Sulfonic acids, C16-alkane hydroxy and C16-alkene, sodium salts

Endpoint summary

Description of key information

Skin irritation

No experimental data are available for the target substance C16 IOS-Na.

 

An in-vitro skin irritation study according to OECD TG 439 is available for two dilutions of the closely related source substance 1 (C16-18 IOS-Na C16-rich).

After exposure of the 2% (v/v) test item solution the mean relative absorbance value decreased to 95.1%. This value is well above the threshold for irritancy of ≤ 50%. With the 20% (v/v) solution the mean relative absorbance value was reduced to 4.9%. As a result,C16-18 IOS-Na C16-rich is irritant to skin if used as 20% (v/v) dilution in deionised water, and is non-irritant if used as 2% (v/v) dilution in deionised water accordingto the criteria of the CLP regulation.

 

Because no information on the irritation potential of the neat substance was available, additional information with another source substance was taken into consideration.

 

An in-vivo skin irritation study with neat test substance according to OECD TG 404 is available for theclosely related source substance 2 (C14-16 AOS-Na).

At 30 min until 72 hours after patch removal the treated skin demonstrated barely perceptible (grade 1) to well defined (grade 2) erythema and very slight (grade 1) to slight (grade 2) edema. The skin surface was dry and rough and demonstrated fine scales which were still visible in 2/3 animals after 7 days, the end of the observation period. Additionally, desquamation of fine scales was observed in 1/3 animals. Based on this study, the neat substance has to be classified asirritating to the skin (Skin Irrit. 2 (H315: Causes skin irritation)) according to the criteria of the CLP regulation.

 

The result for source substance 2 will be also applied for the target substancein a worst-case approach.

 

 

Eye irritation

No experimental data are available for the target substance C16 IOS-Na.

 

An in-vitro eye irritation study according to OECD TG 437 is available for the closely related source substance 1 (C16-18 IOS-Na C16-rich).The test article was tested as a 2% (v/v) and a 20% (v/v) dilution in sterile, deionized water. For both dilutions the in-vitro scores were below 55.1 (IVIS 4.5 for the 20% dilution; IVIS 12.6 for the 2% dilution). According to OECD TG 437, a substance that induces an in vitro score of ≤ 3 would be predicted to be not irritating and a substance that induces an in vitro score of ≥ 55.1 would be predicted to be a corrosive or severe eye irritant. The in vitro scores of a test substance were below 55.1. Therefore the test article is not predicted to be an ocular corrosive or severe irritant. Because the IVIS is 3 <IVIS ≤ 55, no stand-alone prediction could be made based on this test.

 

Because a chemical that is not predicted as causing serious eye damage or as not classified for eye irritation/serious eye damage with the BCOP test method would require additional testing (in vitro and/or in vivo) to establish a definitive classification, further supporting information is taken into account from source substance 2 (C14-16 AOS-Na).

 

For source substance 2 (C14-16 AOS-Na) an in-vivo eye irritation study according to OECD TG 405 is available which was conducted with a test material in powder form containing 90% test substance. Between 1 and 72 hours after application clearly injected vessels up to diffuse, beefy red erythema and slight to severe swellings of the conjunctivae were observed. Furthermore, circumcorneal injection and corneal opacity were noticed. Most of these symptoms were not fully reversible within the observation period of 21 days, and corneal opacity even deteriorated; at the end of the observation period corneal scores of grade 4 (opaque cornea, iris not discernible through the opacity according to the OECD grading table of ocular lesions) were observed (CLP: Eye Dam.1). Concentrations higher than 90% were assumed to induce effects comparable to those observed, according to bridging principles. Therefore, the same classification has to be assumed for the neat substance.

 

According to the criteria of the CLP regulation the substance has to be considered as inducing "severe damage to the eyes" and would have to be classified as: CLP: Eye Dam. 1, H318: Causes serious eye damage

 

The result for source substance 2 will be also applied for the target substancein a worst-case approach.

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:

From 30th April 2013 to 7th June 2013

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Expiry/retest date: 30 June 2013

Test system:

other: EpiSkin Kit, Lot No.: 13-EKIN-021

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes

Vehicle:

other: Deionised water

Details on test system:

-Cell Culture
EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France). The EpiSkin™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.
EpiSkin™ tissues were shipped with ice packs on medium-supplemented agarose gels in a 12-well plate and reached Harlan CCR on June 04, 2013. After receipt EpiSkin™ tissues were transferred to 12-well plates with maintenance medium.

