Reaction mass of 3,7-dimethyloct-6-en-1-yl 2-methylpropanoate and (2E)-3,7-dimethylocta-2,6-dien-1-yl 2-methylpropanoate and (2Z)-3,7-dimethylocta-2,6-dien-1-yl 2-methylpropanoate

Endpoint summary

Description of key information

Skin corrosion: The substance is not corrosive bases on absence of skin and eye irritating properties

Skin irritation: not irritating based on read across from Citronellyl butyrate, which was tested in an OECDTG 439.

Eye irritation: not irritating based on read across from Citronelly butyrate, which was tested in an OECD TG 492.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

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

Adequacy of study:

key study

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across information.

Justification for type of information:

The read across justification is presented in the Endpoint summary Irritation. The accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Irritation / corrosion parameter:

% tissue viability

Value:

106.8

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

other: Not a skin irritant.

Remarks:

according to EU CLP (EC No. 1272/2008 and its amendments).

Conclusions:

Geranyl Isobutyrate is not a skin irritant, based on Citronellyl butyrate tested in OECD TG 439, in which the the cell viability was > 50%.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 April 2016 - 06 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This information is used for read across to Geranyl Isobutyrate MCS.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: MatTek Corporation, 82105 Bratislava, Slovakia

Source strain:

other: Not applicable.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™, 0.6 cm^2
- Tissue batch number: 23339
- 30 μL of the test item was dispensed directly atop the EpiDerm™ tissue and spread to match the surface of the tissue for a complete treatment time of 60 minutes.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5°C

PRE-TEST PROCEDURE:
Assessment of Direct Test Item Reduction of MTT:
MTT Salt Metabolism, Cell Viability Assay:
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells. One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of thecellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction:
Prior to the start of the test, the test item’s colour interference potential had to be evaluated.
For this purpose 30 μL of the test item were mixed with 300 μL of deionised water. This mixture was incubated for 60 minutes at 37 ± 1.5 °C (5 ± 0.5% CO2). The test item was colourless and the colour of test item/water mixture did not change during the incubation period compared with the colour of the pure test item. Therefore, an additional test with viable tissues without MTT addition (step 2 according to study plan) was not necessary.
For correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. For this purpose approximately 30 μL of the test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37 °C for 60 minutes.
Untreated MTT solution was used as control. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer. An additional test with freeze-killed tissues was not necessary.

PRE-INCUBATION:
The plastic bag containing the 24-well plate with epidermal tissues was opened under sterile conditions. Under an airflow using forceps, the gauze was removed and the inserts were taken out. Any remaining agarose that adheres to the outer sides of the inserts was removed by gentle blotting on the sterile filter paper or gauze, and the tissues were placed in the empty, sterile 6-well plate.
0.9 mL of the assay medium (20 – 25 °C) was pipetted into each well of sterile 6-well plates.
The inserts with the EpiDerm™ tissues were placed in the upper wells, and were preincubated for 60 minutes in the incubator (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH). Following, the inserts were transferred from upper wells into the lower wells of the 6-well plates, and, the pre-incubation was continued for further about 18 hours (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH).

TREATMENT:
After pre-incubation of EpiDerm™ tissues was completed, medium was replaced by 0.9 mL of fresh medium per well. The negative and positive control, the vehicle control, and the test item were added into the insert atop the corresponding EpiDerm™ triplicate tissues. The treatment time was 60 minutes in total. Within this period the 6-well plates were put into the incubator for 35 minutes at 37 ± 1.5 °C, 5 ± 0.5 % CO2. In the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment.
After the end of the treatment interval the inserts were removed immediately from the 6-well plate and tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and the outside. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper. The tissues were carefully dried using sterile cotton tipped swap.
The tissues were then transferred into new 6-well plates with 0.9 mL of fresh assay medium in the upper row. The inserts were placed in the prepared holding plate. Tissues were incubated for approximately 24 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. After incubation the inserts were transferred into new 6-wells plates containing fresh medium. Thereafter tissues were incubated for another about 18 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation time was approximately 42 hours.

