Sulfates of potassium, sodium and calcium, byproduct from fermentation

Endpoint summary

Description of key information

Skin irritation: not irritating to skin according to an in vitro study (Reliability 1 key study; GLP compliant; OECD 439 test guideline) (CiToxLAB Hungary Ltd , 2017).

Eye irritation: not irritating to eyes according to an in vitro study (Reliability 1 key study; GLP compliant; OECD 438 test guideline) (CiToxLAB Hungary Ltd , 2017).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 12 April 2017 to 12 September 2017

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:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the Sponsor, EXP LIberica Lote: AI16503047
- Expiration date of the lot/batch: 30 November 2019
- Purity test date: 1 February 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 ºC, below 70 RH%), protected from light and humidity
- Stability under test conditions: not applicable
- Solubility and stability of the test substance in the solvent/vehicle: no vehicle was used
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: the test item did not react with MTT or Medium Assay

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was used as supplied

Test system:

artificial membrane barrier model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: reconstructed epiderdermis

Source strain:

other: not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin
- Tissue batch number(s): 17-EKIN-017
- Production date: 25 April 2017
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 26 April 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C


REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1
- Observable damage in the tissue due to washing: Not specified
- Modifications to validated SOP: It was rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3 mg/mL(stock solution) ; 0.3 mg/mL per well
- Incubation time: 3 hours (± 5 min)
- Spectrophotometer: Plate reader Thermo Fisher Scientific, Catalogue Number: 240 72800
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 0.805
- Barrier function: Yes
- Morphology: Well-differentiated epidermis consisting of a basal monolayer, several spinous and granular layers and thick stratum corneum
- Contamination: Absence of HIV1 and 2, Hepatitic C, Hepatitis B and absence of bacteria, fungus and mycoplasma
- Reproducibility: yes

NUMBER OF REPLICATE TISSUES: triplicates were used

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
killed tissues
- Procedure used to prepare the killed tissues (if applicable): not specified
- N. of replicates : triplicates
- Method of calculation used:
Non Specific Colour % with killed tissues (NSCkilled%):
NSCkilled % = (mean ODCTK / mean ODNC)×100
ODCTK: test substance treated killed tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3 test sequences : Pre incubation / Application and Rinsing / MTT Test

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after after 15 minutes exposure to the test item and 42 hours post incubation is less than 50%
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure to the test item and 42 hours post incubation is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 439: not specified

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
As the test item was solid, first an appropriate amount (10 µL) distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis and then 20 mg of powdered the test item was applied evenly to the epidermal surface. If necessary, the test item was spread gently on the skin surface with a pipette tip (or other appropriate tool) without damaging the epidermis. The amount was sufficient to cover the epidermal surface.

CONTROLS
50 µL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item

Value:

103

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Negative Control

Value:

100

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive Control

Value:

3.4

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No effect
- Direct-MTT reduction: No effect
- Colour interference with MTT: No effect

DEMONSTRATION OF TECHNICAL PROFICIENCY: The positive control treated tissues showed 3.4% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.5.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 4.7.

Table 1: Optical Density (OD) and the calculated relative viability % of the samples

 

Substance		Optical Density (OD)			Viability	SD
			Measured	Blank corrected	(% RV)
Negative Control:		1	0.780	0.734	94.5	-
Phosphate buffered saline		2	0.846	0.799	103.0
		3	0.842	0.796	102.5
		mean	--	0.776	100.0	4.7
Positive Control:		1	0.067	0.021	2.6	-
5%(w/v)SDSsolution		2	0.065	0.019	2.4
		3	0.086	0.040	5.1
		mean	--	0.026	3.4	1.5
Test Item:		1	0.804	0.758	97.6	-
Sulfates of potassium, sodium and calcium,		2	0.859	0.813	104.7
by-product from fermentation		3	0.874	0.828	106.6
		mean	--	0.799	103.0	4.7

Notes:

1.         Mean blank value was0.047.

2.         Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

3.         SD: Standard deviation

 

Table 2 : HISTORICAL CONTROL DATA

(updated11 August 2016)

 

	Negative control (PBS)	Positive control (5% (w/v) SDS solution)
Mean optical density (OD)	0.802	0.094
Standard deviation	0.157	0.048
Minimum optical density (OD)	0.573	0.032
Maximum optical density (OD)	1.362	0.354
Number of cases	111	102

PBS: Phosphate buffered saline SDS: Sodium dodecyl sulphate OD: Optical density (absorbance)

Note: All OD values (measured at 570 ±30 nm) are background corrected values.

 

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in this in vitro EPISKINTM (SM) model test with Sulfates of potassium, sodium and calcium, by-product from fermentation (Batch number: EXP LIberica Lote: AI16503047), the results indicated that the test item was non-irritant to skin. Hence, the test item was not classified for Skin Irritation according to CLP regulation.