-Dose Selection
Each 10 µL of a 2% (v/v) and of a 20% (v/v) dilution of the test item in deionised water were applied to each of triplicate tissues for 15 minutes. The purity of the test item was taken into consideration for dose calculation.
The test was performed two times. In the first experiment, an incorrect dilution ratio for the test item (w/v instead of v/v) was used. The raw data of both experiments are archived, but only the results of the second experiment are reported in the present document.

-Test for Direct MTT Reduction
For correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. To test for this ability approximately 10 µL of the 20% (v/v) dilution of the test item in deionised water were added to 1 mL of MTT solution and the mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT medium was used as control. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.

-Pre-warming of EpiSkin™ Tissues
After incubation period of about 2 hours of the EpiSkin™ tissues were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium under sterile conditions using sterile forceps, the inserts.

-Treatment
The negative and the positive control, and the two different dilutions of the test item were added into the insert atop the concerning EpiSkin™ triplicate tissues. The 12-well plates were placed into the incubator for 15 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2.
After the end of the treatment interval the inserts were removed immediately from the 12-well plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for approximately 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

-MTT Assay
The MTT concentrate was prepared freshly and diluted with the MTT diluent. A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
After the treatment procedure was completed for all tissues of each time point cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (MatTek, USA) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for about 21.5 hours in the refrigerator.
Per each tissue sample 2  200 µL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany) with 570 ± 1 nm filter. Mean values were calculated from the 2 wells per tissue sample.
Some test chemicals may reduce MTT, which will result in a blue colour without any involvement of cellular mitochondrial dehydrogenase. Although in the present assay the test chemicals were rinsed off and the medium beneath the tissues was replaced before contact with MTT medium, some amount of a test chemical may be released by the tissues into the MTT medium and directly reduce the MTT, which would be interpreted as "tissue viability". MTT reducing capability of the test item was tested as described in section “9.5 Test for Direct MTT Reduction”.

-Evaluation of Results
The mean OD of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:

Relative viability (%) = (OD test item / OD mean of negative control) x 100

For the test item and the positive control the mean relative viability  standard deviation of the three individual tissues are calculated and used for classification according to the following prediction model:
For the current test, an irritation potential of a test item according to EU classification R38 (according to directive 67/548/EEC), H315 (according to regulation (EC) 1272/2008) is recommended if the mean relative tissue viability of three individual tissues is reduced below 50% of the negative control.

-Acceptability of the Assay
The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the three tissues is  0.6 till ≤ 1.5.
The relative standard deviations in between tissues of the same treatment group should be ≤ 18%.
An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is  40%.
The data of the quality control (determined by SkinEthic Laboratories, 69007 Lyon, France) of the respective EpiSkin™ lot is mentioned in the present report (the acceptance limit of the IC50 should be between 1.0 and 3.0 mg/mL after 18 hours treatment with SLS).
The historical data (means, standard deviation, and ranges) of the positive control as well as for the negative control obtained with the EpiSkin™ model at Harlan CCR are mentioned in this report.

- Negative Control
Deionised water (produced in-house) was used as negative control. 10 µL were applied to each of triplicate tissues for 15 minutes.

- Positive Control
A 5% SLS (Fluka, Sigma-Aldrich) solution in deionised water, prepared freshly prior to the performance of the experiment, was used as positive control. 10 µL were applied to each of triplicate tissues for 15 minutes.

Control samples:

yes, concurrent negative control

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

Each 10 µL of a 2% (v/v) and of a 20% (v/v) dilution of the test substance

Duration of treatment / exposure:

15 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2% (v/v) dilution of the test substance, mean of 3 replicates

Value:

95.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

20% (v/v) dilution of the test substance, mean of 3 replicates

Value:

4.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≧ 0.6 till ≤ 1.5 for the 15 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 19.2% thus ensuring the validity of the test system.

The relative standard deviations between the % variabilities of the test item, the positive and negative controls were below 13% (threshold of the "OECD TG 439 Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%), thus ensuring the validity of the study.

