Sodium 3,5,5-trimethylhexanoate

Endpoint summary

Description of key information

Skin irritation:

The skin irritation for the K salt was done by OECD 431 and for the Na salt by OECD 439 – both reconstructed human epidermis methods but one is a later version than the other. For the K skin irritation, the undiluted solid substance was used, wetted with saline, for the Na, the 45% product as supplied was used.

The man tissue viability for the Na salt was 7.3% and whilst ‘no prediction’ as to Cat 1 or 2 can be made, it was concluded it is an irritant Cat 2 at 45% active

For the K salt, the relative mean tissue viability after 60 min was reduced to less than 35% but not more than 35% after 3 min treatment. The test item is therefore classified as corrosive in accordance with a combination of optional sub-categories 1B and 1C.

In order to adopt the precautionary principle, the Na salt will also be classified as Cat 1 corrosive.

Eye irritation:

For the K salt BCOP test, the test item was suspended with physiological saline 0.9% NaCl to gain a 20% concentration. For the test item the following mean in vitro irritation score was calculated: 239.46 calculated back to the 100% test substance. The K salt is classified as Cat 1 corrosive

For the Na salt BCOP eye irritation, the ColaTrope INC test was done on the diluted product (45% active but then diluted at 10% in saline – so 4.5% active material for the test). The IVIS score was <1 under these conditions and so it was designated non-irritant. Because this test was done on very dilute Na salt content, it may be useful for the classification of actual product in marketed use, but for the 100% substance, it is likely that the K salt classification is more realistic and should stand.

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:

experimental study

Adequacy of study:

key study

Study period:

17 May 2017 to 21 November 2017

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Test system recommended in OECD Guideline.

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDermTM (MatTek Corporation)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 uL undiluted test article

Duration of treatment / exposure:

35 minutes at 37oC, 5% CO2 and =>95% humidity
Remaining time to 60 minutes in sterile hood
24h after thorough washing and fresh assay medium used

Duration of post-treatment incubation (if applicable):

18h post incubation period followed by:
-Thorough tissue rinse and drying
-Extract solution applied
-Sealed and incubated in the dark overnight
-Samples agitated for 15 minutes
-Each tissue pierced
-Extract was analysed on a microplate reader at 570nm

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Second experiment performed at MatTek laboratories, Run 1

Value:

7.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Second experiment performed at MatTek laboratories, Run 2

Value:

7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Second experiment performed at MatTek laboratories, Run 3

Value:

7.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The first experiment at the test facility failed and so a second experiment at MatTek laboratories was undertaken. The conclusion of this second experiment is that the Test Substance is classed as an Irritant according the GHS classification.

Executive summary:

Test Substance is classed as an Irritant according the GHS classification.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Potassium 3,5,5-trimethylhexanoate
CAS No.: 93918-10-6
Batch No.: PURS151015
Purity: 99.4%
Physical State: solid
Colour: white
Molecular Weight: 196.33 g/mol
Storage Conditions: room temperature
Re-certification Date: 14 October 2016

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Source strain:

other: adult donors

Vehicle:

physiological saline

Details on test system:

The test was carried out with the reconstituted three-dimensional human skin model EPISKIN-SM™ (SkinEthic). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Morphology:
Histology scoring (HES stained vertical paraffin sections, n = 6):
specification ≥ 19.5
result: 21.8 ± 0.3, CV = 1.2%
Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a
thick stratum corneum.
Barrier function:
IC50 determination (SDS concentration, MTT test, n = 14):
specification ≥ 1.5 mg/mL
result: 2.8 mg/mL

The mixture of 20 mg test item per 2 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equaled 0%. The mixture of 10 mg test item per 300 µL Aqua. dest. and per 90 µL isopropanol showed no colouring as compared to the solvent. Therefore NSC equaled 0%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

20 ± 2 mg (52.6 mg/cm2) of the test item was applied directly atop the EPISKIN-SM™ tissue. To ensure good contact with the epidermis surface, the test item was moistened with 100 ± 5 µL 0.9% NaCl solution. The test item was spread to match size of the tissue.

Duration of treatment / exposure:

In the present study Potassium 3,5,5-trimethylhexanoate was applied topically to the EPISKIN-SM™ tissue for 3 min, 60 min and 4 h followed by immediate determination of cytotoxic effects via MTT reduction assay.

Number of replicates:

The test was performed on a total of 6 tissues for each test item and for the negative control, 2 replicates for each treatment period (3 min, 60 min and 4 h exposure time). For the positive control the test was performed with 2 replicates for the 4 h exposure time.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 - 3 min experiment

Value:

104.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2 - 3 min experiment

Value:

101.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 - 60 min experiment

Value:

20

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

4 - 60 min experiment

Value:

23

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

5 - 4h experiment

Value:

16.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

glacial acetic acid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

6 - 4h experiment

Value:

12.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

glacial acetic acid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The controls confirmed the validity of the study. The mean OD570 of the two negative control tissues was between 0.6 and 1.5 for each exposure period. The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3%) after 4 h treatment. Inter tissue viability difference of replicate tissues of all dose groups was ≤ 30% (0.2% - 9.4%).

