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EC number: 939-200-6 | CAS number: -
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- Short-term toxicity to fish
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Endpoint summary
Administrative data
Description of key information
The Reaction mass of lithium 3-hydroxy-2,2,4-trimethylpentanoate and lithium isobutyrate and sodium 3-hydroxy-2,2,4-trimethylpentanoate and sodium isobutyrate was not found to be irritating to skin in an in vitro skin irritation study conducted according to OECD Test Guideline 439 using the EpiSkin model. The substance was slightly irritating to the eyes in studies conducted in vitro using the isolated chicken eye model (OECD Test Guideline 438) and in vivo in rabbits (OECD Test Guideline 405), but did not meet the criteria for classification for eye irritation effects.
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1-3 May 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: An in vitro study, according to OECD test guidelines and GLP compliant, based on the EPISKIN model has been completed. The data are considered reliable for completing this endpoint
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Principles of method if other than guideline:
- In vitro skin irritation test based on reconstructed human epidermis model
- GLP compliance:
- yes (incl. QA statement)
- Species:
- other: iin vitro - reconstructed human epidermis model
- Strain:
- other: not applicable
- Details on test animals or test system and environmental conditions:
- Test model - EPISKIN reconstructed human epidermis model. EPISKIN-SM is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability. Cell viability determination is based on cellular mitochondrial dehydrogenase activity, measured by MTT reduction and conversion into a blue formazan salt that is quantitatively measured after extraction from tissues. The reduction of cell viability in treated tissues is compared to negative controls and expressed as a percentage. The % reduction in viability is used to predict the irritation potential of the material under investigation. The in vitro assay is an accepted alternative to the conduct of a rabbit skin irritation assay.
IN-LIFE DATES: From: 1 May 2013 To: 3 May 2013 - Type of coverage:
- other: in vitro assay
- Preparation of test site:
- other: not applicable
- Vehicle:
- unchanged (no vehicle)
- Controls:
- other: not applicable
- Amount / concentration applied:
- 20 mg of the semi-liquid test material were applied to each epidermal surface in the test wells.
50 µL of phosphate buffered saline negative control applied to three control skin units
50 µL of 5% SDS applied to three positive control skin units - Duration of treatment / exposure:
- On Day -1 maintenance medium was pre-warmed to 37°C and applied to the assay plate wells (2 mL per well). The epidermal constructs were overlaid on the medium and the prepared wells were incubated overnight at 37°C.
On day 0, 20 mg of test material was applied to the epidermal surface of three prepared wells. 50 µL of PBS or SDS applied to each of three wells to act as negative or positive controls. The plates were exposed for 15 minutes at circa 25°Cand then the membranes were removed and rinsed in PBS to remove test material residues. The epidermal surface was then vacuum-cleaned. The epidermal unit was replaced onto fresh medium and incubated for 42 hours at 37°C.
After 42 hours the epidermal units were transferred to wells filled with MTT solution and the units incubated or a further 3 hours at 37°C.
Formazan extraction occurred at the end of the MTT incubation. A disk of epidermis obtained by biopsy punch was separated into the epidermal and collagen matrix fractions and each was placed into 500 µL of acidified isopropanol. Formazan extraction proceeded during a two-hour incubation. Following extraction the absorbance/optical density (OD) was recorded for each sample using spectrophotometry at 540 nm. - Observation period:
- 42 hours
- Number of animals:
- Not applicable. Three replicate wells prepared for test material and three each for the positive and negative controls
- Details on study design:
- Test model - EPISKIN reconstructed human epidermis model. EPISKIN-SM is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability. Cell viability determination is based on cellular mitochondrial dehydrogenase activity, measured by MTT reduction and conversion into a blue formazan salt that is quantitatively measured after extraction from tissues. The reduction of cell viability in treated tissues is compared to negative controls and expressed as a percentage. The % reduction in viability is used to predict the irritation potential of the material under investigation. The in vitro assay is an accepted alternative to the conduct of a rabbit skin irritation assay.
