Phosphoric acid, butyl ester, branched, compd. with 2-ethyl-N-(2-ethylhexyl)-1-hexanamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

04 Sep 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

read-across source

Remarks:

link to target

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

04 Sep 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

read-across

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The present analogue approach contemplates Phosphoric acid, butyl ester, branched, compd. with 2-ethyl-N-(2-ethylhexyl)-1-hexanamine (CAS 98679-19-7) as target substance for read across from the source substance Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates (CAS 96690-34-5). The read-across approach is used to evaluate the hazardous potential of the target substance with respect to ecotoxicological endpoints and human health for REACH Annex VII.
Based on similar chemical structures, read-across based on different compounds having the same type of effect(s) as described in scenario 2 of the Read-Across Assessment Framework document can be used as a basis for assessment.
The target substance (Phosphoric acid, butyl ester, branched, compd. with 2-ethyl-N-(2-ethylhexyl)-1-hexanamine) and the source substance (Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates) are both organic UVCB substances.
In consequence, read-across can be justified due to the high structural similarities as well as common properties, which will be outlined in detail below.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source: Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates (EC 306-227-4, CAS 96690-34-5)
The substance is manufactured from the educts phosphorus pentoxide (CAS 1314-56-3, EC 215-236-1), 2-butanol (CAS 78-92-2, EC 201-158-5), iso-butanol (CAS 78-83-1, EC 201-148-0) and Amines, C12-14-tert-alkyl (CAS 68955-53-3, EC 273-279-1).

Target: Phosphoric acid, butyl ester, branched, compd. with 2-ethyl-N-(2-ethylhexyl)-1-hexanamine (EC 308-859-6, CAS 98679-19-7)
The substance is manufactured from the educts phosphor pentoxide (CAS 1314-56-3, EC 215-236-1), 2-butanol (CAS 78-92-2, EC 201-158-5), iso-butanol (CAS 78-83-1, EC 201-148-0) and bis(2-ethylhexyl)amine (CAS 106-20-7, EC 203-372-4)

Both substances do not contain any impurities which may impact the feasibility of read-across.

3. ANALOGUE APPROACH JUSTIFICATION
Both substances are organic UVCBs and are manufactured from the educts phosphorus pentoxide (CAS 1314-56-3, EC 215-236-1), 2-butanol (CAS 78-92-2, EC 201-158-5), iso-butanol (CAS 78-83-1, EC 201-148-0) in similar ratios, the only slight difference is the amine part. In the source chemical, Amines, C12-14-tert-alkyl is used, and in the target bis(2-ethylhexyl)amine.
So, the reaction products have a similar distribution pattern with regard to varied alkyl phosphate species (mainly dialkyl phosphates). Both used amines have similar molecular weights, i.e. 185.35-213.41 g/mol (CAS 68955-53-3) and 241.46 g/mol (CAS 106-20-7), which are hence not expected to alter their toxicokinetic behaviour essentially. The fact that one amine is mono-substituted and the other di-substituted, is also not considered to impact the suitability for read-across, as amines are considered to be rather stable functional groups, e.g. hydrolytically stable, and are not considered to be altered to a relevant extent in the body. Both amines are not covalently bound to the phosphates, so they are expected to dissociate into similar dissociation products, too.
Last but not least, both substances show similar physico-chemical properties. They are both liquid at all relevant handling temperatures, both are characterized by negative glass transition temperatures they do not boil but decompose, have similar densities and low vapour pressures indicating that they are no volatile liquids. They have both logPow values and water solubilities which, despite slight variations, are not expected to alter essentially their uptake and distribution through the body.
As there is not sufficient data on both source and target substance available, and the amine is considered to be the main difference and also not covalently bound to the phosphates, data on human health relevant endpoints is retrieved from publically available data sources, i.e. ACToR (https://actor.epa.gov/actor/searchidentifier.xhtml) and RTECS (http://ccinfoweb.ccohs.ca/rtecs/search.html). Further, ecotoxicological properties were estimated via US EPA EpiSuite ECOSAR Class Program v1.11.
Data indicate that both amines produce severe irritating reactions when applied into the eye or onto the skin. Further, the acute dermal toxicity LD50 values are nearly identical in the rabbit (acute tox. Cat. 4), and when applied orally, the available LD50 values similarly indicate that a classification as acute toxic Cat. 4 is triggered. With regard to ecotoxicological properties, the estimated values slightly differ. However, they consistently trigger the same classification, as both substances are not biodegradable; the source chemical is not, so the same can be applied to the amines.
So in consequence, taking into account the similar manufacturing process, i.e. identical and similar educts, and so similar reaction products, similar physico-chemical properties of the source and target chemical and similar (eco)toxicological properties of the source and target amines, the read-across from Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates to Phosphoric acid, butyl ester, branched, compd. with 2-ethyl-N-(2-ethylhexyl)-1-hexanamine is scientifically justified

