Reaction products of alcohols, C11-14-iso, C13 rich and phosphorus pentoxide

Endpoint summary

Description of key information

Skin irritation: irritant (OECD 439)

Skin corrosive: non-corrosive (OECD 431)

Eye irritation: causing serious eye damage (OECD 437)

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:

16 Dec 2017- 04 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EPI-200-SIT

Justification for test system used:

Recommended in vitro model

Details on test system:

Dose Groups
1. Negative control 30 µL DPBS
2. Positive control 30 µL 5% SDS solution
3. Test Item 30 µL
The test was performed on a total of 3 tissues per dose group.
SDS sodium dodecyl sulfate
DPBS Dulbecco's phosphate buffered saline

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 µL Test item

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

47 hours

Number of replicates:

3

Details on study design:

The test was performed on EpiSkin,a three-dimensional model of the human epidermis. 3 replicate tissues are dosed with the test
item, the negative control (30 µL DPBS) and the positive control (300µL 5% SDS), respectively. After 60 minutes treatment period at room temperature the test item and the controls are rinsed off with DPBS and the tissues are post-incubated for 47 h (based on RhE model). Then the tissues are stained via MTT for 3 hours. The MTT was extracted from the tissues for 2 hours at room temperature with gentle shaking on a plate shaker. MTT extracts are measured photometrically at 570 nm.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

negative control

Value:

>= 96.1 - < 102.6

Negative controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

positive control

Value:

>= 4.7 - < 4.9

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item

Value:

>= 7.2 - < 7.8

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Based on the results obtained under the laboratory testing conditions, the test item has been categorized as irritant to Reconstructed Human Epidermis (RhE) in accordance with UN GHS Category 2 or Category 1, as the mean percentage after 60 minutes of exposure and 45 hours and 10 minute post incubation, tissue viability was less than 50% of the negative control.

Executive summary:

The skin irritation potential of the submission study was evaluated using Reconstructed Human Epidermal Model - EpiDerm (EPI-200 -SIT) according to OECD Guideline 439.

Based on the results obtained under the laboratory testing conditions, the test item has been categorized as irritant to Reconstructed Human Epidermis (RhE) in accordance with UN GHS Category 2 or Category 1, as the mean percentage after 60 minutes of exposure and 45 hours and 10 minute post incubation, tissue viability was less than 50% of the negative control.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-09-19 to 2019-09-19

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:

adopted on 18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Amount/concentration applied:

The Reconstructed Human Epidermal Model - EpiDerm™ (EPI-200-SCT) was used as test system.
Source of the Test System: MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05, Bratislava II, Slovak Republic, www.mattek.com; Phone: [phone]; Fax: [phone].

Duration of treatment / exposure:

3min, 1hour

Number of replicates:

two

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3min

Value:

50.8

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of corrosive

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour

Value:

34.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of corrosive

Other effects / acceptance of results:

The mean OD of the negative control tissues is 1.537 (3 minutes exposure) and 1.201 (1 hour exposure) which was within the range of ≥0.8 and ≤2.8, hence the tissues were considered as viable after shipping and storing procedures and under specific conditions of use.
The mean percentage viability of positive control treated tissues was 6.1 after 3 minutes and 6.4 after 1 hour exposure which were <15% of the negative control clearly represents the irritation potential of positive control.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

together with another study according to OECD 439

Conclusions:

Based on the results obtained under the laboratory testing conditions, the test item Hostacor ITD is considered to be non-corrosive, as the mean percentage tissue viability was greater than 50% after 3 minutes exposure and greater than 15% after 1 hour exposure of the negative control.

Executive summary:

The objective of this study was to evaluate the in vitro skin corrosion potential of Hostacor ITD by measurement of tissue viability in the Epidermal Model - Epiderm™ (EPI-200-SCT) as per the OECD Guideline for the testing of chemicals No. 431,  “In vitro skin corrosion: reconstructed human epidermis (RHE) test method”, adopted on 18 June 2019.

The test item did not develop any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution.

After receipt of the tissues, visual inspection was done to verify the defects. There were no tissue defects, air bubble or excess moisture observed. All the tissue inserts were used for the study. Tissue inserts were transferred to upper row of 6 well plates prefilled with 0.9 mL of assay medium and incubated in CO2 incubator for 65 minutes.