EPISKIN-SM (Source: SkinEthic, France, Batch No.: 13-EKIN-016, Expiry date: 06 May 2013) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeIts use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viabilityded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
The negative control use was phosphate buffered saline (PBS). The positive control was 5% sodium dodecyl sulphate in distilled water.
Control checks were included fordetermination of false viability, for possible direct MTT reduction by the test material and for any colouring potential arising directly from coloured test materials that mayhave affected the OD from the spectrophotometry fluid or by direct staining of the tissues.. - Irritation / corrosion parameter:
- other: other: mean OD as an indicator of cell viability
- Value:
- 98
- Remarks on result:
- other:
- Remarks:
- Basis: other: percent relative viability. Time point: 42 hours. Max. score: 100.0. Reversibility: other: not applicable. Remarks: Following exposure to Sodium Isobutyrate Solution, the mean relative viability value of the treated skins was 98% ; the substance was not irritating. All validity criteria were within acceptable limits and therefore the study can be considered as valid. (migrated information)
- Irritant / corrosive response data:
- Following exposure to Sodium Isobutyrate Solution, the mean relative viability value of the treated skins was 98%. THe test substance was not therefore irritating to skin. All validity criteria were within acceptable limits and therefore the study can be considered as valid
- Interpretation of results:
- study cannot be used for classification
- Remarks:
- Migrated information
- Conclusions:
- Following exposure to Sodium Isobutyrate Solution, the mean relative viability value of the treated skins was 98%; the test substance was not therefore irritating to skin
- Executive summary:
The reconstructed human epidermis model EPISKIN-SM is designed to predict and classify the skin irritant potential of chemicals, by measuring its cytotoxic effect, as reflected in the MTT (Thiazolyl blue) cell viability assay, on the EPISKIN reconstituted human epidermis.
Disks of EPISKIN (three units / chemical) were treated with sodium isobutyrate solution and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS. Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified. SDS 5% and PBS treated epidermis were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes of exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test substance is considered to be irritating to skin.
Following exposure to Sodium Isobutyrate Solution, the mean relative viability of the treated skin was 98% and therefore the substance was not considered to be irritating to skin. All validity criteria were within acceptable limits and therefore the study can be considered as valid. Based on this study, Sodium Isobutyrate Solution was not found to be irritating to the skin and does not meet the criteria for classification for skin irritation.
Reference
The results of the optical density (OD) measured at 540 nm of each extract and the calculated % viability of the cells is presented in Table 1:
Table 1: Optical Density (OD) and the calculated % viability of the cells
Substance |
Optical Density (OD) |
Viability (% RV) |
|
Negative Control: |
1 |
0.680 |
102 |
PBS |
2 |
0.630 |
95 |
|
3 |
0.687 |
103 |
|
mean |
0.666 |
100 |
|
Standard deviation |
4.36 |
|
Positive Control: |
1 |
0.047 |
7.1 |
5%SDS |
2 |
0.052 |
7.8 |
|
3 |
0.038 |
5.7 |
|
mean |
0.046 |
6.9 |
|
Standard deviation |
1.07 |
|
Test Item: |
1 |
0.692 |
104 |
Sodium Isobutyrate Solution |
2 |
0.517 |
78 |
|
3 |
0.737 |
111 |
|
mean |
0.649 |
98 |
|
Standard deviation |
17.39 |
The OD value for the test item treated skin was a viability of 98%.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation: in vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 27th May 2013 to 15th August 2013.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted according to GLP and OECD Guideline.
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 405 (Acute Eye Irritation / Corrosion)
- Deviations:
- not specified
- Principles of method if other than guideline:
- Not applicable.
- GLP compliance:
- yes (incl. QA statement)
- Species:
- rabbit
- Strain:
- New Zealand White
- Details on test animals or tissues and environmental conditions:
- TEST ANIMALS
- Source: S&K-LAP Kft., 2173 Kartal, Czászár út 135, Hungary.