Table. Results after treatment with Internal olefin sulfonate (IOS) and comtrols

Dose Group	Treatment Interval	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Absorbance 570 nm Tissue 3*	Mean Absorbance of 3 Tissues	Relative Absorbance [%] Tissue 1, 2 + 3**	Relative Standard Deviation [%]	Rel. Absorbance [% of Negative Control]***
Negative Control	15 min	1.030	1.165	1.199	1.131	91.0, 102.9, 106.0	7.9	100.0
Positive Control	15 min	0.236	0.209	0.207	0.217	20.9, 18.4, 18.3	7.5	19.2
Test Item2% (v/v)	15 min	1.098	1.086	1.043	1.076	97.1, 96.0, 92.2	2.7	95.1
Test Item20% (v/v)	15 min	0.051	0.053	0.064	0.056	4.5, 4.7, 5.6	12.4	4.9

* Mean of two replicate wells after blank correction

** relative absorbance per tissue [rounded values]: 100×(absorbancetissue)/(mean absorbancenegative control)

*** relative absorbance per treatment group [rounded values]: 100×(mean absorbancetset item)/(mean absorbancenegative control)

 

 

Table. HISTORICAL DATA

Positive Control		Negative Control
Mean Viability	19.2%	Mean OD	1.004
Standard Deviation	10.0%	Standard Deviation	0.219
Range of Viabilities	1.7% - 35.4%	Range of ODs	0.607 – 1.521

Data of 206 studies performed from October 2007 until March 2013

Interpretation of results:

other: Non irritant if used as 2% (v/v) dilution in deionised water, and irritant to skin if used as 20% (v/v) dilution in deionised water

Conclusions:

In this study and under the experimental conditions reported, the 2% (v/v) dilution of Internal olefin sulfonate (IOS) in deionised water is not irritant to skin, whereas the 20% (v/v) dilution possesses a clear irritating potential according to UN GHS and EU CLP regulation. The purity of the test item was taken into consideration for dose calculation.

Executive summary:

This in vitro study was performed to assess the irritation potential of Internal olefin sulfonate (IOS) by means of the Human Skin Model Test according to OECD TG 439. Three tissues of the human skin model EpiSkin™ were treated with two different dilutions of the test item, the negative or the positive control for 15 minutes. Each 10 µL of a 2% (v/v) and of a 20% (v/v) dilution of the test item in deionised water, of the positive and the negative control were applied to each tissue, spread to match the surface of the tissue.

 

After treatment with the negative control (deionised water) the absorbance values were well within the required acceptability criterion of mean OD ≧ 0.6 till ≦ 1.5 for the 15 minutes treatment interval thus showing the quality of the tissues. Treatment with the positive control (5% SDS in deionised water) induced a sufficient decrease in the relative absorbance as compared to the negative control for the 15 minutes treatment interval thus ensuring the validity of the test system.

 

After exposure of the 2% (v/v) test item solution the mean relative absorbance value decreased to 95.1%. This value is well above the threshold for irritancy of ≤ 50%. With the 20% (v/v) solution the mean relative absorbance value was reduced to 4.9%.

 

In conclusion, it can be stated that in this study and under the experimental conditions reported, Internal olefin sulfonate (IOS) is irritant to skin if used as 20% (v/v) dilution in deionised water, and is non irritant if used as 2% (v/v) dilution in deionised water according to UN GHS and EU CLP regulation.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05. July 1994 to 19. July 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Only 7 days of observation period.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

adopted July 17, 1992

Deviations:

yes

Remarks:

Only 7 days observation period

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

31 July 1992

Deviations:

yes

Remarks:

Only 7 days observation period

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): α-Olefinsulfonate (4192), C14-C16-α-Olefinsulfonate, Sodium salt
- Physical state: faint yellow powder
- Analytical purity: 95% active
- Composition of test material, percentage of components: 3% Na2SO4; 2% residual oil
- Purity test date: 18 May 1994
- Lot/batch No.: E06184192 from 01 March 1994
- Expiration date of the lot/batch: 31 December 1994
- Storage condition of test material: dark at room temperature under extractor hood
- Other: pH 5 (1% in water)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Chemisch pharmazeutische Fabrik, Dr. Karl Thomae GmbH, 88400 Biberach, SPF breeding
- Age at study initiation: ca. 3 - 5 months
- Weight at study initiation: 2.8 - 3.8 kg
- Housing: individually in fully air conditioned rooms
- Diet: Altromin 2123 maintenance diet for rabbits, Altromin-GmbH, Lage/Lippe, ad libitum, and hay, ca. 15 g daily)
- Water: tap water from automatic drinking trough, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ±3
- Humidity (%): 50 ± 20
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

physiological saline

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 500 mg, moistened with 0.4 ml 0.9% NaCl-solution

