	1	1	Slight desquamation
915
1 hour	2	1
24 hour	2	2
48 hour	2	2
72 hour	2	2
168 hour	1	1
917
1 hour	2	1
24 hour	2	2
48 hour	2	2
72 hour	2	2
168 hour	1	1

 

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Undiluted dimyrcetol is rated as a skin irritant in the rabbit.

Executive summary:

In a key study in rabbits, the undiluted analogue chemical, dimyrcetol, applied under semi-occlusive wrap was rated a skin irritant according to Annex V of EEC Directive 78/831 based on a group mean erythema irritation score of 2 or higher for the 24-, 48- and 72-hour observations. Minimal erythema and edema (scores of 0 to 1) persisted to the 168 hour observation. A 50% dilution in diethyl phthalate was not an irritant under the same conditions. All irritant effects noted in the study with 50% test material were resolved by the 168-hour observation point.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1980

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Principles of method if other than guideline:

The study was conducted according to the procedure originally suggested by Draize (Draize, H.J. "Appraisal of the Safety of Chemical in Foods, Drugs and Cosmetics", Assoc. of Food and Drug Officials of the U.S., Austin, Texas, 1959).

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Not stated
- Weight at study initiation: 2.0 to 3.5 kg at the preliminary eye examination
- Housing: Singly under standard laboratory conditions; suspended pans beneath cages were littered with prepared bedding (Easi Litter)
- Diet: Daily 100 g of Purina Rabbit Chow supplemented by whole oats
- Acclimation period: Yes, but the period not stated
- Assurance of animal health: Animals were examined by a veterinarian during quarantine and all animals assigned to the study were in good health prior to the start of the study and were selected to be free of apparent ocular defects

ENVIRONMENTAL CONDITIONS
The criteria used for housing and maintenance were those specified in the "Guide for the Care and Use of Laboratory Animals", DHEW Publication No. (NIH) 78-23, Revised 1978.

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated eye of each rabbit served as control

Amount / concentration applied:

0.1 mL

Duration of treatment / exposure:

single application

Observation period (in vivo):

10 days

Number of animals or in vitro replicates:

6

Details on study design:

METHOD

24 hours prior to dosing, both rabbit eyes were examined using a hand held ophthalmoscope and then by fluorescein staining. One drop of Fluorescein Sodium Ophthalmic Solution USP was dropped onto the cornea which was then irrigated with Sodium Chloride Solution USP. Eyes were examined for corneal ulceration using an ultraviolet light.

On the day of dosing, the lower lid was everted from the eyeball of the right eye of each animal to form a cup into which the test article was instilled. The eyelids were then gently held shut for one second. The eye was not washed after instillation. Any untoward reaction to instillation of the test substance was recorded and the animal returned to its cage.

Both eyes of each rabbit were scored according to the method of Draize (table 1) at 24, 48 and 72 hours and at 4 and 7 days after dosing. In addition, at 24 hours and 7 days, both eyes were examined with fluorescein stain under an ultra violet light.

Any persistent changes observed on Day 7 were re-evaluated on Day 10 using a hand-held ophthalmoscope and the fluorescein method described previously.

Irritation parameter:

chemosis score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0.66

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0.66

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.66

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #6

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 4 days

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #5

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #4

Time point:

24/48/72 h

Score:

2.33

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.66

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.33

Max. score:

3

Reversibility:

fully reversible within: 4 days

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

2

Reversibility:

fully reversible within: 24 hours

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #6

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 10 days

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0.66

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #4

Time point:

24/48/72 h

Score:

1.66

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 10 days

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

All of the six animals developed corneal opacity within 24 hours of being dosed. Opacity had cleared in one of the six animals at 72 hours, and in four of the six animals by Day 7. The eyes of the last two animals had cleared by Day 10.

See the table below for individual animal irritation results.

Table 2: Primary eye irritation data for individual animals and observation times

 