MTT ASSAY:
On the day of testing the MTT concentrate was diluted with the MTT diluent (1 mg/mL). The 24-well plates were prepared before the end of the tissue pre-warming period. A volume of 300 μL of the MTT solution was added to each well and the plates were kept in an incubator (37 ± 1 °C, 5 ± 0.5 % CO2) until further use.
After the 42-hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT-plates. After a 3-hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2), the MTT solution was aspirated from the wells, and the wells were rinsed three times with DPBS. Inserts were transferred onto new 24-well plates. The inserts were immersed into extractant solution by gently pipetting 2 mL extractant solution (isopropanol) in each insert. The level rose above the upper edge of the insert, thus tissues were completely covered from both sides. The 24-well plate was sealed to inhibit the isopropanol evaporation.
The formazan salt was extracted for about 69 hours without shaking in the refrigerator.
After the extraction period was completed, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken. Afterwards the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96- well flat bottom microtiter plate from the 15 minutes exposure. OD was read in a microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1) with a 570 nm filter. Mean values were calculated from the 3 wells per tissue.

DECISION CRITERIA:
For the current test, a test item is considered irritant if the mean relative tissue viability of three individual tissues is reduced to ≤ 50% of the negative control.
For the current test, a test item is considered non-irritant if the mean relative tissue viability of three individual tissues is > 50% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 μL

Duration of treatment / exposure:

60 minutes exposure period and 42 hours post-exposure incubation period.

Number of replicates:

A total of 9 tissues were used: Triplicate tissues were treated with: test substance, positive control or negative control.

Irritation / corrosion parameter:

% tissue viability

Value:

106.8

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Direct MTT Reduction and colour interference:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.
Test Item:
The mean relative absorbance value of the test item, corresponding to the cell viability, did not decrease to (106.8%; threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues.
- Acceptance criteria met for positive control: Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.3% thus ensuring the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The relative standard deviations between the % variability values of the test item and negative controls in the main test were below 14% , thus ensuring the validity of the study. Since the blank corrected absorption values of the positive control were very low (values between 0.061 and 0.089) compared with the test item and negative control values, the acceptance criteria “% variability values in the main test were < 18%” was slightly exceeded (19%). Due to the clear positive result of the positive control, this circumstance does not affect the integrity of the study.

Mean OD570 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Item	OD570 of tissues	Mean OD570 of triplicate tissues	Relative SD (%)	Relative individual tissue viability (%)	Relative mean viability (%)
Negative Control Item	1.525	1.811	13.7	84.2	100
	1.957			108.1
	1.950			107.7
Positive Control Item	0.061	0.078	19.0	3.4	4.3
	0.082			4.6
	0.089			4.9
Test Item	2.034	1.934	5.9	112.3	106.8
	1.958			108.1
	1.811			100.0

OD = Optical Density

SD = Standard deviation

Interpretation of results:

other: Not a skin irritant

Remarks:

according to EU CLP (EC No. 1272/2008 and its amendments).

Conclusions:

Since the mean relative tissue viability for the substance was above 50% the substance is considered not to be a skin irritant.

Executive summary:

The possible skin irritation potential of the substance was tested in an in vitro test using a human skin model through topical application for 60 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 30 μL test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 4.3%. The relative mean tissue viability obtained after 60 minutes treatment with the substance compared to the negative control tissue was 106.8%. Since the mean relative tissue viability of the substance was above 50% after 60 minutes treatment, the substance is considered not to be a skin irritant.

Reference 3

Endpoint:

skin corrosion: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

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:

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

Adequacy of study:

key study

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across information.

Justification for type of information:

The read across justification is presented in the Endpoint summary Irritation. The accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

other: % tissue viability

Value:

98.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

other: Not an eye irritant.

Remarks:

according to EU CLP (EC No. 1272/2008 and its amendments).

Conclusions:

Geranyl Isobutyrate is not an eye irritant based on the information from Citronellyl butyrate which was tested in an OECDTG 492 study. The mean relative tissue viability in this test is above 60% and therefore also for Geranyl Isobutyrate the cell viability will be > 60% and therefore not showing eye irritation.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 June 2016 - 17 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This information is used for read across to Geranyl Isobutyrate MCS.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: epidermal keratinocytes

Details on test animals or tissues and environmental conditions:

EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 μL

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Duplicate tissues were treated with: test substance, positive control or negative control.

Details on study design:

RhCE tissue construct used, including batch number :
EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation, batch 23714 (82105 Bratislava, Slovakia). The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts..