Duration of treatment / exposure:

4 hours

Observation period:

30 to 60 min, 24, 48 and 72 hours, 7 days

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: dorsal area of the trunk was shaved on an area of 25 cm² with electric clippers. Only animals with intact skin were used.
- Type of wrap if used: Moistened test substance was applied to 2.5 x 2.5 cm cellulose patches covered with adhesive tape. The patch was secured to the prepared skin site and covered with a semiocclusive bandage.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, substance remnants were carefully removed with lukewarm tap water
- Time after start of exposure: 4 hours

SCORING SYSTEM: Draize, evaluations were done 30 - 60 min and 24, 48 and 72 hours after patch removal. As effects were observed 72 hours after removal additional readings were performed after 7 days.

Irritation parameter:

erythema score

Basis:

animal: #1, #2, #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 2 days

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: fine scales observed at the end of observation period

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: no edema observed

Remarks on result:

other: dry rough skin after 3 and 7 days

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.7

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: dry rough skin after 2 and 3 days, fine scales with desquamation after 7 days

Irritant / corrosive response data:

30 min until 72 hours after patch removal the treated skin demonstrated barely perceptible (grade 1) to well defined (grade 2) erythema and very slight (grade 1) to slight (grade 2) edema. The skin surface was dry and rought and demonstrated fine scales which were still visible in 2/3 animals after 7 days, the end of the observation period. Additionally, desquamation of fine scales was observed in 1/3 animals.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In this study, the test substance has irritating effects on the skin and has to be classified as Skin Irrit. 2 (H315: Causes skin irritation) according to the criteria of the CLP regulation.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar (eco)toxicological properties because
Source and target substances share structural similarities (predominantly linear aliphatic hydrocarbon chain) with a common functional group: (polar sulfonate group). The molecular structure is almost identical.
They are manufactured from similar resp. identical precursors under similar conditions. Therefore, common breakdown products via physical and biological processes, which result in structurally similar chemicals are evident. Target and source substances are both a mixture of linear long chain Sulfonic acids, alkene, sodium salts and Sulfonic acids, alkane hydroxy, sodium salts and share an identical counter ion. A constant pattern in the changing of the potency of the properties across the target and source substances by chain-length and is not observed, because the distribution is too narrow.

Therefore, read-across from the existing toxicity studies on the source substance, is considered as an appropriate adaptation to the standard information requirements of Annex VII, 8.1, 8.2, 8.3, 8.4, 8.5 as well as 9.1 and 9.2 of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see cross reference to assessment report: Justification for read-across document attached to section 13

3. ANALOGUE APPROACH JUSTIFICATION
see cross reference to assessment report: Justification for read-across document attached to section 13

4. DATA MATRIX
see cross reference to assessment report: Justification for read-across document attached to section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Irritation parameter:

erythema score

Basis:

animal: #1, #2, #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 2 days

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: fine scales observed at the end of observation period

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: no edema observed

Remarks on result:

other: dry rough skin after 3 and 7 days

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.7

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: dry rough skin after 2 and 3 days, fine scales with desquamation after 7 days

Irritant / corrosive response data:

30 min until 72 hours after patch removal the treated skin demonstrated barely perceptible (grade 1) to well defined (grade 2) erythema and very slight (grade 1) to slight (grade 2) edema. The skin surface was dry and rought and demonstrated fine scales which were still visible in 2/3 animals after 7 days, the end of the observation period. Additionally, desquamation of fine scales was observed in 1/3 animals.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Based on the result, Sulfonic acids, C16-alkane hydroxy and C16-alkene, sodium salts has to be classified as irritating to the skin (Skin Irrit. 2 (H315: Causes skin irritation)) according to the criteria of the CLP regulation.