- Age at study initiation: approximately 12 weeks old.
- Weight at study initiation: 2766 - 3236g
- Housing: Rabbits were individually housed in AAALAC approved metal wire cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbit(s) in adjoining cages.
- Diet (e.g. ad libitum): UNI diet for rabbits ad libitum.
- Water (e.g. ad libitum): Municipal tap water ad libitum from an automatic system.
- Acclimation period: At least 13 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.1°C - 21.6°C.
- Humidity (%): 37 - 72%
- Air changes (per hr): 15-20 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours daily (from 6am to 6pm).
IN-LIFE DATES: From: 4th June 2013 To: 13th June 2013. - Vehicle:
- unchanged (no vehicle)
- Controls:
- other: Contralateral eye of test animal acted as control.
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1mL - Duration of treatment / exposure:
- 24 hours.
- Observation period (in vivo):
- 72 hours.
- Number of animals or in vitro replicates:
- 3
- Details on study design:
- REMOVAL OF TEST SUBSTANCE
- Washing (if done): Treated eyes were rinsed with physiological saline solution.
- Time after start of exposure: 24 hours.
SCORING SYSTEM: The eye irritation scores were evaluated according to the scoring system by Draize and OECD 405. - Irritation parameter:
- conjunctivae score
- Basis:
- other: all test animals
- Time point:
- other: One hour after application
- Score:
- 2
- Max. score:
- 2
- Reversibility:
- fully reversible within: 72 hours
- Irritation parameter:
- chemosis score
- Basis:
- other: all test animals
- Time point:
- other: One hour after application
- Score:
- 2
- Max. score:
- 2
- Reversibility:
- fully reversible within: 72 hours
- Irritation parameter:
- other: Discharge
- Basis:
- other: all test animals
- Time point:
- other: One hour after application
- Score:
- 2
- Max. score:
- 3
- Reversibility:
- fully reversible within: 72 hours
- Irritation parameter:
- conjunctivae score
- Basis:
- other: all test animals
- Time point:
- other: 24 hours after treatment
- Score:
- 2
- Max. score:
- 2
- Reversibility:
- fully reversible within: 72 hours
- Irritation parameter:
- chemosis score
- Basis:
- other: all test animals
- Time point:
- other: 24 hours after treatment
- Score:
- > 1 - < 2
- Max. score:
- 2
- Reversibility:
- fully reversible within: 72 hours
- Irritation parameter:
- other: Discharge
- Basis:
- other: all test animals
- Time point:
- other: 24 hours after treatment
- Score:
- > 1 - < 3
- Max. score:
- 3
- Reversibility:
- fully reversible within: 72 hours
- Irritation parameter:
- conjunctivae score
- Basis:
- other: all test animals
- Time point:
- other: 48 hours after treatment
- Score:
- 1
- Max. score:
- 1
- Reversibility:
- fully reversible within: 72 hours
- Irritation parameter:
- chemosis score
- Basis:
- animal: 2 test animals
- Time point:
- other: 48 hours after application
- Score:
- 1
- Max. score:
- 1
- Reversibility:
- fully reversible within: 72 hours
- Irritation parameter:
- other: Discharge
- Basis:
- animal: 2 test animals
- Time point:
- other: 48 hours after treatment
- Score:
- 1
- Max. score:
- 1
- Reversibility:
- fully reversible within: 72 hours
- Irritation parameter:
- conjunctivae score
- Basis:
- other: all test animals
- Time point:
- other: 72 hours
- Score:
- 0
- Max. score:
- 0
- Reversibility:
- fully reversible
- Irritation parameter:
- chemosis score
- Basis:
- other: all test animals
- Time point:
- other: 72 hours
- Score:
- 0
- Max. score:
- 0
- Reversibility:
- fully reversible
- Irritation parameter:
- cornea opacity score
- Basis:
- other: all test animals
- Time point:
- other: 72 hours
- Score:
- 0
- Max. score:
- 0
- Reversibility:
- fully reversible
- Irritant / corrosive response data:
- At 72 hours after treatment, no clinical signs or conjunctival or corneal effects were observed.