Assessment of Direct Test Item Reduction by MTT:
Test items may have the ability to directly reduce MTT and to form a blue/purple reaction product which could have an impact on the quantitative MTT measurement. Therefore, it was necessary to assess this ability for the test item prior to conducting any assays with viable tissues. For this purpose approximately 50 μl of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a 6-well plate and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for three hours. A control (50 μL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was run concurrently. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT. Since the MTT solution colour did not turn blue/purple, the test item is not presumed to be a MTT reducer. An additional test with freeze-killed tissues did not have to be performed.

Assessment of Coloured or Staining Materials:
Coloured test items or test items which become coloured after application to the tissues could interfere with the quantitative photometric MTT measurement if the colorant bound to the tissue and would be extracted together with MTT. Therefore, each test item had to be checked for its colorant properties.
Since the test item was colourless additional tests had to be performed to assess, if it will dye water or isopropanol. For this purpose each 50 μL of the test item were added to 1 mL of water and to 2 ml isopropanol in 6-well plates. The water mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for one hour, the isopropanol mixture 3 hours at room temperature Since the test item did not dye water or isopropanol, additional tests with viable tissues did not have to be performed.

Experimental Performance:
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca++Mg++free-DPBS. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH) for 30 minutes.
Test item exposure: After the 30 minute Ca++Mg++free-DPBS pre-treatment, the test and control items were tested by applying 50 μL topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions for 30 minutes.
At the end of the 30 minutes treatment time, the test item was removed by extensively rinsing the tissues with Ca++Mg++-free DPBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of Ca++Mg++-free DPBS were used per test item. Each test item utilized a different set of three beakers. The inserts containing the tissues were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).
After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for 12 minutes immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.
At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 mL of warm Assay Medium. The tissues are incubated for approximately 120 minutes at standard culture conditions (post-treatment incubation).

MTT Assay:
After post-treatment incubation of 120 minutes the MTT assay was performed.
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues are placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions and rinsed 3 times with DPBS afterwards.
Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2-8 °C in the dark and then shaken for 2-3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
The extract solution was mixed and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
The absorbance at 570 nm (OD570) of each well was measured with a plate reader reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1). No reference wavelength measurement was used.

Data Evaluation:
1) The mean OD value of the blank control wells (ODBlk) for each experiment were calculated.
2) The ODBlk from each OD value of the same experiment (blank corrected values) were subtracted.
3) The mean value of the two aliquots for each tissue (= corrected test item OD) were calculated.
4) The mean value of the two relating tissues for each control and test item (= corrected mean OD) were calculated. For further calculations only the corrected mean negative control OD value was needed.
5) The corrected OD value of the negative control corresponds to 100% viability.
Corrected negative control OD = Negative Control OD - ODBlk = 100% Viability

Calculations for Viability Tests:
1) The percent viability of each of the two relating tissues for each control and test item relative to the negative control (100% control) were calculated.
Viability [%]= 100 x corrected test item OD/corrected mean negative control OD
2) The difference of the viability between duplicate tissues was calculated. If the difference is >20% the test is considered as non-qualified.
3) The mean test item viability (TI viability) was calculated and the test item was classified according to the decision criteria.

Decision criteria:
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant.

Acceptability of the Assay:
The results are acceptable if:
1) The negative control OD is > 0.8 and < 2.5,
2) The mean relative viability of the positive control is below 50% of the negative control viability.
3) The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the killed controls (items and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Irritation parameter:

other: % tissue viability

Value:

98.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Direct MTT Reduction and colour interference:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not led to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue colour.
Test item:
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 98.6% (threshold for irritancy: ≤ 60%), consequently the test item was not irritant to eye.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD was > 0.8 and < 2.5 (2.118 and 2.013).
- Acceptance criteria met for positive control: The mean relative viability of the positive control was below 50% of the negative control viability (34.9%).
The difference of viability between the two relating tissues of a single item was < 20% (values between 5.1% and 17.7%) in the same run (for positive and negative control tissues and tissues of single test items).

Mean OD570 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Item	OD570 of tissues	Mean OD570 of triplicate tissues	Absolute value of the difference of the rel. absorbances (%)	Relative individual tissue viability (%)	Relative mean viability (%)
Negative Control Item	2.118	2.066	5.1	102.5	100
	2.013			97.5
Positive Control Item	0.538	0.721	17.7	26.0	34.9
	0.904			43.7
Test Item	2.140	2.037	9.9	103.6	98.6
	1.935			93.6

OD = Optical Density

Interpretation of results:

other: Not an eye irritant

Remarks:

according to Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

Since the mean relative tissue viability for the substance was above 60% the substance is considered not to be an eye irritant.