Executive summary:

This read-across is based on the hypothesis that source and target substances have similar (eco)toxicological properties because

Source and target substances share structural similarities (predominantly linear aliphatic hydrocarbon chain) with a common functional group: (polar sulfonate group). The molecular structure is almost identical.

They are manufactured from similar resp. identical precursors under similar conditions. Therefore, common breakdown products via physical and biological processes, which result in structurally similar chemicals are evident. Target and source substances are both a mixture of linear long chain Sulfonic acids, alkene, sodium salts and Sulfonic acids, alkane hydroxy, sodium salts and share an identical counter ion. A constant pattern in the changing of the potency of the properties across the target and source substances by chain-length and is not observed, because the distribution is too narrow.

 

Therefore, read-across from the existing skin irritation study on the source substance, is considered as an appropriate adaptation to the standard information requirements of Annex VII, 8.1 of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 5th Feb 2013 to 17th Apr 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Details on test animals or tissues and environmental conditions:

-Bovine Eyes
Bovine eyes were obtained from a local abattoir as a by-product from freshly slaughtered animals (J.W. TREUTH & SONS, Inc., Baltimore, MD). The eyes were excised and then placed in Hanks' Balanced Salt Solution, containing Penicillin/Streptomycin (HBSS), and transported to the laboratory on ice packs. Immediately upon receipt of the eyes into the laboratory, preparation of the corneas was initiated.

-Preparation of Corneas
The eyes were grossly examined for damage and those exhibiting defects were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised such that a 2 to 3 mm rim of sclera was present around the cornea. The isolated corneas were then stored in a petri dish containing HBSS until they were mounted in a corneal holder. The corneas were mounted in the holders with the endothelial side against the O-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and the screws were tightened. Starting with the posterior chamber, the two chambers were then filled with Minimum Essential Medium (EMEM) without phenol red, containing 1% fetal bovine serum and 2 mM L glutamine (Complete MEM (without phenol red)). Each corneal holder was uniquely identified with a number written in permanent marker, on both the anterior and posterior chambers. The corneal holders were incubated at 32 ± 1ºC for a minimum of 1 hour.

Vehicle:

water

Controls:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test article was tested as a 2% (v/v) and a 20% (v/v) dilution in sterile, deionized water. An aliquot of 750 µL of each test article dilution, positive control (ethanol), or negative control (deionized water) was introduced into the anterior chamber.

Duration of treatment / exposure:

Three corneas were incubated in the presence of the positive control at 32 ± 1ºC for 10 minutes. Three corneas were incubated in the presence of the negative control at 32 ± 1ºC for 10 minutes. Five corneas were incubated in the presence of each test article dilution at 32 ± 1ºC for 10 minutes.

Number of animals or in vitro replicates:

Three corneas were incubated in the presence of the positive and negative control.
Five corneas were incubated in the presence of each test article dilution.

Details on study design:

-Controls
The positive control used in this study was ethanol (Pharmco). The negative control used in this study was sterile, deionized water (Quality Biological).

-Test Article Preparation
The test article was administered to the test system as a 2% (v/v) and a 20% (v/v) dilution in sterile, deionized water. The test article was supplied at a concentration of 35% and was warmed to 37°C prior to dilution preparation. The 20% (v/v) test article dilution was prepared by adding 4 mL of test article to 3 mL of water in a prelabeled conical tube. The 2% (v/v) test article dilution was prepared by adding 0.4 mL of test article to 6.6 mL of water in a prelabeled conical tube. The dilutions were vortexed for approximately 1 minute prior to application. The 20% (v/v) dilution was described as a slightly cloudy yellow semi-viscous liquid. The 2% (v/v) dilution was described as a clear colorless non-viscous solution.

-Bovine Corneal Opacity and Permeability Assay
After a minimum of 1 hour of incubation, the corneas were removed from the incubator. The medium was removed from both chambers and replaced with fresh Complete MEM (without phenol red). The initial opacity was determined for each cornea using an Electro Design OP-KIT opacitometer. Any cornea whose initial opacity was greater than 7 was not used in the assay. The treatment of each cornea was identified with the test article number written in permanent marker on colored tape, affixed to each holder. The medium was then removed from the anterior chamber and replaced with the test article, positive control, or negative control.