4. DATA MATRIX

Table: Data Matrix, source and target chemical
Endpoint Source: CAS 96690-34-5 Target: CAS 98679-19-7
Physical state at 20°C, 1013 hPa liquid liquid
Glass transition temperature -44.95 °C -72.1 °C (onset)
Decomposition 243.36 °C 167.1°C (onset)
Density 0.97 g/cm³ 0.968 g/cm³
Vapour pressure ≤ 54 Pa at 20°C < 6.7 Pa at 20°C
logPow ≥ 0.69 - ≤ 5.6 (estimated) - 0.61 at 23°C
Water solubility 1322 mg/L at 25 °C and pH 3.57 17.14 g/l at 20°C
Surface tension 42.9 mN/m at 20°C and 0.66g/l 37.8 mN/m ≤ST ≤ 41.2 mN/m at 20°C and 1g/l
Flash point 1 35°C at 101.325 kPa 107.5°C at 101.3 kPa

Table: Data Matrix, source and target amine
Endpoint Source amine: CAS 68955-53-3 Target: CAS 106-20-7
Acute oral toxicity LD50 = 552 mg/kg (m/f mice, 470 - 719 mg/kg) (ACToR) LD50 = 800µL/kg (mouse, intraperitoneal) (ACToR, RTECS)
LD50 = 320 mg/kg (male rats) (ACToR; RTECS) LD50 = 1640mg/kg (rat) (ACToR, RTECS)
LD50 = 300 mg/kg (rat) (RTECS)
Acute dermal toxicity LD50 = 251 mg/kg (m/f rats, 190 - 322 mg/kg) (ACToR) LD50 = 1190uL/kg (rabbit) (ACToR, RTECS)
LD50 = 1.12 g/kg (rabbits, 0.83 - 1.51) (ACToR, RTECS)
Acute toxicity inhalation LC50 = 157 ppm (female rats, 1.19 mg/L; 90 - 249 ppm) (ACToR, RTECS)
LC50 > 231 ppm (male rats, 1.75 mg/L) (ACToR, RTECS) No data
LC50 > 940 mg/m³/4h (rats) (RTECS)
Skin irritation Severe reaction (rabbit, Draize test) (RTECS) Severe reaction (rabbit, Draize test) (RTECS)
Severe reaction (rabbit, open irritation test) (RTECS)
Mild reaction (rabbit, open irritation test) (RTECS)
Eye irritation Severe reaction (rabbit, Draize test) (RTECS) Severe reaction (rabbit, Draize test) (RTECS)
Genetic Toxicity No data Negative ±S9 (Ames test) (ACToR)
Fish 96-hr LC50 0.644 mg/L* 0.047 mg/L*
0.632 mg/L** 0.016 mg/L**
Daphnid 48-hr LC50 0.104 mg/L* 0.010 mg/L*
0.458 mg/L** 0.014 mg/L**
Green Algae 96-hr EC50 0.047 mg/L* 0.003 mg/L*
0.939 mg/L** 0.059 mg/L**
Fish ChV 0.015 mg/L* 0.000546 mg/L*
0.082 mg/L** 0.003 mg/L**
Daphnid ChV 0.012 mg/L* 0.00131 mg/L*
0.088 mg/L** 0.004 mg/L**
Green Algae ChV 0.019 mg/L* 0.00134 mg/L*
0.424 mg/L** 0.039 mg/L**
* Aliphatic Amines
** Neutral Organic SAR (Baseline Toxicity)

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

yes

Remarks:

no positive control was included in the assay, but reliability of the assay was checked on a regular basis in blinded positive/negative control studies of the laboratory; no data on individual OD570 values from replicate measurements were reported.

GLP compliance:

yes

Specific details on test material used for the study:

TEST SKIN MODEL
- Source: CellSystems, Troisdorf, Germany

TEST METHOD
As mentioned in the study report, Human full thickness skin models and reconstituted epidermal equivalents are in vitro engineered tissue cultures that provide a three dimensional architecture which is biochemically, morphologically and functionally comparable to human epidermal tissue/skin in vivo. While the testing for corrosives usually involves the rabbit skin as a predictive in vivo test method, substances may be classified according to their corrosive potential by the determination of their cytotoxic effects on an in vitro reconstructed human epidermis. The viability of the living cells in the model must be sufficiently high to discriminate well between the positive and negative control substances. Cell viability was measured by the amount of MTT reduction, i.e. an OD value following exposure to the negative control substance or the test item.