Rabbit		Cornea			Iris		Conjunctivae				Total
Number	Hr/Day	A (Opacity)	B (Area)	(A x B x 5)	A	A X 5	A (Redness)	B (Chemosis)	C (Discharge)	(A + B + C) x 2	Score
A-1126	24 hours	2	4	40	0	0	2	1	2	10	50
	48 hours	2	4	40	0	0	1	0	0	2	42
	72 hours	2	3	30	0	0	0	0	0	0	30
	day 4	1	3	15	0	0	0	0	0	0	15
	day 7	1	2	10	0	0	0	0	0	0	10
	day 10	0	0	0	0	0	0	0	0	0	0
	Mean=	1.33	2.67		0.00		0.50	0.17	0.33
Mean	(24,48 and 72 hr)	2.00	3.67		0.00		1.00	0.33	0.67
A-1127	24 hours	2	3	30	1	5	3	2	3	16	51
	48 hours	2	3	30	0	0	1	0	0	2	32
	72 hours	2	1	10	0	0	1	0	0	2	12
	day 4	2	1	10	0	0	0	0	0	0	10
	day 7	0	0	0	0	0	0	0	0	0	0
	day 10	0	0	0	0	0	0	0	0	0	0
	Mean=	1.33	1.33		0.17		0.83	0.33	0.50
Mean	(24,48 and 72 hr)	2.00	2.33		0.33		1.67	0.67	1.00
A-1128	24 hours	2	4	40	0	0	2	1	2	10	50
	48 hours	2	4	40	0	0	2	0	0	4	44
	72 hours	2	4	40	0	0	1	0	0	2	42
	day 4	2	4	40	0	0	1	0	0	2	42
	day 7	0	0	0	0	0	0	0	0	0	0
	day 10	0	0	0	0	0	0	0	0	0	0
	Mean=	1.33	2.67		0.00		1.00	0.17	0.33
Mean	(24,48 and 72 hr)	2.00	4.00		0.00		1.67	0.33	0.67
A-1129	24 hours	2	3	30	0	0	3	2	2	14	44
	48 hours	2	3	30	0	0	2	0	0	4	34
	72 hours	1	1	5	0	0	2	0	0	4	9
	day 4	1	1	5	0	0	1	0	0	2	7
	day 7	0	0	0	0	0	0	0	0	0	0
	day 10
	Mean=	1.2	1.6		0		1.6	0.4	0.4
Mean	(24,48 and 72 hr)	1.67	2.33		0.00		2.33	0.67	0.67
A-1139	24 hours	1	2	10	0	0	2	2	1	10	20
	48 hours	1	2	10	0	0	1	0	0	2	12
	72 hours	0	0	0	0	0	0	0	0	0	0
	day 4	0	0	0	0	0	0	0	0	0	0
	day 7	0	0	0	0	0	0	0	0	0	0
	day 10	0	0	0	0	0	0	0	0	0	0
	Mean=	0.33	0.67		0.00		0.50	0.33	0.17
Mean	(24,48 and 72 hr)	0.67	1.33		0.00		1.00	0.67	0.33
A-1140	24 hours	1	4	20	0	0	1	1	0	4	24
	48 hours	1	4	20	0	0	1	0	0	2	22
	72 hours	1	4	20	0	0	1	0	0	2	22
	day 4	1	3	15	0	0	0	0	0	0	15
	day 7	1	1	5	0	0	0	0	0	0	5
	day 10	0	0	0	0	0	0	0	0	0	0
	Mean=	0.83	2.67		0.00		0.50	0.17	0.00
Mean	(24,48 and 72 hr)	1.00	4.00		0.00		1.00	0.33	0.00
Mean by sample time =
	24 hours	1.67	3.33		0.17		2.17	1.50	1.67		39.83
	48 hours	1.67	3.33		0.00		1.33	0.00	0.00		31.00
	72 hours	1.33	2.17		0.00		0.83	0.00	0.00		19.17
	day 4	1.17	2.00		0.00		0.33	0.00	0.00		14.83
	day 7	0.33	0.50		0.00		0.00	0.00	0.00		2.50
	day 10	0.00	0.00		0.00		0.00	0.00	0.00		0.00

 

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The primary ocular irritation scores were 39.83 at 24 hours, 31.00 at 48 hours, 19.17 at 72 hours, 14.83 at 4 days, 2.5 at 7 days and 0 at 10 days. Under the conditions of this study, the test article was classified as moderately irritating to the eye of the albino rabbit.

Executive summary:

In a study in rabbits, 0.1 mL of undiluted test item was applied to six rabbit eyes followed by examinations at 24-, 48 and 72-hours and at days 4 and 7 days following treatment. Eyes were examined with fluorescein stain under ultraviolet light at 24-hours and 7 days. Any persistent changes noted at day 7 were re-evaluated on day 10. All animals developed corneal opacity by 24 hours (scores of 1 to 2). Opacity had cleared in one animal at 72 hours and in 4 of 6 animals by day 7. The eyes of the last 2 animals cleared by day 10. Based on the Draize scoring system, the material would be rated as moderately irritating to eyes. Mean overall scores at 24-, 48- and 72-hours were as follows: opacity, 1.56; iris, 0.06; conjunctival redness, 1.44; conjunctival chemosis, 0.50; and conjunctival discharge, 0.56. In a supporting study in rabbits, undiluted dimyrcetol was rated an eye irritant based on the Draize scoring system. Mean overall scores at 24-, 48- and 72-hours were as follows: opacity, 0.33; iris, 0.17; conjunctival redness, 0.83; conjunctival chemosis, 0.56; and conjunctival discharge, 0.56. Under the conditions of this study, the test article was classified as moderately irritating to the eye of the albino rabbit.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

