Reaction mass of 3,4,5-trichlorophthalic acid, strontium salt and 3,4,6-trichlorophthalic acid, strontium salt and 3,4-dichlorophthalic acid, strontium salt and 3,5-dichlorophthalic acid, strontium salt and 3,6-dichlorophthalic acid, strontium salt and 3-chlorophthalic acid, strontium salt and 4,5-dichlorophthalic acid, strontium salt and 4-chlorophthalic acid, strontium salt

Endpoint summary

Description of key information

The potential of strontium dichlorophthalate to induce skin irritation (OECD 439) and eye irritation (OECD 492) was tested in suitable in vitro test methods. Based on the results, the target substance can be considered non-irritant to the skin and eye.

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:

2019-06-24 to 2019-09-10

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:

adopted on 18th June 2019

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 number of test material:Dynamit Nobel GmbH Explosivstoff- und Systemtechnik, WE50352354
- Expiration date of the lot/batch: 28 November 2019
- Purity test date: 2 May 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 5 °C – 40 °C, protected from light, separated from oxidizers, strong acids and alkalis; do not heat
- Stability under storage conditions: stable under normal conditions

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item (25 mg) was directly applied atop the tissue

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multi-layered, highly differentiated model of the human epidermis and therefore the EpiDerm™ epidermis model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Standard Model (EPI-200-SIT, MatTek)
- Tissue batch number(s): Lot No.: 30807
- Date of initiation of testing: 24th June 2019
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 5 min
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm
NUMBER OF REPLICATE TISSUES: 3
PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue viability after exposure and post-incubation is less than or equal to 50%
- The test substance is considered to be non-irritant to skin if the viability after exposure and post-incubation is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL 5% SDS solution

Duration of treatment / exposure:

60 ± 1 min

Duration of post-treatment incubation (if applicable):

42 h post-incubation

Number of replicates:

3 per dose group

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three tissues

Value:

86.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (1.646)
- Acceptance criteria met for positive control: Yes, the mean relative tissue viability of the positive control was ≤ 20% (7.4%)
- Acceptance criteria met for variability between replicate measurements: Yes, the standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (1.1%).

For detailed results see Table 1 in box "Any other information on results incl. tables".

Results of the Pre-Experiments:

The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

The mixture of 25 mg of the test item per 300 μL aqua dest. and per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.

The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.

 

Results of the main experiment:

 

Name	Negative Control			Positive Control			Test Item
Replicate Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.716	1.750	1.419	0.138	0.171	0.173	1.368	1.380	1.487
	1.737	1.803	1.453	0.160	0.152	0.193	1.375	1.469	1.482
Mean Absolute OD570	1.646****			0.164			1.427
OD570(Blank Corrected)	1.669	1.704	1.372	0.091	0.125	0.126	1.321	1.333	1.441
	1.691	1.757	1.406	0.114	0.105	0.147	1.328	1.423	1.435
Mean OD570of the Duplicates (Blank Corrected)	1.680	1.730	1.389	0.102	0.115	0.136	1.325	1.378	1.438
Total Mean OD570of the 3 Replicate Tissues (Blank Corrected)	1.600*			0.118			1.380
SD of Mean OD570of the 3 Replicate Tissues (Blank Corrected)	0.184			0.017			0.057
Relative Tissue Viability [%]	105.0	108.2	86.8	6.4	7.2	8.5	82.8	86.1	89.9
Mean Relative Tissue Viability [%]	100.0			7.4**			86.3
SD of Relative Tissue Viability [%]***	11.5			1.1			3.5
CV [% Viabilities]	11.5			14.6			4.1

 * Blank-corrected mean OD₅₇₀ of the negative control corresponds to 100% absolute tissue viability.

** Mean relative tissue viability of the three positive control tissues is 20%.

*** Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.

**** The mean absolute OD₅₇₀ of the negative control i s≥ 0.8 and ≤ 2.8.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in this in vitro skin irritation study (OECD 439), strontium dichlorophthalate is considered to no be irritant to the skin (UN GHS “No category”).

Executive summary:

In a primary dermal irritation study conducted according to OECD guideline 439, the EpiDerm™-Model (EPI-200-SIT) was topically exposed to strontium dichlorophthalate for 60 min and 42 h post incubation period. Irritant potential of the test item was not predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS. The mean relative tissue viability (% negative control) was > 50% (86.3 %) after 60 min treatment and 42 h post-incubation. Therefore, the test item is not considered to be irritating to the skin in accordance with UN GHS "No Category”.