-Method for Testing Liquid or Surfactant Materials
The test article was tested as a 2% (v/v) and a 20% (v/v) dilution in sterile, deionized water. An aliquot of 750 µL of each test article dilution, positive control, or negative control was introduced into the anterior chamber while slightly rotating the holder to ensure uniform distribution over the cornea. Due to its viscous nature, the 20% (v/v) dilution of the test article was administered by direct addition onto the epithelial surface of the cornea using a positive displacement pipet (open chamber window technique). In the open chamber window technique, the glass window was removed from the anterior chamber immediately prior to treatment. Each cornea was completely covered with test article. After dosing, the glass window was replaced on the anterior chamber. Three corneas were incubated in the presence of the positive control at 32 ± 1ºC for 10 minutes. Three corneas were incubated in the presence of the negative control at 32 ± 1ºC for 10 minutes. Five corneas were incubated in the presence of each test article dilution at 32 ± 1ºC for 10 minutes. After the 10-minute exposure time, the control or test article treatments were removed. The epithelial side of the corneas was washed at least three times with Complete MEM (containing phenol red) to ensure total removal of the control or test articles. The corneas were then given a final rinse with Complete MEM (without phenol red). For the corneas treated using the open chamber window technique, the glass window was removed from the anterior chamber, and the test article was rinsed from the treated cornea and the anterior chamber with Complete MEM (with phenol red). Care was taken not to spray the corneas directly. The chamber windows were returned to the chambers when most or all of the test article had been removed. The rinsing process continued in the same manner as the positive and negative control corneas. The corneas were then given a final rinse with Complete MEM (without phenol red). The anterior chambers were refilled with fresh Complete MEM (without phenol red) and an opacity measurement was performed. The corneas were returned to the incubator for approximately 2 hours after which a final measure of opacity was obtained.

After the final opacity measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was filled with fresh Complete MEM (without phenol red) and 1 mL of a 4 mg/mL fluorescein solution was added to the anterior chamber. The corneas were then incubated in a horizontal position (anterior side up) for approximately 90 minutes at 32 ± 1ºC. At the end of the 90-minute incubation period, the medium was removed from the posterior chambers and placed into tubes numbered corresponding to chamber number. Aliquots of 360 µL from the numbered tubes were placed into their designated wells on a 96 well plate. The optical density at 490 nm (OD490) was determined using a Molecular Devices Vmax kinetic microplate reader. If the OD490 value of a control or test article sample was greater than 1.500, a 1:5 dilution of the sample was prepared in Complete MEM (without phenol red) (to bring the OD490 value within the linear range of the platereader). A 360 µL sample of each 1:5 dilution was transferred to its specified well on the 96 well plate. The plate was read again and the final reading was saved to a designated print file.

-Criteria for Determination of a Valid Test
The BCOP assay was accepted when the positive control (ethanol) caused an in vitro score that fell within two standard deviations of the historical mean.

-Bovine Corneal Opacity and Permeability Assay
According to OECD TG 437, a substance that induces an in vitro score of ≥55.1 would be predicted to be a corrosive or severe eye irritant. If the substance is not predicted to be an ocular corrosive or severe irritant, additional testing should be conducted for regulatory classification or labeling purposes.

Irritation parameter:

in vitro irritation score

Run / experiment:

20% of the test item

Value:

4.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2% of the test item

Value:

12.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Since the results of the positive control fell within two standard deviations of the historical mean (within a range of 39.2 to 63.9), the assay was considered valid.

Table 1 summarizes the opacity, permeability, and in vitro score for each test article dilution. Table 2 summarizes the opacity, permeability, and in vitro score for the positive control.

 

Table 1

BCOP Results of the Test Article

	Assay Date	IIVS Test Article Number	Conc. (v/v)	Exposure Time	Mean Opacity Value	Mean OD490 Value	In Vitro Score	pH
14 Feb 2013		13AA85	20%	10 minutes	-0.9	0.357	4.5	8.5
			2%	10 minutes	4.5	0.534	12.6	6.0

 

Table 2

BCOP Results of the Positive Control

Assay Date	Positive Control	Exposure Time	Mean Opacity Value	Mean OD490 Value	In Vitro Score
14 Feb 2013	Ethanol	10 minutes	32.0	0.941	46.1

 

Interpretation of results:

other: The in vitro scores of a test substance were below 55.1. Therefore the test article is not predicted to be an ocular corrosive or severe irritant. However for the classification purpose, further testing is required.