The potential of the test item to induce skin corrosion was analysed by using the three-dimensional human skin model EPISKIN-SM™, comprising a reconstructed epidermis with a functional stratum corneum. In the present study Potassium 3,5,5-trimethylhexanoate was applied topically to the EPISKIN-SM™ tissue for 3 min, 60 min and 4 h followed by immediate determination of cytotoxic effects via MTT reduction assay. Corrosivity potential of the test item was predicted from the relative mean tissue viabilities compared to the corresponding negative control tissues concurrently treated with 0.9% NaCl. The test item showed no non-specific MTT-reducing or water-colouring potential. The test item showed corrosive effects. The mean relative tissue viability (% negative control) was reduced below 35% (20%) after 60 min treatment and to not more than 35% (103%) after 3 min treatment. Relative mean tissue viability was reduced to 15% after 4 h treatment. The controls confirmed the validity of the study. The mean OD570 of the two negative control tissues was between 0.6 and 1.5 for each exposure period. The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3%) after 4 h treatment. Inter tissue viability difference of replicate tissues of all dose groups was ≤ 30% (0.2% - 9.4%).

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

In this study under the given conditions the test item showed corrosive effects. The relative mean tissue viability after 60 min was reduced to less than 35% but not more than 35% after 3 min treatment. The test item is therefore classified as corrosive in accordance with a combination of
optional sub-categories 1B and 1C.

Executive summary:

In the present study the skin corrosivity potential of Potassium 3,5,5-trimethylhexanoate was analysed. Since corrosive chemicals are cytotoxic after a short time exposure to the stratum corneum of the epidermis the cytotoxic effects of the test item on EPISKIN-SM™, a reconstituted three-dimensional human epidermis model, were determined. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity

measured by formazan production from MTT after a 3 min, 60 min and 4 h exposure period and compared to those of the concurrent negative controls. In this study under the given conditions the test item showed corrosive effects. The relative mean tissue viability after 60 min was reduced to less than 35% but not more than 35% after 3 min treatment. The test item is therefore classified as corrosive in accordance with a combination of optional sub-categories 1B and 1C.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

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:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Potassium 3,5,5-trimethylhexanoate
CAS No.: 93918-10-6
Batch No.: PURS151015
Purity: 99.4%
Physical State: solid
Colour: white
Molecular Weight: 196.33 g/mol
Storage Conditions: room temperature
Re-certification Date: 14 October 2016

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany. On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated. The eyes were carefully examined for defects and any defective eyes were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was suspended with physiological saline 0.9% NaCl to gain a 20% concentration.

Duration of treatment / exposure:

750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 4 hours ± 5 minutes incubation at 32 +- 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed.
After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 +- 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

Number of animals or in vitro replicates:

3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive control treated with imidazole 20% in physiological saline 0.9% NaCl

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

239.46

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

The eye irritancy potential of Potassium 3,5,5-trimethylhexanoate was investigated in the bovine corneal opacity and permeability assay. The test item was suspended with physiological saline 0.9% NaCl to gain a 20% concentration.

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid. The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

For the test item the following mean in vitro irritation score was calculated: 239.46

For the positive control the following mean in vitro irritation score was calculated: 106.03

For the positive control the following mean in vitro irritation score was calculated: 1.14

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

According to the evaluation criteria the test item Potassium 3,5,5-trimethylhexanoate is classified into UN GHS Category 1.

Executive summary:

The eye irritancy potential of Potassium 3,5,5-trimethylhexanoate was investigated in the bovine corneal opacity and permeability assay. The test item was suspended with physiological saline 0.9% NaCl (see 10.3) to gain a 20% concentration. The following mean in vitro irritation score was calculated: 239.46

Therefore the test item was classified into UN GHS Category 1. The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 August 2017 to 4 December 2017

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine eyes were
Obtained from: ENA Meat Packing, Paterson, NJ
Date of harvest: 29 August 2017 (approx 10:00 am)
Method of transport: Plastic container in Hank's balanced salt solution (HBSS)
Used: Day of harvest, approximately 2.5h post harvest

Eyes were assessed for damage under magnification and any damaged eyes were rejcted. Accepted corneas were dissected from the eye including a 2-3 mm sclera surround. Excised cornea were held in HBSS until required.

Test
Corneas were held in cornea holders at 32oC in Eagle's Minimum Essnetial Media (EMEM) with 1% Fetal Bovine Serum (FBS) and incubated/equilibrated for 1 hour.

After equilibration media was removed from the cornea holding chambers and replaced with fresh media. Initial opacity readings were taken and those cornas with initial readings greater than 008 were discrded.

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

10% in saline

Duration of treatment / exposure:

10 minutes

Number of animals or in vitro replicates:

3

Details on study design:

Opacity Measurement
After 10 minutes exposure test solutions were removed and the corneas washed.

The corneas were incubated in EMEM for 2 hours. EME was then replaced and opacity measurements were taken.

Permeability
Following opacity measurements, fluorescein in Dubelcco's Phosphate Buffered Saline (DPBS) was added to one side of the chamber. Spectrophotometric analysis of the opposing chamber determined permeability.

Irritation parameter:

cornea opacity score

Run / experiment:

Mean Corrected

Value:

0.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Optical density

Run / experiment:

Mean corrected

Value:

0.019

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Mean opacity and permeability

Article	Mean Corrected Opacity score	Mean Corrected Optical Density (OD490)	Mean In-vitro score
Test	0.7	0.019	1.0
Positive Control	27.3	1.244	46.0
Control	(1.0)	(0.004)	-

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Clearly, sodium and potassium 3,5,5-trimethylhexanoateare closely related, being the respective acid and salts. The only difference between the two substances is the alkali metal cation, which generally exhibit similar properties as a salt once associated with an acid, in this case3,5,5-trimethylhexanoic acid.They can therefore be considered together in terms of safety assessment and the K salt has been assessed as being a good read-across substance for the Na salt.