An Initial Pain Reaction score of 1 was observed in 2 animals tested. - Other effects:
- There was no mortality observed during the study.
The body weights of the animals were considered to be within the normal range of variability.
During the daily general examinations, there were no clinical signs observed that could be related to treatment and the general state and behaviour of the animals were normal throughout the study period.
During the study, the control eye of each animal was symptom-free. - Interpretation of results:
- not irritating
- Remarks:
- Migrated information Criteria used for interpretation of results: EU
- Conclusions:
- The test item, Sodium Isobutyrate Solution, applied to rabbit eye mucosa caused conjunctival irritatnt effects at one hour which were reduced at 48 hours after application. The effects were fully reversible within 72 hours.
Under the conditions of this study and based on its results, the test material, Sodium Isobutyrate Solution is not considered to be an eye irritant and does not require classification according to Regulation (EC) No 1272/2008. - Executive summary:
An acute eye irritation study was conducted to determine the potential of Sodium Isobutyrate Solution to cause eye irritation in New Zealand White rabbits. The study was conducted in accordance with OECD Guideline 405 and was scored according to the Draize method. Three male test animals were used in the study, with the test material placed in the conjunctival sac of the left eye of each animal, with the right eye serving as the control. A single volume of 0.1 mL of test material was administered as a single dose.
The eyes were examined 1, 24, 48 and 72 hours after application of the test material. An Initial Pain Reaction of 1 was observed in 2 animals and one hour after application, conjunctival redness (score of 2), chemosis (score of 2) and discharge (score of 2 and 3) were observed in the treated eye of all test animals. 24 hours after application, conjunctival redness (score of 2), chemosis (score of 1 or 2) and discharge (score of 1, 2 and 3) were observed in the treated eye of all test animals. 48 hours after treatment, conjunctival redness (score of 1) was observed in all animals. Chemosis (score 1) and discharge (score of 1) were sen in 2 of the test animals.
In the absence of clinical signs 72 hours after treatment, the study was terminated. At all times during the study, the control eye of each animal was symptom free and the general state and behaviour of the animals was normal for the duration of the study.
Under the conditions of this study and based on its results, the test material, Sodium Isobutyrate Solution is not considered to be an eye irritant and does not require classification according to Regulation (EC) No 1272/2008.
Reference
Mean Values of Eye Irritation:
Animal |
Cornea Opacity |
Iris |
Conjunctivae |
||
Redness |
Chemosis |
Discharge |
|||
1 |
0.00 |
0.00 |
1.00 |
0.67 |
1.00 |
2 |
0.00 |
0.00 |
1.00 |
0.33 |
0.33 |
3 |
0.00 |
0.00 |
1.00 |
1.00 |
1.33 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Skin irritation/corrosion
The potential for the Reaction mass of lithium 3-hydroxy-2,2,4-trimethylpentanoate and lithium isobutyrate and sodium 3-hydroxy-2,2,4-trimethylpentanoate and sodium isobutyrateto cause skin irritation has been investigated in an in vitro skin irritation assay conducted according to OECD Test Guideline 439 (Kiss, 2013). In the study, the skin irritant potential of the substance was assessed by measuring its cytotoxic effect, as reflected in the MTT (Thiazolyl blue) cell viability assay on the EpiSkin reconstructed human epidermis.
Disks of EpiSkin (three units / chemical) were treated with the test material, sodium isobutyrate solution, and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS. Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2, protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified. SDS 5% and PBS treated epidermis were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a % relative to negative control. If the mean relative viability after 15 minutes of exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test substance is considered to be irritating to skin.
Following exposure to sodium isobutyrate solution, the mean relative viability of the treated skin was 98% and therefore the substance is not considered to be irritating to skin. All validity criteria were within acceptable limits and therefore the study can be considered as valid. Based on this study, the Reaction mass of lithium 3-hydroxy-2,2,4-trimethylpentanoate and lithium isobutyrate and sodium 3-hydroxy-2,2,4-trimethylpentanoate and sodium isobutyrateis not irritating to skin and does not meet the criteria for classification for skin irritation.