Conclusions:

According to OECD TG 437, a substance that induces an in vitro score of ≥55.1 would be predicted to be a corrosive or severe eye irritant. The in vitro scores of a test substance were below 55.1. Therefore the test article is not predicted to be an ocular corrosive or severe irritant.

Executive summary:

The purpose of this study was to evaluate the potential ocular irritancy of the test article as measured by changes in opacity and permeability (to fluorescein) in isolated bovine corneas.

 

The test article was tested as a 2% (v/v) and a 20% (v/v) dilution in sterile, deionized water. Three corneas were incubated in the presence of the positive control and the negative control each at 32 ± 1ºC for 10 minutes. Five corneas were incubated in the presence of each test article dilution at 32 ± 1ºC for 10 minutes. After the 10-minute exposure time, the control or test article treatments were removed then the corneas were returned to the incubator for approximately 2 hours. An in vitro score was determined for each test article based on the induction of opacity and permeability (to fluorescein) in the isolated bovine corneas. According to OECD TG 437, a substance that induces an in vitro score of ≥55.1 would be predicted to be a corrosive or severe eye irritant.

 

The in vitro scores of a test substance were below 55.1. Therefore the test article is not predicted to be an ocular corrosive or severe irritant.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28. August 1984 to 18. September 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted 1981

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): Hostapur OS Pulver; alpha-Olefinsulfonate, sodium salt
- Molecular weight (if other than submission substance): approx. 310
- Physical state: white powder
- Analytical purity: approx. 90 %
- Impurities (identity and concentrations): approx. 5 % Na2SO4, 3 % Na2CO3, 2 % residual oil
- Composition of test material, percentage of components: C-chainlengths: ≤ C12: max. 1 %, C14: approx. 65 %, C16: approx. 30 %, ≥ C18: max. 1.5 %
- Lot/batch No.: E0617179, dated 1984-06-05
- Storage condition of test material: dark at 22°C
- Other: Source: Hoechst AG

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG, Kastengrund, conventional breeding
- Weight at study initiation: 2.9 - 3.2 kg
- Housing: individually
- Diet (e.g. ad libitum): Altromin 2123 maintenance diet - rabbit, Altromin GmbH, Lage/Lippe, ad libitum
- Water (e.g. ad libitum): deionised chlorinated water, automatic supply, ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2
- Humidity (%): 55 ± 10
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

physiological saline

Controls:

other: untreated eyes served as control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg, moistened with physiological saline, into conjunctival sac of left eye


VEHICLE
- Amount(s) applied (volume or weight with unit): 0.08 mL 0.9 % physiological saline

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

21 days

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, with 37°C warm physiological saline
- Time after start of exposure: 24 hours


SCORING SYSTEM: Draize


TOOL USED TO ASSESS SCORE: additionally fluorescein (0.01 %), UV light at 24 and 72 hours

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: opacity becoming worse during observation period, score of "4" after 21 days

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: opacity becoming worse during observation period, score of "4" after 21 days

Irritation parameter:

iris score

Basis:

animal: #1 and #2

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 14 days

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.67

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not reversible

Remarks on result:

other: score of "2" after 21 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.67

Max. score:

3

Reversibility:

not reversible

Remarks on result:

other: score of "2" after 21 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: Score of "2" after 21 days

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: Score of "1" after 21 days

Irritant / corrosive response data:

Between 1 and 72 hours after application clearly injected vessels up to diffuse, beefy red erythemae and slight to severe swellings of the conjunctivae were observed. Furthermore, circumcorneal injection of the iris and corneal opacity were noticed. Additionally, the treated eyes demonstrated discharge, clear and colourless in the beginning, white and viscous later on.

One animal was free of symptoms after 7 days, the remaining 2 animals still suffered from distinct edema and erythema, ranging from diffuse, crimson red up to diffuse, beefy red. Additionally, white, viscous discharge was observed.

After 14 and 21 days these animals still demonstrated obvious edema and clearly injected vessels or diffuse, crimson red erythema of the conjunctivae, and complete corneal opacity with advanced vascularisation.