Executive summary:

A GLP compliant in vitro skin irritation test of Sulfates of potassium, sodium and calcium, by-product from fermentation test item was performed in a reconstructed human epidermis model. EPISKINTM (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) assay (detailed in 3.6. section). The irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

Disks of EPISKINTM (SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Following exposure with Sulfates of potassium, sodium and calcium, by-product from fermentation, the mean cell viability was 103.0% compared to  the negative  control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In  conclusion,   in   this   in   vitro   EPISKINTM   (SM)   model   test   with   Sulfates of potassium, sodium and calcium, by-product from fermentation (Batch number: EXP LIberica Lote: AI16503047), the results indicated that the test item was non-irritant to skin. Hence, the test item was not classified for Skin Irritation according to CLP regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

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:

From 5 April 2017 To 24 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: EXP LIberica Lote: AI16503047
- Expiration date of the lot/batch: November 2019
- Purity test date: 1 February 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 ºC, below 70 RH%),
protected from light and humidity.
- Solubility and stability of the test substance in the solvent/vehicle: Solubility of the test item in physiological saline was tested prior to the experiment (30 mg test item in 1 mL physiological saline). The test item was dissolved in physiological saline.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was used as supplied, no formulation was required.

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT (slaughterhouse)
- Number of animals: 7 eyes were used for ICE assay, 3 were used for test item and positive control and one for physiological saline condition
- Characteristics of donor animals (e.g. age, sex, weight): Approximately 7 weeks old and mean weight 2.40kg in each experiment
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Heads were collected by a slaughter house technician and heads transported to testing facility at ambient temperature at the earliest convenience. They were processed within two hours of collection.
- Time interval prior to initiating testing: Eyes were checked, and acclimatization period was conducted for approximately 45-60 minutes
- indication of any existing defects or lesions in ocular tissue samples: No indication of lesions in ocular tissue samples. Samples were discarded in the case of lesions.
- Indication of any antibiotics used: not specified

Vehicle:

unchanged (no vehicle)

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):30 mg
- Concentration (if solution): not applicable

CONTROL
- Amount(s) applied (volume or weight with unit): 30µL of physiological saline or 30mg powdered imidazole

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

3 replicates for positive and test item condition, one replicate for physiological saline solution

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES

After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein- treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit

The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

Eyes were examined for slit lamp again when placed in the steel clamp for corneal opacity and fluorescein staining.

EQUILIBRATION AND BASELINE RECORDINGS

At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time. No changes in thickness (0.0%) were observed in the eyes in each experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES

Triplicates were used.

NEGATIVE CONTROL USED

Physiological Saline Solution

POSITIVE CONTROL USED

Powdered Imidazole

APPLICATION DOSE AND EXPOSURE TIME

30 mg of test item or 30 mg of powdered imidazole

OBSERVATION PERIOD

The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The time of application was observed, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed throughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual of the test item if possible.
Additional gentle rinsing with 20 mL saline was performed at each time point when the test item or positive control material remaining on the cornea was observed. The test item treated eyes were rinsed additional gentle rinsing with 3x20 mL saline after treatment in each experiment.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: slit lamp microscope [Haag-Streit BP 900 slit lamp microscope, setting measurements with depth-measuring device no.I; slit-width setting: 9.5)
- Damage to epithelium based on fluorescein retention: 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein- treated cornea was examined with a hand-held slit lamp or slit lamp microscope
- Swelling: measured on a slit-lamp microscope
- Macroscopic morphological damage to the surface: Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test substance to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator.
- Others (e.g, histopathology): at the termination of the observation period, eyes were kept in neutral buffered formalin.

SCORING SYSTEM:
See attached tables 1 ,2 and 3 below

DECISION CRITERIA: The descision criteria used was the same as used in the OECD Guideline 438 method for ICE Assay

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment I : at 75 minutes

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment I : at 240 minutes

Value:

0.5

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment I

Value:

0.17

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment I

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment II : at 75 minutes

Value:

0.5

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment II : at 240 minutes

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment II

Value:

0.17

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment II

Value:

0.17

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 30 minutes after the post-treatment rinse.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The positive control (Imidazole) was used and classified as inducing serious eye damage (Category 1)

ACCEPTANCE OF RESULTS:
The results from all eyes used met the quality control standards. The negative control and positive control results were in line with historical control data. This experiment was considered to be valid.

Table4 :Summary of Experiment I

Experiment I
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.5%	I
Mean maximum corneal opacity	0.17	I
Mean fluorescein retention	0.00	I
Other Observations	Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 30 minutes after the post-treatment rinse.
Overall ICE Class	3xI

 

Table5 :Summary of Experiment II

 

Experiment II
Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.5%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity	0.17	I
Mean fluorescein retention	0.17	I
Other Observations	Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 30 minutes after the post-treatment rinse.
Overall ICE Class	3xI

 

Table 6 SUMMARY TABLE FOR UN GHS CLASSIFICATION EXPERIMENT I AND EXPERIMENT II

 

 

Criteria for “No category” (all true)
3 endpoints classed as I or 2 endpoints classed as I and 1 endpoint classed as II:	True
No severe corneal morphological changes:	True
Test item was not stuck to the cornea at 240 minutes after the post- treatment rinse:	True