Eye irritation
The eye irritation effects of the Reaction mass of lithium 3-hydroxy-2,2,4-trimethylpentanoate and lithium isobutyrate and sodium 3-hydroxy-2,2,4-trimethylpentanoate and sodium isobutyrate have been investigated in an in vitro eye irritation test conducted according to OECD Test Guideline 438 using the isolated chicken eye test method for identifying ocular corrosives and severe irritants (Kiss, 2013).
Following suitable acclimatisation, preparation and zero reference measurements, the test eyes were held in a horizontal position and 30 μL of the test substance, sodium isobutyrate solution, was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface of the cornea was rinsed with saline. The positive control eyes were treated with 30 μL Trichloroacetic acid 30 (w/v) %. The negative control eye was treated with 30 μL of Saline (Salsol solution, NaCl 0.9% w/v).
Based on this study, the Reaction mass of lithium 3-hydroxy-2,2,4-trimethylpentanoate and lithium isobutyrate and sodium 3-hydroxy-2,2,4-trimethylpentanoate and sodium isobutyrate was slightly irritating to eyes, but did not meet the criteria for classification as a severe eye irritant.
A further in vivo confirmatory eye irritation study was carried out.
The eye irritation effects of the Reaction mass of lithium 3-hydroxy-2,2,4-trimethylpentanoate and lithium isobutyrate and sodium 3-hydroxy-2,2,4-trimethylpentanoate and sodium isobutyrate was investigated in an acute eye irritation study in New Zealand White rabbits conducted using the Draize method (OECD Test Guideline 405; Matting, 2013b). In the study, the test substance, sodium isobutyrate solution was placed into the conjuctival sac of the left eye of three rabbits respectively. The eyes were examined at 1, 24, 48 and 72 hours after application. An initial pain reaction score of 1 was observed in 2/3 rabbits. One hour after the application, conjuctival redness (score 2), chemosis (score 2) and discharge (score 2 or 3) were seen in all animals. At 24 hours after treatment, conjuctival redness (score 2), chemosis (score 1or 2) and discharge (score 1,2 or 3) were seen in all animals. At 48 hours after treatment, conjuctival readness (score 1) was noted in all animals. Chemosis (score 1) and discharge (score 1) were seen in two animals. At 72 hours after treatment, no clinical signs and no conjuctival or corneal effects were observed. The test item, sodium isoburyrate solution, when applied to rabbit eye mucosa, caused conjuctival irritant effects at one hour which were reduced at 38 hours after application and were fully reversible within 72 hours. The substance did not therefore meet the criteria for classification for eye irritation effects.
Justification for selection of skin irritation / corrosion endpoint:
Sole study providing data from a guideline compliant study. Under the conditions of this study, the test substance was not determined to be irritating to skin.
Justification for selection of eye irritation endpoint:
Study providing data from a guideline compliant study. Under the conditions of this study, the test substance was not determined to be irritating to eyes.
Justification for classification or non-classification
Skin irritation/corrosion
The Reaction mass of lithium 3-hydroxy-2,2,4-trimethylpentanoate and lithium isobutyrate and sodium 3-hydroxy-2,2,4-trimethylpentanoate and sodium isobutyratedid not cause skin irritation in an in vitro skin irritation study. The substance does not meet the criteria for classification for skin irritation according to Directive 67/548/EEC or Regulation 1272/2008/EC.
Eye irritation
The Reaction mass of lithium 3-hydroxy-2,2,4-trimethylpentanoate and lithium isobutyrate and sodium 3-hydroxy-2,2,4-trimethylpentanoate and sodium isobutyratedid not cause eye irritation in studies in vitro or in vivo. The substance does not meet the criteria for classification for eye irritation according to Directive 67/548/EEC or Regulation 1272/2008/EC.
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