Other effects:

non reported

Interpretation of results:

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

Conclusions:

According to the criteria of the CLP regulation the substance has to be considered as inducing "severe damage to the eyes" and would have to be classified as:

CLP: Eye Dam. 1, H318: Causes serious eye damage

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar (eco)toxicological properties because
Source and target substances share structural similarities (predominantly linear aliphatic hydrocarbon chain) with a common functional group: (polar sulfonate group). The molecular structure is almost identical.
They are manufactured from similar resp. identical precursors under similar conditions. Therefore, common breakdown products via physical and biological processes, which result in structurally similar chemicals are evident. Target and source substances are both a mixture of linear long chain Sulfonic acids, alkene, sodium salts and Sulfonic acids, alkane hydroxy, sodium salts and share an identical counter ion. A constant pattern in the changing of the potency of the properties across the target and source substances by chain-length and is not observed, because the distribution is too narrow.

Therefore, read-across from the existing toxicity studies on the source substance, is considered as an appropriate adaptation to the standard information requirements of Annex VII, 8.1, 8.2, 8.3, 8.4, 8.5 as well as 9.1 and 9.2 of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see cross reference to assessment report: Justification for read-across document attached to section 13

3. ANALOGUE APPROACH JUSTIFICATION
see cross reference to assessment report: Justification for read-across document attached to section 13

4. DATA MATRIX
see cross reference to assessment report: Justification for read-across document attached to section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Controls:

other: untreated eyes served as control

Number of animals or in vitro replicates:

3

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: opacity becoming worse during observation period, score of "4" after 21 days

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: opacity becoming worse during observation period, score of "4" after 21 days

Irritation parameter:

iris score

Basis:

animal: #1 and #2

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 14 days

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.67

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not reversible

Remarks on result:

other: score of "2" after 21 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.67

Max. score:

3

Reversibility:

not reversible

Remarks on result:

other: score of "2" after 21 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: Score of "2" after 21 days

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: Score of "1" after 21 days

Irritant / corrosive response data:

Between 1 and 72 hours after application clearly injected vessels up to diffuse, beefy red erythemae and slight to severe swellings of the conjunctivae were observed. Furthermore, circumcorneal injection of the iris and corneal opacity were noticed. Additionally, the treated eyes demonstrated discharge, clear and colourless in the beginning, white and viscous later on.

One animal was free of symptoms after 7 days, the remaining 2 animals still suffered from distinct edema and erythema, ranging from diffuse, crimson red up to diffuse, beefy red. Additionally, white, viscous discharge was observed.

After 14 and 21 days these animals still demonstrated obvious edema and clearly injected vessels or diffuse, crimson red erythema of the conjunctivae, and complete corneal opacity with advanced vascularisation.

Other effects:

non reported

Interpretation of results:

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

Conclusions:

According to the criteria of the CLP regulation Sulfonic acids, C16-alkane hydroxy and C16-alkene, sodium salts has to be considered as inducing "severe damage to the eyes" and would have to be classified as:

CLP: Eye Dam. 1, H318: Causes serious eye damage

Executive summary:

This read-across is based on the hypothesis that source and target substances have similar (eco)toxicological properties because

Source and target substances share structural similarities (predominantly linear aliphatic hydrocarbon chain) with a common functional group: (polar sulfonate group). The molecular structure is almost identical.

They are manufactured from similar resp. identical precursors under similar conditions. Therefore, common breakdown products via physical and biological processes, which result in structurally similar chemicals are evident. Target and source substances are both a mixture of linear long chain Sulfonic acids, alkene, sodium salts and Sulfonic acids, alkane hydroxy, sodium salts and share an identical counter ion. A constant pattern in the changing of the potency of the properties across the target and source substances by chain-length and is not observed, because the distribution is too narrow.

 

Therefore, read-across from the existing eye irritation study on the source substance, is considered as an appropriate adaptation to the standard information requirements of Annex VII, 8.2 of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation

Based on the results of the in-vitro study with a 20% (v/v) dilution and in the vivo study with neat substance, the substance has to be classified as irritating to the skin (Skin Irrit. 2 (H315: Causes skin irritation)) according to the criteria of the CLP regulation.

Eye irritation

According to the criteria of the CLP regulation the substance has to be considered as inducing "severe damage to the eyes" and would have to be classified as: CLP: Eye Dam. 1, H318: Causes serious eye damage