Storax (balsam)

Endpoint summary

Description of key information

Skin:

OECDTG 439: in vitro, undiluted. Conclusion: not irritating

4 HMT irritation pre-tests are available according to Kligman, A. M. (1966) method. Conclusion: No irritation was observed in all 4 studies performed with 8% test substance.

Three primary irritation tests were performed with a closed patch test on human volunteers with doses of 20%, 2% and 0,05%. Conclusion: Not irritating

Eye:

OECDTG 437: ex vivo, undiluted. Conclusion: not irritating

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:

24 Apr 2017 - 01 May 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)

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes

Specific details on test material used for the study:

Identification: Styrax resinoid oil
Appearance: Orange viscous liquid
Batch: K17 031-1
Test item storage: At room temperature
Stable under storage conditions until: 02 February 2019

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: multiple donors

Source strain:

other: not applicable

Details on animal used as source of test system:

- Source: SkinEthic Laboratories, Lyon, France.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model TM
- Tissue batch number(s): 17-Ekin-017, 16-Ekin-047, 17-Ekin-003
- Test System: This model is a three-dimensional human epidermis model, which consists of adult humanderived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Test System Set Up: On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for approximately 22 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.
- Killed tissues: Living epidermis was transferred to 12 well plates and incubated with 2 ml Milli-Q for 48 ± 1 hours. After incubation, killed epidermis was stored at ≤ -15°C. Killed tissues were thawed by placing them for 1 hour at room temperature in 12 well plates on 2 ml maintenance medium. Further use of killed tissues was similar to living tissues.
- MTT medium: MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).
- Environmental conditions: All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 74 - 93%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3 - 37.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 36.3 - 37.4°C

APPLICATION AND REMOVAL OF TEST MATERIAL AND CONTROLS
The test was performed on a total of 3 tissues per test item together with negative and positive controls. Twenty five μl of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. Since the test item reacted with the MTT medium, in addition three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
Test for Color Interference by the Test Item: Styrax resinoid oil was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, at least 10 mg of the test item was added to 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently. At the end of the incubation period a color check was performed.

Test for Reduction of MTT by the Test Item: After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-solution (0.3 mg/ml in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labeled microtubes and extracted with 500 μl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 69 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
17-EKIN-017:
- Passage: 2
- Histology scoring (HES stained vertical paraffin sections): 21.6 ± 1.1 (CV = 5.2%), satisfactory
- IC50 determination (SDS concentration, MTT test): 1.8 mg/L
- Statistical analysis: Histology, probability 0.95 that 100% of the batch > 20. IC50, probability 0.95 that IC50≥ 1.7 mg/mL (threshold value)
- Biological safety: absence of HIV1 and 2 antibodies, Hepatitis C antibodies, Hepatitis B antigen HBs, bacteria, fungus and mycoplasma.

16-EKIN-047:
- Passage: 2
- Histology scoring (HES stained vertical paraffin sections): 22.6 ± 0.2 (CV = 0.9%), satisfactory
- IC50 determination (SDS concentration, MTT test): 1.9 mg/L
- Statistical analysis: Histology, probability 0.95 that 100% of the batch > 20. IC50, probability 0.95 that IC50≥ 1.9 mg/mL (threshold value)
- Biological safety: absence of HIV1 and 2 antibodies, Hepatitis C antibodies, Hepatitis B antigen HBs, bacteria, fungus and mycoplasma.

17-EKIN-003:
- Passage: 2
- Histology scoring (HES stained vertical paraffin sections): 22.8 ± 0.3 (CV = 1.1%), satisfactory
- IC50 determination (SDS concentration, MTT test): 1.9 mg/L
- Statistical analysis: Histology, probability 0.95 that 100% of the batch > 20. IC50, probability 0.95 that IC50≥ 1.6 mg/mL (threshold value)
- Biological safety: absence of HIV1 and 2 antibodies, Hepatitis C antibodies, Hepatitis B antigen HBs, bacteria, fungus and mycoplasma.

NUMBER OF REPLICATE TISSUES:
The test was performed on a total of 3 tissues per test item together with negative and positive controls.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Styrax resinoid oil was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the color interference, at least 10 mg of Styrax resinoid oil was added to 90 μl Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μl Milli-Q water was tested concurrently. At the end of the shaking period a color check was performed.

PREDICTION MODEL / DECISION CRITERIA
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.

A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 μL
- Concentration: undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μL, PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μL, SDS (re-spread after 7 minutes contact time.)
- Concentration (if solution): 5%

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Remarks:

after 15 min exposure

Run / experiment:

mean

Value:

80

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Standard deviation 9.8%

Other effects / acceptance of results:

RESULT:
The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Styrax resinoid oil compared to the negative control tissues was 80%. Since the mean relative tissue viability for Styrax resinoid oil was above 50% Styrax resinoid oil is considered to be non-irritant.