Exposure with the test item was performed for 1 hour and 3 minutes separately. All the treatments were maintained in duplicates. For 3 minutes treatment, quantity of 50 µL of sterile distilled water (NC) was dispensed into the first insert atop the tissue. After 60 seconds the procedure was repeated with second tissue and continued for all other tissues. Similar procedure was followed in the same manner until all the tissues were treated. Tissues were treated with 50 µL test item and 50 µL of positive control (glacial acetic acid).

For 1 hour treatment, 50 µL of test item, 50 µL of negative control (sterile distilled water) and 50 µL of positive control were dispensed directly atop Epiderm™ tissues at 1 minute intervals to facilitate rinsing after exposure. The tissues were incubated at standard culture conditions for 1 hour.

At the end of treatment time tissue inserts were rinsed with sterile PBS (fill and empty insert 20 times in a constant soft stream of 1xPBS) to remove any residual test item. Post rinsing procedure, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into 24-well plate containing 0.3 mL of MTT solution (1 mg/mL) and incubated for 3 hours at 37±1°C and 5±1% CO2. Post incubation, the tissue inserts were removed and blotted onto the tissue paper and transferred to a prelabeled 24-well plate containing 2.0 mL of isopropanol in each designated well. The plates were placed on an orbital plate shaker and shaken (̴ 120 rpm/minute) for 2 hours at room temperature. At the end of the extraction period, the tissue was pierced with an injection needle and the extract was allowed to run into the well from which the insert was taken. The punctured inserts were discarded and solution was placed on mixer for 15 minutes until it became homogenous. The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm. Viability of tissues was calculated.

After 3 minutes exposure, the percentage viability of the negative control, positive control and test item was 100±5.5, 6.1±0.2 and 50.8±1.6 respectively. The percentage viability of the test item was thus greater than 50% of the negative control. The percentage viability of the positive control (PC) is less than 50% of the negative control and thus clearly represents the irritation potential of the positive control.

After 1 hour exposure, the percentage viability of negative control, positive control and test item was 100±1.3, 6.4±0.1 and 34.7±0.7 respectively. As the percentage viability of the test item was greater than 15% of the negative control. The percentage viability of the positive control (PC) was less than 15% of negative control and thus clearly represents the irritation potential of the positive control. Positive and negative controls showed the expected results. The experiments were considered to be valid.

Based on the results obtained under the conditions of this study, the test item  Hostacor ITDis considered asnon-corrosive in accordance with UN GHS,as the mean percentage tissue viability was greater than 50% after 3 minutes exposure and greater than 15% after 1 hour exposure of the negative control.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

opacity & permeability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 January 2018 to 07 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: No. 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”, adopted on 9th October 2017

Version / remarks:

9th October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Slaughter House
- Age at study initiation: 3.5 to 4.5 years

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 Hours

Number of animals or in vitro replicates:

Triplicates

Details on study design:

QUALITY CHECK OF THE ISOLATED CORNEAS: Done

NUMBER OF REPLICATES: Triplicates

NEGATIVE CONTROL USED: Normal saline

POSITIVE CONTROL USED: Ethanol

APPLICATION DOSE AND EXPOSURE TIME : 750 µL & 10 mins
TREATMENT METHOD: [closed chamber]

POST-INCUBATION PERIOD: yes, duration: 2 hours

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3 times
- POST-EXPOSURE INCUBATION: 2 hours

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Done
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of spectrophotometry] (OD490) : Done
- Others: histopathology

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1
SELECTION AND PREPARATION OF CORNEAS

QUALITY CHECK OF THE ISOLATED CORNEAS

NUMBER OF REPLICATES

NEGATIVE CONTROL USED

SOLVENT CONTROL USED (if applicable)

POSITIVE CONTROL USED

APPLICATION DOSE AND EXPOSURE TIME

TREATMENT METHOD: [closed chamber / open chamber]

POST-INCUBATION PERIOD: yes/no. If YES please specify duration

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
- POST-EXPOSURE INCUBATION:

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [ spectrophotometry] (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean-Test item

Value:

95

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean-Positive control

Value:

116.4

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: No

Other effects:

- Histopathological findings: Epithelium: Mild focal squamous cell coagulation
Stroma: Apparently normal
Endothelium: Apparently normal

Interpretation of results:

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

Conclusions:

Based on the results obtained in the Bovine Corneal Opacity Test, the test item induced an IVIS of 95.0 after 10 minutes of treatment. The results indicated an appreciable increase in both the endpoints “permeability” as well as “opacity”. As the test item resulted in IVIS >55, considered as severe irritant causing serious eye damage and classified as UN GHS category 1, and no further testing will be carried out.

