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

Endpoint summary

Description of key information

Skin corrosion in vitro (OECD 431): corrosive

Skin irritation in vivo (OECD 404): irritating
Eye irritation in vitro (OECD 437): corrosive

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18 May - 01 June 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

28 July 2015

Deviations:

yes

Remarks:

occlusive coverage

GLP compliance:

yes (incl. QA statement)

Remarks:

Umweltministerium Baden-Württemberg, Stuttgart, Germany

Species:

rabbit

Strain:

other: albino

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. Zimmermann, Untergröningen/Aalen, Germany
- Weight at study initiation: 2988 - 3090 g
- Housing: rabbits were housed individually in wire grating cages (area: 2450 cm², height: 42 cm) without bedding.
- Diet: Raiffeisen-Ringfutter, Kaninchen Trockenfutter 52/P (Raiffeisen Kraftfutterwerk Kehl, Germany), ad libitum
- Water: community tap water from Karlsruhe (Germany), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

other: the untreated shaved area close to the exposure area served as control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.5 mL

Duration of treatment / exposure:

4 h

Observation period:

14 days
Reading time points: 1, 24, 48 and 72 h, Days 6 - 10, Day 12 and Day 14

Number of animals:

3 (2 males and 1 female)

Details on study design:

TEST SITE
- Area of exposure: dorsolumbar area of the trunk (3 x 5 cm² on the trunk were shaved before treatment)
- Type of wrap if used: the treated region was protected with gauze and with an occlusive bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: residual test substance was removed by gently washing with water.
- Time after start of exposure: 4 h

SCORING SYSTEM: Draize scoring system

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 6 days

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 6 days

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 6 days

Irritant / corrosive response data:

At the 1 h reading time point, very slight (1/3 animals, score 1) to well-defined erythema (2/3 animals, score 2) were observed. At the same time point, 2 animals showed very slight edema (score 1), whereas 1 animal showed moderate edema (score 3). Well-defined erythema (score 2) and very slight edema (score 1) were observed in all animals at the 24, 48 and 72 h reading time point after exposure of the test substance. In all animals, edema were fully reversible within 6 days after application, whereas very slight erythema (score 1) were still evident in all animals at the end of the 14-day observation period.

Other effects:

No further local or systemic effects were observed.

Table 1: Results of skin irritation study

Observation time	Rabbit no.
	1		2		3
	Erythema	Edema	Erythema	Edema	Erythema	Edema
1 h	1	1	2	1	2	3
24 h	2	1	2	1	2	2
48 h	2	1	2	1	2	1
72 h	2	1	2	1	2	0
Mean value 24 + 48 + 72 h	2,00	1,00	2,00	1,00	2,00	1,00

Interpretation of results:

other: Skin Irrit. 2, H315 according to Regulation (EC) No 1272/2008

Conclusions:

CLP: Cat. 2, H315

Reference 2

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

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

Table 1. Results of the MTT assay (3 min and 60 min exposure to the test item)

Sample No.	Test item	Time [min]	OD570 mean*	SD	% Viability
1-3	Negative control NaCl 0.9%	60	2.51	0.07	100.00
4-6	Test item	60	0.31	0.18	12.38
7-9	Negative control NaCl 0.9%	3	2.67	0.27	100.00
10-12	Test item	3	1.12	0.17	41.84

*mean value from 6 measurements; SD = standard deviation

Interpretation of results:

other: Skin Corr. 1B, 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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Sep 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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 slautherhouse 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.