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:

2019-09-30 to 2019-11-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 18 June 2019

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 number of test material:Dynamit Nobel GmbH Explosivstoff- und Systemtechnik, WE50352354
- Expiration date of the lot/batch: 28 November 2019
- Purity test date: 2 May 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under storage conditions: stable under normal conditions

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item (50 mg) was directly applied atop the EpiOcular™ tissue

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method: This test uses the three-dimensional RhCE EpiOcular™ (MatTek). It consists of normal, human-derived epidermal keratinocytes and mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium. The MatTek EpiOcular™ model has been widely used as a research and testing model for many years.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg

Duration of treatment / exposure:

6 ± 0.25 h

Duration of post- treatment incubation (in vitro):

Post exposure post-soak plate: 25 ± 2 min at room temperature
Post exposure post-treatment plate: 18 ± 0.25 h at 37 ± 1 °C

Number of animals or in vitro replicates:

2 tissues per dose group

Details on study design:

- Details of the test procedure used:
Upon receipt of the EpiOcular™, the tissues were equilibrated in the 24-well shipment plate to room temperature for about 15 min. Then, the EpiOcular™ tissues were transferred into 6-well plates containing 1 mL pre-warmed assay medium per well and incubated for 1 h in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% air. Then the inserts were transferred into new 6-well plates containing 1 mL fresh assay medium per well and pre-incubated in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% air for 16 - 24 h.
After the overnight incubation the tissues were pre-treated with 20 μL of DPBS-buffer and incubated for 30 ± 2 min in a humidified incubator at 37 ± 2 °C, 5.0% CO2 / 95% air to mimic the wet conditions of the human eye.
Afterwards, the tissues were treated with each dose group in duplicate, starting with the negative and positive control. While the test item was applied, the tissue inserts were placed on a sterile surface. After dosing, the inserts were placed back into the culture medium. Then the 6-well plate(s) were incubated for 6 ± 0.25 h at 37 ± 2 °C, 5.0% CO2 / 95% air. At the end of the exposure period the test item and control substances were removed by extensively rinsing the tissue with DPBS. Excess DPBS was removed by decanting the insert and blotting bottom with blotting paper. After rinsing the inserts were transferred to and immersed in a prepared 12-well “post-soak plate“, containing 5 mL fresh pre-warmed assay medium per well and incubated for 25 ± 2 min at room temperature. Afterwards, the inserts were removed from the assay medium, the medium was decanted off the tissue and the tissues were blotted on blotting paper. The inserts were transferred to a new 6-well plate (post-treatment plate) containing 1 mL pre-warmed assay medium. The tissues were incubated for 18 ± 0.25 h at 37 ± 2°C, 5.0% CO2 / 95% air.
After this incubation period excess medium was removed by blotting bottom on absorbent paper before the inserts were transferred in a prepared 24-well “MTT assay plate” containing 0.3 mL pre-warmed MTT medium and further incubated for 3 h ± 15 min at 37 ± 2 °C, 5.0% CO2 / 95% air.
After the 3 h MTT incubation period the inserts were removed, the bottom of the inserts blotted on blotting paper, and then transferred into new 6-well “extraction plates“, containing 2 mL of isopropanol to extract only the bottom of the tissues. The extraction plates were sealed to inhibit isopropanol evaporation. Extraction was carried out immediately by shaking on an orbital plate shaker for 2 - 3 h at room temperature. At the end of the extraction period the tissues were not pierced to avoid contamination of the extract with remaining test item.
Then the inserts were discarded and the extracts were mixed three times using a pipette. If any visible cell/tissue fragments were in suspension, extracts were centrifuged to eliminate the fragments and avoid further possible interference with the absorbance readings.
For each tissue 2 x 200 μL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer using isopropanol as a blank.
- RhCE tissue construct used, including batch number:
EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek), consisting of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.
The EpiOcular™ tissues were provided as kits (e.g. OCL-200-EIT; MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing 24 inserts with EpiOcular™ tissues on agarose (Lot No.: 30632)
1x bottle EpiOcularTM assay medium (Lot No.: 102819ISA)
1x bottle Ca2+/Mg2+-free DPBS buffer (Lot No.: 092419MSA)

- Doses of test chemical and control substances used:
1. Negative Control: 50 µL Aqua dest. (Sigma, Lot No.: RNBG4913)
2. Positive Control: 50 µL methyl acetate (CAS 79-20-9, Merck, Lot No. S6943111)
3. Test Item: 50 mg

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable):
Exposure: 6 ± 0.25 h at 37 ± 1 °C, 5.0% CO2 / 95% air.
Post exposure post-soak plate: 25 ± 2 min at room temperature
Post exposure post-treatment plate: 18 ± 0.25 h at 37 ± 1 °C, 5.0% CO2 / 95% air

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
See section "Pre-experiments" in box "Any other information on materials and methods incl. tables"