WoE, skin irritation in vitro, RL1

This in vitro study according to OECD guideline 439 was performed to assess the skin irritation potential of the test item by means of the Human Skin Model Test. The test item did not prove to be a MTT reducer in the MTT interference pre-experiment. Also, its intrinsic color was not intensive and the OD of the test item in deionised water or isopropanol at 570 nm after blank correction was < 0.08. Therefore, additional tests with freeze-killed tissues or viable tissues (without MTT addition) did not have to be performed. Three tissues each of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes. After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the viability as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system. After treatment with the test item, the mean relative viability value was 3.14% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is irritant to skin. The test item is identified as requiring classification and labelling according to UN GHS/ EU CLP “Category 2” or “Category 1”, since the tissue viability after exposure and post-incubation is less or equal to 50%. Further testing is required to resolve between UN GHS Categories 1 and 2 and decide on the final classification of the test item.

 

WoE, skin corrosion in vitro, RL1

This in vitro study according to OECD guideline 431 and GLP was performed to assess the corrosive potential of the test item by means of the Human Skin Model Test with EpiDerm™tissues models. The test item did not reduce MTT (pre-test for direct MTT reduction), and it did not change colour when mixed with deionised water and isopropanol (pre-test for colour interference). Consequently, additional tests with freeze-killed and viable tissues and non-specific killed tissues to determine correction factors for calculating the true viability in the main experiment were not necessary. Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (Dulbecco’s Phosphate-Buffered Saline (DPBS)) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively. After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period. The 1 hour exposure caused a decrease of the cell viability < 15% of the negative control. The CV in the range 20 – 100% viability between the tissue replicates is ≤ 30%, thus the validity of the test system and the specific batch of the tissue models is confirmed. After exposure of the tissues to the test item the relative absorbance value was 95.97% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was 49.69%. Both values did not reach the threshold for corrosivity which is defined to be < 50% after the 3 minutes exposure or ≥ 50% after 3 minutes exposure and < 15% after the 1 hour exposure. Therefore, the test item is considered to be non-corrosive. In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item is non-corrosive to skin according to EU CLP and UN GHS.

 

WoE (Read-across), skin irritation in vivo, RL2

In a study in rabbits, the undiluted analogue chemical, dimyrcetol, applied under semi-occlusive wrap was rated a skin irritant according to Annex V of EEC Directive 78/831 based on a group mean erythema irritation score of 2 or higher for the 24-, 48- and 72-hour observations. Minimal erythema and edema (scores of 0 to 1) persisted to the 168 hour observation. A 50% dilution in diethyl phthalate was not an irritant under the same conditions. All irritant effects noted in the study with 50% test material were resolved by the 168-hour observation point.

 

Eye Irritation in vivo, RL1

In a key study in rabbits, 0.1 mL of undiluted test item was applied to six rabbit eyes followed by examinations at 24-, 48 and 72-hours and at days 4 and 7 days following treatment. Eyes were examined with fluorescein stain under ultraviolet light at 24-hours and 7 days. Any persistent changes noted at day 7 were re-evaluated on day 10. All animals developed corneal opacity by 24 hours (scores of 1 to 2). Opacity had cleared in one animal at 72 hours and in 4 of 6 animals by day 7. The eyes of the last 2 animals cleared by day 10. Based on the Draize scoring system, the material would be rated as moderately irritating to eyes. Mean overall scores at 24-, 48- and 72-hours were as follows: opacity, 1.56; iris, 0.06; conjunctival redness, 1.44; conjunctival chemosis, 0.50; and conjunctival discharge, 0.56. In a supporting study in rabbits, undiluted dimyrcetol was rated an eye irritant based on the Draize scoring system. Mean overall scores at 24-, 48- and 72-hours were as follows: opacity, 0.33; iris, 0.17; conjunctival redness, 0.83; conjunctival chemosis, 0.56; and conjunctival discharge, 0.56.

 

Please refer to Section 13 of this IUCLID file for read-across documentation and rationale for the selection of representative analogue chemicals.

 

Skin irritation/corrosion: irritating

Eye irritation: irritating

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
Skin Irritation

The available experimental test data for the substances are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the available data, the test substance is classified for skin irritation (category 2) according to Regulation (EC) No 1272/2008 (CLP), as amended for sixteenth time in Regulation (EU) No 2021/743.

 

Eye irritation

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the available data, the test substance was an eye irritant and is thus classified for eye irritation (category 2) according to Regulation (EC) No 1272/2008 (CLP), as amended for sixteenth time in Regulation (EU) No 2021/743.