Executive summary:

Eyes of cattle were collected from slaughter house by immersing them in the Hank’s Balanced Salt Solution (HBSS) with antibiotics (penicillin and streptomycin) in a suitable container and transported to the test facility by placing on cool packs. Eye balls free of defects were selected for the experiment. Empty cornea holder’s opacity with pre-warmed Eagle’s Minimum Essential Medium was measured and the mean opacity value obtained was determined as I₀.Cornea holders with selected Corneas were equilibrated at 32±1ºC for 1 hour with Eagle’s Minimum Essential Medium with       1% Fetal Bovine Serum supplemented with 1% antibiotics and baseline opacity was recorded for each cornea. Corneas with opacity units less than 7 were selected and used for the study and distributed for the treatment groups.Quantity of 750 µL of test item, normal saline (negative control) and ethanol (positive control) was introduced into anterior chamber in triplicates to the designated cornea holders and incubated at 32±1ºC for 10 minutes. Treated corneas were washed with EMEM with phenol red. Opacity was measured with the aid of opacitometer and permeability was determined spectrophotometrically at 490 nm (OD₄₉₀) using 4 mg/mL sodium fluorescein, post incubation of 90 min at 32±1ºC. Baseline opacity and permeability values obtained for negative control (normal saline) treated corneas were used for correction.The mean corrected opacity and mean corrected permeability values of test item is 71.31 and 1.581 respectively. The in vitro Irritancy Score (IVIS) of test item resulted in 95.0 indicating corrosivity or severe irritancy to Bovine corneas.

Whereas the positive control resulted in the mean corrected opacity and mean corrected permeability values is 91.91 and 1.633 respectively. Thein vitroIrritancy Score (IVIS) of positive control resulted in 116.4, indicatingcorrosivity or severe irritancy to Bovine corneas.

In histopathological examination, the corneas treated with negative control did not show any abnormalities, whereas in thehistopathological examination ofthe corneas treated with test item resulted in mild focal squamous cell coagulation in epithilium. Stroma and endothelium of all corneas were apparently normal. 

In positive control, histopathology data of epitheliumshowed mild diffuse and moderate multifocal cytoplasmic and nuclear vacuolization in the wing and basal layers.In stroma minimal multifocal expansion of the superficial collagen was observed. Endothelium was apparently normal in all three corneas.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The skin irritation potential of the submission substance was evaluated using Reconstructed Human Epidermal Model - EpiDerm (EPI-200 -SIT) according to OECD Guideline 439.

Based on the results obtained under the laboratory testing conditions, the test item has been categorized as irritant to Reconstructed Human Epidermis (RhE), as the mean percentage after 60 minutes of exposure and 45 hours and 10 minute post incubation, tissue viability was less than 50% of the negative control.

The skin corrosive potential of the substance was evaluated by measurement of tissue viability in the Epidermal Model - Epiderm™ (EPI-200-SCT) as per the OECD Guideline for the testing of chemicals No. 431, “In vitroskin corrosion: reconstructed human epidermis (RHE) test method”. Based on the results obtained under the conditions of this study, the test item  Hostacor ITDis considered as non-corrosive, as the mean percentage tissue viability was greater than 50% after 3 minutes exposure and greater than 15% after 1 hour exposure of the negative control.

The eye irritation potential of the submission substance was evaluated in a Bovin Corneal Ipacity Test according to OECD Guideline 437.

Based on the results obtained in the Bovine Corneal Opacity Test, the test item induced an IVIS of 95.0 after 10 minutes of treatment. The results indicated an appreciable increase in both the endpoints “permeability” as well as “opacity”. As the test item resulted in IVIS >55, considered as severe irritant causing serious eye damage and classified as UN GHS category 1, and no further testing will be carried out.

Justification for classification or non-classification

Based on the results obtained in a studies according to OECD 439 and OECD 431, the test item has been categorized as non-corrosive but irritant to Reconstructed Human Epidermis (RhE) in accordance with UN GHS Category 2.

Based on the results obtained in the Bovine Corneal Opacity Test, the test item induced an IVIS of 95.0 after 10 minutes of treatment. The results indicated an appreciable increase in both the endpoints “permeability” as well as “opacity”. As the test item resulted in IVIS >55, considered as severe irritant causing serious eye damage and classified as Category 1 (irrevesible effects on the eye) based on GHS criteria.