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 2 tissues per group

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm ± 30 nm

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
Mean tissue viability (% negative control) <= 60 %: Irritant (I): UN GHS “Category 1” or “Category 2”
Mean tissue viability (% negative control) > 60%: Non-Irritant (NI): UN GHS “No Category”

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria:
Historical control data were generated from 2014-2018:
Absolute OD570 ± 30 nm NK: Mean: 1.697; SD: 0.275, n= 50
Relative Viability PC [%]: Mean: 24.9, SD: 12.9, n= 50
Difference of Viability [%]: Mean: 6.6, SD: 7.2, n= 216

Test Acceptance Criteria:
- mean absolute OD570 nm of the negative control is > 0.8 and < 2.8
- mean relative tissue viability of the positive control is < 50%
- relative tissue viability difference of replicate tissues is < 20%

Irritation parameter:

other: Relative Tissue Viability [%]

Run / experiment:

Mean of replicates

Value:

92.4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

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: see in box "Any other information on materials and methods incl. tables"

For individual results see Table 1 in box "Any other information on results incl. tables"

Table 1: Result of the Test Item Strontium dichlorophthalate

Name	Negative Control		Positive Control		Test Item
Replicate Tissue	1	2	1	2	1	2
Absolute OD570	1.146	1.172	0.358	0.333	1.088	1.061
	1.145	1.182	0.359	0.334	1.086	1.072
Mean Absolute OD570	1.161****		0.346		1.077
OD570 (Blank Corrected)	1.098	1.123	0.309	0.284	1.040	1.012
	1.096	1.133	0.310	0.286	1.037	1.023
Mean OD570 of the Duplicates	1.097	1.128	0.310	0.285	1.038	1.018
(Blank Corrected)
Total Mean OD570 of the 2 Replicate Tissues (Blank Corrected)	1.112*		0.297		1.028
SD of Mean OD570 of the Duplicates (Blank Corrected)	0.022		0.018		0.015
Relative Tissue Viability [%]	98.6	101.4	27.9	25.6	93.4	91.5
Difference [%]***	2.8		2.3		1.9
Mean Relative Tissue Viability [%]	100.0		26.7**		92.4

^*             Corrected mean OD₅₇₀of the negative control corresponds to 100% absolute tissue viability

^**            Mean relative tissue viability of the positive control is < 50%

^***           Relative tissue viability difference of replicate tissues is < 20%

^****       Mean absolute OD₅₇₀of the negative control is > 0.8 and < 2.8

Table 2: Test Acceptance Criteria

	Value	Cut off	pass/fail
Mean Absolute OD570 nm NC	1.161	0.8 < NC < 2.8	pass
Mean Relative Viability PC [%]	26.7	< 50%	pass
Max. Difference of % Viability [%]	2.8	< 20%	pass

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category” for eye irritation.

Executive summary:

In the present study the eye irritant potential of strontium dichlorophthalate was analysed according to OECD 492 using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium. Hereby, 50 mg of the test item was applied directly atop the EpiOcular™ tissue. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 6 hours exposure and 18 hours post-incubation period and compared to those of the concurrent negative controls. The test item showed non-specific reduction of MTT, but no colouring potential. Therefore, killed tissue controls were included and used for quantitative correction of results. The test item showed no irritant effects. The mean relative tissue viability of two replicates (% negative control) was > 60% (92.4%). Therefore, the test item is considered to be non-irritating to the eye in accordance with UN GHS “No Category”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a primary dermal irritation study conducted according to OECD guideline 439, the EpiDerm™-Model (EPI-200-SIT) was topically exposed to strontium dichlorophthalate for 60 min and 42 h post incubation period. Irritant potential of the test item was not predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS. The mean relative tissue viability (% negative control) was > 50% (86.3 %) after 60 min treatment and 42 h post-incubation. Therefore, the test item is not considered to be irritating to the skin in accordance with UN GHS "No Category”.

In the eye irritation study the eye irritant potential of strontium dichlorophthalate was analysed according to OECD 492 using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium. Hereby, 50 mg of the test item was applied directly atop the EpiOcular™ tissue. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 6 hours exposure and 18 hours post-incubation period and compared to those of the concurrent negative controls. The test item showed non-specific reduction of MTT, but no colouring potential. Therefore, killed tissue controls were included and used for quantitative correction of results. The test item showed no irritant effects. The mean relative tissue viability of two replicates (% negative control) was > 60% (92.4%). Therefore, the test item is considered to be non-irritating to the eye in accordance with UN GHS “No Category”.

Thus, strontium dichlorophthalate is not considered to be an irritant for skin and eye.

Justification for classification or non-classification

Based on available data, no classification for skin and/or eye irritation is warranted based on the results from suitable in vitro tests (OECD 439, 492).