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

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 Sep 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

read-across source

Remarks:

link to target

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

05 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 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

26 July 2013

Deviations:

yes

Remarks:

no data on values (ranges) of historical positive and negative control are provided in the report

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Attenberger Fleisch GmbH & Co. KG, Germany
- Storage, temperature and transport conditions of ocular tissue: On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories.
- Time interval prior to initiating testing: Immediately after arrival of the eyes, cornea preparation was initiated.
- indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were discarded
- Indication of any antibiotics used: Yes

Vehicle:

other: sesame oil

Controls:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied in the test: 750 µL
- Concentration: 20% in sesame oil

VEHICLE
- Substance: sesame oil (Caelo)
- Amount(s) applied in the test: 750 µL
- Lot/batch no.: 12054501

Duration of treatment / exposure:

4 h ± 5 min

Number of animals or in vitro replicates:

number of eyes for the test item: 3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
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.

QUALITY CHECK OF THE ISOLATED CORNEAS
Before the corneas were mounted in corneal holders with the endothelial side against the O-ring of the posterior chamber, a visual check was examined for defects and any defective cornea was discarded.

NUMBER OF REPLICATES
3 eyes for each test group

NEGATIVE CONTROL USED
0.9% physiological saline (AlleMan Pharma, Lot/batch no.: 111214)

SOLVENT CONTROL USED
sesame oil (Caelo, Lot/batch no.: 12054501)

POSITIVE CONTROL USED
20% Imidazole (Sigma, Lot/batch no.: 109K5306V) solution in physiological saline (0.9% NaCl)

APPLICATION DOSE AND EXPOSURE TIME
750 µL and 4 h ± 5 min for each test group

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times with Minimal Essential Medium (MEM)
- POST-EXPOSURE INCUBATION: 90 min

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: decreased light transmission with the aid of a microtiter plate reader (OD490)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
According to OECD 437, liquids are usually tested undiluted and corneas are exposed to the liquids for 10 min. However, the test item was too viscous to be directly applied to the corneas. On the demand of the sponsor, the test item was suspended in sesame oil and tested according to the protocol for solids. Therefore, imidazole was used as positive control, sesame oil was used as an additional negative control and the exposure period was expanded to 4 h instead of 10 min.

Irritation parameter:

in vitro irritation score

Remarks:

mean value of 3 eyes

Run / experiment:

4 h of exposure

Value:

222.1

Positive controls validity:

valid

Other effects / acceptance of results:

Since the IVIS was 222.1, the test substance was defined as a corrosive. The IVIS of the positive control was 158.55, thus higher than that of the historical control values (out of the two standard deviations of the current historical mean), but still regarded valid.
No further lesions of the isolated eyes were reported.

Interpretation of results:

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

Conclusions:

CLP: Cat. 1, H318

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

26 July 2013

Deviations:

yes

Remarks:

no data on values (ranges) of historical positive and negative control are provided in the report

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

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

Specific details on test material used for the study:

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

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Attenberger Fleisch GmbH & Co. KG, Germany
- Storage, temperature and transport conditions of ocular tissue: On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories.
- Time interval prior to initiating testing: Immediately after arrival of the eyes, cornea preparation was initiated.
- indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were discarded
- Indication of any antibiotics used: Yes

Test system

Vehicle:

other: sesame oil

Controls:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied in the test: 750 µL
- Concentration: 20% in sesame oil

VEHICLE
- Substance: sesame oil (Caelo)
- Amount(s) applied in the test: 750 µL
- Lot/batch no.: 12054501

Duration of treatment / exposure:

4 h ± 5 min

Number of animals or in vitro replicates:

number of eyes for the test item: 3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
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.

QUALITY CHECK OF THE ISOLATED CORNEAS
Before the corneas were mounted in corneal holders with the endothelial side against the O-ring of the posterior chamber, a visual check was examined for defects and any defective cornea was discarded.

NUMBER OF REPLICATES
3 eyes for each test group

NEGATIVE CONTROL USED
0.9% physiological saline (AlleMan Pharma, Lot/batch no.: 111214)

SOLVENT CONTROL USED
sesame oil (Caelo, Lot/batch no.: 12054501)

POSITIVE CONTROL USED
20% Imidazole (Sigma, Lot/batch no.: 109K5306V) solution in physiological saline (0.9% NaCl)

APPLICATION DOSE AND EXPOSURE TIME
750 µL and 4 h ± 5 min for each test group

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times with Minimal Essential Medium (MEM)
- POST-EXPOSURE INCUBATION: 90 min

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: decreased light transmission with the aid of a microtiter plate reader (OD490)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
According to OECD 437, liquids are usually tested undiluted and corneas are exposed to the liquids for 10 min. However, the test item was too viscous to be directly applied to the corneas. On the demand of the sponsor, the test item was suspended in sesame oil and tested according to the protocol for solids. Therefore, imidazole was used as positive control, sesame oil was used as an additional negative control and the exposure period was expanded to 4 h instead of 10 min.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

mean value of 3 eyes

Run / experiment:

4 h of exposure

Value:

222.1

Positive controls validity:

valid

Other effects / acceptance of results:

Since the IVIS was 222.1, the test substance was defined as a corrosive. The IVIS of the positive control was 158.55, thus higher than that of the historical control values (out of the two standard deviations of the current historical mean), but still regarded valid.
No further lesions of the isolated eyes were reported.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

CLP: Cat. 1, H318