 

Criteria for “Category 1” (one or more true)
2 or more endpoints classed as IV:	False
Corneal opacity ≥ 3 at 30 min (in at least 2 eyes):	False
Corneal opacity = 4 at any time point (in at least 2 eyes):	False
Severe loosening of epithelium (in at least 1 eye):	False

 

Criteria for “No prediction can be made” (one or two true)
Based on the endpoints not classifiable for No Category, or for Category 1:	False
Particles of test item were stuck to the cornea and could not be washed off during the study:	False

 

Interpretation of results:

GHS criteria not met

Conclusions:

Based on this in vitro eye irritation assay in the isolated chicken eyes with Sulfates of potassium, sodium and calcium, by-product from fermentation, the test item is not classified for eye irritation or serious eye damage according to GHS and CLP criteria.

Executive summary:

The purpose of this GLP compliant study was to evaluate the potential eye irritation effect of the test substance when tested in an Isolated Chicken's Eye Assay (performed according to OECD 438 guideline method).

After the zero reference measurements,  the eye  was held  in  horizontal position and 30 mg of powered test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg  powdered Imidazole. The negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution). In the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were in good correlation with the  historical control data. Thus, the experiment was considered to be valid.

Experiment I: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No significant cornea opacity change (severity 0.5) was observed on one eye. No fluorescein retention change was noted on three eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Experiment II: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No significant cornea opacity change (severity 0.5) was observed on one eye. No significant fluorescein retention change (severity 0.5) was noted on one eye. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Based on this in vitro eye irritation assay in the isolated chicken eyes  with Sulfates of potassium, sodium and calcium, by-product from fermentation, the test item is not classified for eye irritation or serious eye damage according to GHS and CLP criteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

One in vitro GLP study is available for the registered substance (OECD 439, CiToxLAB Hungary Ltd , 2017). After exposure of disks of EPISKIN TM (SM) with Sulfates of potassium, calcium and sodium, by-products from fermentation, no decrease in tissue viability was observed (103%). Hence, the test item was not considered as a skin irritant according to the CLP criteria (mean cell viability above the threshold of 50%).

Reliable experimental data for skin irritation are also available on the analogue substances Calcium sulfate (CAS 7778-18-9) and Sodium sulfate (CAS 7757-82-6). Two key studies (OECD 404: Centre of Research and Applied Development, 1990/ NIER, 2002) on calcium sulfate (CAS 7778-18-9) and one (OECD 404, Bayer AG, 1991) on the sodium sulfate (CAS 7757-82-6) are available. Rabbits were exposed to the test item for 4 hours. No skin irritation or corrosion was observed. Hence, Sodium sulfate and Calcium sulfate were not considered to be irritating to skin.

The results of the in vitro and in vivo skin irritation studies on both the source substances and the target substance are indicative of a similar behavior regarding the skin irritation/corrosion endpoint. The very similar behavior for skin local effects further reinforces the read across justification applied for the systemic toxicity endpoints.

Eye irritation:

A GLP in vitro study in isolated chicken’s eyes (OECD 438, CiToxLAB Hungary Ltd, 2017) was performed to assess the eye irritation potential of the registered substance. Sulfates of potassium, sodium and calcium, by-product from fermentation showed no significant corneal effect in the first experiment. As the test item is a solid, the negative results were confirmed by a second experiment according to the recommendations of the OECD 438 guideline. The second experiment confirmed the negative results. Based on the results of this in vitro eye irritation test, Sulfates of potassium, sodium and calcium, by-product from fermentation, is not considered to be irritating to eyes and thus is not classified for eye irritation or serious eye damage according to the CLP criteria.

Reliable experimental data for eye irritation are also available on the analogue substances Calcium sulfate (CAS 7778-18-9) and Sodium sulfate (CAS 7757-82-6)

OECD 405 studies are available for Calcium sulfate (CAS 7778-18-9) and Sodium sulfate (CAS 7757-82-6) (Centre of Research and Applied Development, 1990; Bayer AG, 1991 respectively). Eyes of each rabbit were exposed to the test substances Slight and reversible effects were observed in all three studies. Thus, these analogous substances were not considered to be irritant to eyes.

The results of the in vitro and in vivo eye irritation studies on both the source substances and the target substance are indicative of a similar behavior regarding the eye irritation/corrosion endpoint. The very similar behavior for eye local effects further reinforces the read across justification applied for the systemic toxicity endpoints.

Justification for classification or non-classification

Skin irritation: According to the negative results of the key study (OECD 439, CiToxLAB Hungary Ltd, 2017), the test item Sulfates of potassium, sodium and calcium, by-products from fermentation is not classified for skin irritation according to the CLP criteria.

Eye irritation: According to the negative results of the key study (OECD 438, CiToxLAB Hungary Ltd, 2017), the test item Sulfates of potassium, sodium and calcium, by-products from fermentation is not classified for eye irritation according to the CLP criteria.