DATA EVALUATION

- Calculation of opacity values:

The opacity value was calculated according to the following steps:

i) opacity change = final opacity – initial opacity

ii) corrected opacity change = opacity change – mean opacity change of the negative control

iii) mean opacity value = mean of all corrected opacity changes per group

 

- Calculation of OD490 (optical density at 490 nm) values:

The permeability value was calculated according to the following steps:

iv) OD490 change = OD490 – mean blank OD490

Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor.

v) corrected OD490 change = OD490 change – mean OD490 change of negative control

vi) mean OD490 value = mean of all corrected OD490 changes per group

 

- Calculation of the IVIS (in-vitro irritancy score) values:

The In-Vitro Irritancy Score (IVIS) was calculated as follows:

IVIS per cornea per group = mean corrected opacity change + (15 x mean corrected OD490 change)

 

- Evaluation criteria:

Test substance with an IVIS ≥ 55.1 was regarded as severe irritant/corrosive and labelled Category 1.

 

- Acceptance criteria:

OECD 437: “A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean, which is to be updated at least every three months, or each time an acceptable test is conducted in laboratories where tests are conducted infrequently (i.e., less than once a month). The negative or solvent/vehicle control responses should result in opacity and permeability value that are less than the establisher upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control.”

RESULTS

Table 1. Opacity scores

Treatment group	Cornea no.	Initial opacity	Final opacity	Opacity change	Corrected opacity change	Mean corrected opacity change
Test substance (20% in sesame oil)	7 8 9	3 4 4	195 216 210	192 212 206	188.33 208.33 202.33	199.67
Negative control (0.9% NaCl)	1 2 3	4 4 4	7 9 9	3 5 5	NA NA NA	NA
Negative control (sesame oil)	10 11 12	4 5 5	8 10 7	4 5 2	NA NA NA	NA
Positive control (20% imidazole in 0.9% NaCl)	4 5 6	5 5 5	151 174 136	146 169 131	141.67 164.67 126.67	144.33

NA = not applicable

 

Table 2. Permeability score

Treatment group	Cornea no.	OD490 value	Corrected OD490 value	Mean corrected OD490 value
Test substance (20% in sesame oil)	7 8 9	1.355 1.618 1.632	1.316 1.579 1.593	1.496
Negative control (0.9% NaCl)	1 2 3	0.074 0.098 0.107	NA NA NA	NA
Negative control (sesame oil)	10 11 12	0.043 0.020 0.053	NA NA NA	NA
Positive control (20% imidazole in 0.9% NaCl)	4 5 6	1.034 0.878 1.211	0.941 0.785 1.118	0.948

NA = not applicable

 

Table 3. In vitro irritancy score (IVIS)

Treatment group	Cornea no.	Corrected opacity change	Corrected OD490	IVIS per group
				mean
Test substance (20% in sesame oil)	7 8 9	188.33 208.33 202.33	1.316 1.579 1.593	222.11
Positive control (20% imidazole in 0.9% NaCl)	4 5 6	141.67 164.67 126.67	0.941 0.785 1.118	158.55

 

Interpretation of results:

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

Conclusions:

CLP: Cat. 1, H318

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data on in vivo and in vitro skin irritation/corrosion and in vitro eye irritation are available for Amines C12-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 96690-34-5) to fulfill the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VIII, 8.1 and 8.2. In accordance with Section 3.2.2.2.3 of Annex I to Regulation (EC) No. 1272/2008 an acute dermal toxicity study conducted with C12-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 96690-34-5) was also taken into account.

Skin Irritation

In vivo

The skin irritation potential of Amines, C12-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 96690-34-5) was investigated in a GLP-conform study according to OECD guideline 404 (WoE, BioChem GmbH, 1993). The undiluted test substance (0.5 mL) was applied for 4 h to the shaved skin of 3 albino rabbits (2 males and 1 female) under occlusive conditions. The untreated site of the animals served as control. The scoring of skin reactions (erythema and edema) was performed 1, 24, 48 and 72 h as well as on Days 6-10, Day 12 and Day 14 after removal of the dressing. At the 1 h reading time point, very slight (1/3 animals) to well-defined erythema (2/3 animals) were observed. At the same time point, 2 animals showed very slight edema (score 1), whereas 1 animals showed moderate edema (score 3). Well-defined erythema (score 2) and very slight edema (score 1) were observed in all animals 24, 48 and 72 h after exposure. In all animals, edema were fully reversible within 6 days after application, whereas very slight erythema (score 1) were still evident in the animals at the end of the 14-day observation period. During the study period, no signs of systemic toxicity were observed in treated animals. Over the 24, 48 and 72 h reading time points, the mean erythema scores were 2/2/2 and the mean edema scores were 1/1/1.33 for the 3 animals, respectively. Based on the irreversibility of skin reactions during the 14-day observation period, the test substance is considered to be irritating to the skin.