ADAPTATION TO CELL CULTURE CONDITIONS
Upon arrival, 6-well culture plates were pre-filled with 1mL of fresh and cool maintenance medium. The reconstructed tissues were placed into the prepared cell culture plates (1 insert/well) and were adapted to the recommended tissue culture conditions (5% CO2, 37°C, max. humidity) afterwards for at least 6 hours before use.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37 ± 2
- CO2 gas concentration (%): 5
- Humidity: maximum

Test system:

human skin model

Remarks:

reconstructed human epidermis EST-1000

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: intact reconstructed human epidermis

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS
- Tissue batch number: EST-120812-001

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 2 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Carefully washed in PBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: Triplicates

DECISION CRITERIA
- 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 1 hour exposure is less than 15%.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL

NEGATIVE CONTROL SUBSTANCE
- Negative control substance: 0.9% NaCl
- Amount(s) applied: 50 µL

Duration of treatment / exposure:

3 and 60 min

Number of replicates:

Triplicates for each treatment and control group.

Vehicle:

unchanged (no vehicle)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

41.84

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min

Value:

12.38

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Remarks:

Skin Corr. 1, H314 according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the test, the test substance was shown to have a corrosive potential towards reconstructed human epidermis tissue in the human epidermis EST-1000 model.

CLP: Skin Corr. 1B, H314

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

yes

Remarks:

no positive control was included in the assay, but reliability of the assay was checked on a regular basis in blinded positive/negative control studies of the laboratory; no data on individual OD570 values from replicate measurements were reported.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates

EC Number:

306-227-4

EC Name:

Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates

Cas Number:

96690-34-5

Molecular formula:

C4H11O4P to C8H20O7P2 as representative molecular formula of the composition as specified in section 1.2

IUPAC Name:

Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates

Details on test material:

- Name of test material (as cited in study report): Additin RC 3740
- Physical state: light yellow, viscous liquid
- Analytical purity: 100% (UVCB)
- Lot/batch No.: 0900215946
- Expiration date of the lot/batch: 2015-01-01
- Storage condition of test material: at room temperature
- pH: 5-6 (in TBN solvents)

Specific details on test material used for the study:

TEST SKIN MODEL
- Source: CellSystems, Troisdorf, Germany

TEST METHOD
As mentioned in the study report, Human full thickness skin models and reconstituted epidermal equivalents are in vitro engineered tissue cultures that provide a three dimensional architecture which is biochemically, morphologically and functionally comparable to human epidermal tissue/skin in vivo. While the testing for corrosives usually involves the rabbit skin as a predictive in vivo test method, substances may be classified according to their corrosive potential by the determination of their cytotoxic effects on an in vitro reconstructed human epidermis. The viability of the living cells in the model must be sufficiently high to discriminate well between the positive and negative control substances. Cell viability was measured by the amount of MTT reduction, i.e. an OD value following exposure to the negative control substance or the test item.

ADAPTATION TO CELL CULTURE CONDITIONS
Upon arrival, 6-well culture plates were pre-filled with 1mL of fresh and cool maintenance medium. The reconstructed tissues were placed into the prepared cell culture plates (1 insert/well) and were adapted to the recommended tissue culture conditions (5% CO2, 37°C, max. humidity) afterwards for at least 6 hours before use.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37 ± 2
- CO2 gas concentration (%): 5
- Humidity: maximum


Physical state at 20°C, 1013 hPa: liquid
Glass transition temperature: -44.95 °C
Decomposition: 243.36 °C
Density: 0.97 g/cm³
Vapour pressure: ≤ 54 Pa at 20°C
log Pow: ≥ 0.69 - ≤ 5.6 (estimated)
Water solubility: 1322 mg/L at 25 °C and pH 3.57
Surface tension: 42.9 mN/m at 20°C and 0.66g/l
Flash point: 135°C

In vitro test system

Test system:

human skin model

Remarks:

reconstructed human epidermis EST-1000

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: intact reconstructed human epidermis

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS
- Tissue batch number: EST-120812-001

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 2 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Carefully washed in PBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: Triplicates

DECISION CRITERIA
- 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 1 hour exposure is less than 15%.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL

NEGATIVE CONTROL SUBSTANCE
- Negative control substance: 0.9% NaCl
- Amount(s) applied: 50 µL

Duration of treatment / exposure:

3 and 60 min

Number of replicates:

Triplicates for each treatment and control group.

Test system

Vehicle:

unchanged (no vehicle)

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

41.84

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min

Value:

12.38

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes

Applicant's summary and conclusion

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Remarks:

Skin Corr. 1, H314 according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the test, the test substance was shown to have a corrosive potential towards reconstructed human epidermis tissue in the human epidermis EST-1000 model.

CLP: Skin Corr. 1B, H314