Executive summary:

The possible eye irritation potential of the substance was tested in an in vitro test. Two type of tissues can be used either primary human epidermal keratinocytes or human immortalized corneal epithelial cells. The epidermal keratinocytes were used in the present test. The study procedures described in this report were according to OECD TG 492 guideline and GLP principles. Skin tissue was treated by topical application of 50 μL test substance for 30 minutes. After 120 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Eye irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 34.9%. The relative mean tissue viability obtained after 30 minutes treatment with the substance compared to the negative control tissue was 98.6% presenting absence of eye irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

The skin corrosion, skin and eye irritation of Geranyl Isobutyrate are derived from read across from Citronellyl Butyrate. The executive summary of tests carried out with this source substance are first presented followed by the read-across rationale.

Citronellyl butyrate skin irritation information:

The possible skin irritation potential of the substance was tested in an in vitro test using a human skin model through topical application for 60 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 30 μL test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 4.3%. The relative mean tissue viability obtained after 60 minutes treatment with the substance compared to the negative control tissue was 106.8%. Since the mean relative tissue viability of the substance was above 50% after 60 minutes treatment, the substance is considered not to be a skin irritant.

Citronellyl butyrate eye irritation:

The possible eye irritation potential of the substance was tested in an in vitro test. Two type of tissues can be used either primary human epidermal keratinocytes or human immortalized corneal epithelial cells. The epidermal keratinocytes were used in the present test. The study procedures described in this report were according to OECD TG 492 guideline and GLP principles. Skin tissue was treated by topical application of 50 μL test substance for 30 minutes. After 120 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Eye irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 34.9%. The relative mean tissue viability obtained after 30 minutes treatment with the substance compared to the negative control tissue was 98.6% presenting absence of eye irritation.

Skin and eye irritation of Geranyl Isobutyrate MCS based on read across from data available for Citronellyl butyrate (CAS #141-16-2)

 

Introduction and hypothesis for the analogue approach

Geranyl Isobutyrate MCS consists of 4 constituents which areIsobutyrate esters of a 3,7-dimethyloctanol chain and can be divided into two subgroups based on the number of unsaturated bonds. The Geranyl-type subgroup consists of two isomers of Geranyl  Isobutyrate which have two double bonds in the chain and are present at a total of 70%. The Citronellyl-type subgroup consists of Citronellyl Isobutyrate, which has one double bond in the chain and is present at ca. 25% and a Citronellyl like component without a double bond which is present at <5%.

For Geranyl  Isobutyrate MCS there are no skin and eye irritation data available.In accordance with Article 13 of REACH, lacking information can be generated by means of alternative methods such as in vitro tests, QSARs, grouping and read-across.For assessing the skin and eye irritation potential of Geranyl Isobutyrate MCS, the analogue approach is selected because for a structural analogue, Citronellyl butyrate, skin irritation and eye irritation data is available which can be used for read across.

Hypothesis:The irritating potential of Geranyl Isobutyrate MCS is the same as from Citronellyl butyrate.

Available information:For Citronellyl butyrate in vitro skin and eye studies are available performed according to OECD TG 439 and OECD TG 492 (both Rel. 1), respectively. In the skin irritation study, the mean relative absorbance value of the test article, corresponding to the cell viability, did not decrease (> 100%) and therefore not a skin irritant. In the eye irritation study, the mean relative absorbance value of the test article, corresponding to the cell viability, minimally decreased to 98.6% and therefore not an eye irritant.

Target chemical and source chemical(s)

Chemical structures of the target chemical and the source chemical are shown in the data matrix, including physico-chemical properties and available toxicologicalinformation. Furthermore, a full list of constituents of Geranyl Isobutyrate MCS, including information relevant for read-across, is given in Appendix 1.

Purity / Impurities

The unidentified impurities of Geranyl Isobutyrate MCS are not considered to have a significant influence on the irritating potential.

Analogue approach justification

According to Annex XI 1.5 read across can be used to replace testing when the similarity can be based on a common backbone and a common functional group. When using read across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation.