In vitro

Furthermore, an in vitro skin corrosion test using an artificial human skin model was performed with the test substance according to OECD guideline 431 and under conditions of GLP (WoE, Bayer Pharma AG, 2013). Within this assay, three replicates of intact reconstructed human epidermis (EST-1000) were treated with 50 µL of the undiluted test substance for 3 min and 1 h, respectively. Cells treated with 0.9% NaCl served as controls. No concurrent positive control was included in the assay, but reliability of the assay was checked on a regular basis. After exposure to the test substance, cell viability was determined compared to the control. The cell viability after 3 min exposure was below 50% (42%) and the cell viability after 1 h exposure was lower than 15% (12%). Therefore, the substance was considered to be corrosive to reconstructed human epidermis.

Acute dermal toxicity study

An acute dermal toxicity study (limit test) was performed with the test substance according to OECD guideline 402 and GLP conditions (Envigo Research Limited, 2017). 5 male and 5 female Wistar rats were exposed to 2000 mg test substance /kg bw for 24 h on the back skin under semi-occlusive conditions. The observation period was 14 days. Signs of dermal irritation noted included very slight to well defined erythema, slight edema, scattered areas of green necrosis, scattered areas of blanching, small scattered superficial scabs, hardened light and dark brown colored scabs, scab cracking, scab lifting to reveal glossy skin, dried blood and/or bleeding, crust formation and desquamation. Adverse dermal reactions prevented the accurate evaluation of erythema and edema at the treatment sites of two male and two female animals. Due to the observed necrosis in one male and one female, respectively, the test substance can be regarded as corrosive to the skin.

Conclusively, the test substance showed irritant properties towards the skin of rabbits in an in vivo skin irritation study according to OECD guideline 404 and corrosive properties towards the skin of rats in an acute dermal toxicity study according to OECD guideline 402. Based on the results of the in vitro skin corrosion test according to OECD guideline 431 using an artificial human skin model the substance was considered to be corrosive to reconstructed human epidermis. Overall and in a weight of evidence approach, the test substance was considered to be corrosive towards the skin.

Eye Irritation

In vitro

A GLP-conform Bovine Corneal Opacity and Permeability (BCOP) test was performed to identify the potential of Amines, C12-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 96690-34-5) to induce eye corrosivity and severe eye irritation (BSL Bioservice, 2012). Since the test substance was too viscous to be directly applied to the epithelium of the isolated corneas, the testing protocol for solids based on the principles of OECD 437 was selected for testing. According to this procedure, the test substance was suspended in sesame oil at a concentration of 20% and applied to each of three corneas for a period of 4 h. In addition each 3 corneas were treated with the respective positive (imidazole) and negative control substances (physiological saline and sesame oil) using the same treatment duration. After exposure, significant changes in opacity and permeability were observed in treated corneas compared to the negative control. Based on the measurements for opacity and permeability, an In Vitro Irritancy Score (IVIS) of 222.1, which is significantly exceeding the threshold value of ≥ 55 for a positive result, was calculated after treatment with the test substance. The IVIS of the positive control (158.55) was higher than that of the historical control values (out of the two standard deviations of the current historical mean), but still regarded valid. Based on the result of this study, the test substance was considered to be a severe eye irritant.

Justification for classification or non-classification

The available data on skin and eye irritation of the substance meet the criteria for classification as Skin Corr. Cat. 1B (H314) and Eye Dam. Cat. 1 (H318) according to Regulation (EC) 1272/2008.