Analogue selection:Geranyl Isobutyrate MCS share a similar backbone and functional groups with Citronellyl butyrate and for the latter skin and eye information was available. Therefore Citronellyl butyrate can be used as an analogue.

Structural similarities and differences:Geranyl Isobutyrate MCS is structurally similar to Citronellyl  butyrate: Both have a 3,7-dimethyloctanol chain with at least one double bond a both have a butyl ester group. The difference is that Geranyl Isobutyrate MCS has one additional double bond in the alkene chain compared to Citronellyl butyrate and it has an Isobutyrate ester instead of a straight butyl chain.

Bioavailability: Geranyl Isobutyrate MCS and Citronellyl butyrate have the same molecular weights, are both liquids and the log Kow are very similar (around log Kow 5.5) both exceeding the favourable range for dermal penetration (< 4).  

Reactivity: The reactivity of Geranyl Isobutyrate MCS and of Citronellyl butyrate is considered similar in view of the similar functional groups. The reactive site in both is the ester group, which is present in all constituents of Geranyl Isobutyrate MCS. Geranyl Isobutyrate (MCS) has some constituents that have a conjugated double bond with the ester, which might be slightly more reactive. In view of the limited dermal bioavailability this limited additional reactivity will not change the skin and eye irritation potential also because the results are clearly negative and not borderline.

Uncertainty of the prediction:In view of the reasoning above there are no additional uncertainties.

Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented in the data matrix below.

Conclusions per endpoint for hazard and risk assessment  

In absence of skin and eye irritation information for Geranyl Isobutyrate MCS the experimental data from Citronellyl butyrate are used for read across. When using read across the result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation. This documentation is presented in the current document. Citronellyl butyrate is not a skin or eye irritant in OECD TG 439 and OECD TG 492, tests (Rel. 1) and based on this it is also not corrosive. Using the read across also Geranyl Isobutyrate MCS is not corrosive or irritating to skin and eye.

Final conclusion on hazard risk assessment: Geranyl Isobutyrate MCS is not corrosive and not irritating to skin and eye.  

 

Data matrix for the read across from Citronellyl butyrate to Geranyl  Isobutyrate MCS

Common name	Geranyl  Isobutyrate MCS	Citronellyl butyrate
	Target	Source
Chemical name	n.a.	3,7-dimethyloct-6-en-1-yl butanoate
Chemical structures	For a full list of constituents, see Appendix 1.
CAS #	--	141-16-2
EINECS	905-357-4	205-463-4
REACH registration	2018	Registered
Empirical formula	n.a.	C14H26O2
SMILES	n.a.	CCCC(=O)OCCC(C)CCC=C(C)C
Physico-chemical data
Molecular weight	n.a.	226
Physical state	Liquid	Liquid
Log Kow	5.7 (exp.)	5.54 (est.)
Ws (mg/L)	17.4 (exp.)	1.63 (est.)
Vp (Pa)	1.2 (exp.)	6.2 (est.)
Human health endpoints
Skin irritation	Read-across: Not irritating	Not irritating  (OECD 439)
Eye irritation	Read-across: Not irritating	Not irritating(OECD 492)

 

 

  

Appendix 1. Data matrix for the constituents of Geranyl  Isobutyrate MCS and Citronellyl butyrate

Common names	Geranyl  Isobutyrate MCS				Citronellyl butyrate
Constituent	1	2	3	4
Constituents	3,7-dimethyloctyl 2-methylpropanoate	3,7-dimethyloct-6-en-1-yl 2-methylpropanoate	(2Z)-3,7-dimethylocta-2,6-dien-1-yl 2-methylpropanoate	(2E)-3,7-dimethylocta-2,6-dien-1-yl 2-methylpropanoate	3,7-dimethyloct-6-en-1-yl butanoate
Chemical structures
CAS no	71662-25-4	97-89-2	2345-24-6	2345-26-8	141-16-2
Concentration range	3.7	25	14	56
Empirical formula	C14H28O2	C14H26O2	C14H24O2	C14H24O2	C14H26O2
Molecular weight	228	226	224	224	226
Physico-chemical data*
Water solubility, mg/l	0.56	0.68	0.82	0.82	0.59
Log Kow	5.6	5.5	5.4	5.4	5.5

* Episuite v4.11

 

 

Justification for classification or non-classification

Based on the results, the substance does not need to be classified for skin irritation or eye irritation according to EU CLP (1272/2008 and its amendments).