OTHER EFFECTS:
Styrax resinoid oil was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because a color change was observed by adding MTT-medium it was concluded that Styrax resinoid oil did interact with the MTT endpoint. In addition to the normal procedure, three killed tissues treated with test item and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by Styrax resinoid oil was 7.8% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 20%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 11%, indicating that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, Styrax resinoid oil is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations and according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

To evaluate Styrax resinoid oil for its ability to induce skin irritation, a human three dimensional epidermal model (EPISKIN Small model (EPISKINSMTM)) was used according to OECDTG 439. Styrax resinoid oil was applied undiluted (25μl), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Styrax resinoid oil did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by Styrax resinoid oil was 7.8% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.

 

The positive control had a mean cell viability of 20% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range.The standard deviation value of the percentage viability of three tissues treated identically was less than 11%, indicating that the test system functioned properly. Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Styrax resinoid oil compared to the negative control tissues was 80%.

 

In conclusion, Styrax resinoid oil is non-irritant in the in vitro skin irritation test under the experimental conditions described and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.and according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Principles of method if other than guideline:

Kligman, A. M. (1966). "The identification of contact allergens by human assay. III. The maximization test: a procedure for screening and rating contact sensitizers". J. Invest. Dermatol. 47: 393–409.

GLP compliance:

no

Specific details on test material used for the study:

TEST MATERIAL
STYRAX ALC. SOL. TURKISH IN DEP. 78-8-8283

Species:

other: human

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

Details on test animals and environmental conditions 29 healthy male and female volunteers were screened and 25 completed the study.
Age 20 - 69
Gender: 10 males, 15 females

Type of coverage:

occlusive

Preparation of test site:

not specified

Vehicle:

other: petrolatum

Amount / concentration applied:

8%

Duration of treatment / exposure:

48 hours

Number of animals:

25

Details on study design:

In a pre-test for a human maximization study, a patch of the test material in petrolatum was applied to normal sites on the backs of 25 healthy male and female volunteers for 48 hours under occlusion. The skin irritation of Styrax alcohol soluble Turkish in DEP in a concentration of 8% was evaluated prior to a human maximization test and to determine whether sodium lauryl sulfate pretreatment was necessary.

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

48 h

Score:

<= 0

Reversibility:

other: not relevant

Remarks on result:

no indication of irritation

Remarks:

Styrax alcohol soluble Turkish in DEP in a concentration of 8%

Irritation parameter:

erythema score

Basis:

mean

Time point:

48 h

Score:

<= 0

Reversibility:

other: not relevant

Remarks on result:

no indication of irritation

Remarks:

Styrax alcohol soluble Turkish in DEP in a concentration of 8%

Irritant / corrosive response data:

No skin irritation observed.

Interpretation of results:

GHS criteria not met

Conclusions:

8% Styrax alcohol soluble Turkish in DEP was not irritating to human skin under the conditions of this test.

Executive summary:

In a pre-test for a human maximization study, a patch of the test material in petrolatum was applied to normal sites on the backs of 25 healthy male and female volunteers for 48 hours under occlusion. The skin irritation of Styrax alcohol soluble Turkish in DEP in a concentration of 8% was evaluated prior to a human maximization test and to determine whether sodium lauryl sulfate pretreatment was necessary. No skin irritation observed. 8% Styrax alcohol soluble Turkish in DEP was not irritating to human skin under the conditions of this test.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Principles of method if other than guideline:

Kligman, A. M. (1966). "The identification of contact allergens by human assay. III. The maximization test: a procedure for screening and rating contact sensitizers". J. Invest. Dermatol. 47: 393–409.

GLP compliance:

no

Specific details on test material used for the study:

TEST MATERIAL
STYRAX HONDURAS EX HEXANE 81-8-PROD-1

Species:

other: human

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

29 healthy male and female volunteers were screened and 25 completed the study.

Type of coverage:

occlusive

Preparation of test site:

not specified

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

8%

Duration of treatment / exposure:

48 hours

Number of animals:

25

Details on study design:

In a pre-test for a human maximization test, a 48 hour closed patch test with test material was conducted on the backs of 25 healthy, male and female volunteers. The skin irritation of Styrax honduras EX hexane was evaluated prior to a human maximization test and to determine whether sodium lauryl sulfate pretreatment was necessary.

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

48 h

Score:

<= 0

Reversibility:

other: not relevant

Remarks on result:

no indication of irritation

Remarks:

Styrax honduras EX hexane

Irritation parameter:

erythema score

Basis:

mean

Time point:

48 h

Score:

<= 0

Reversibility:

other: not relevant

Remarks on result:

no indication of irritation

Remarks:

Styrax honduras EX hexane

Irritant / corrosive response data:

No skin irritation was observed.

Interpretation of results:

GHS criteria not met

Conclusions:

8% Styrax honduras EX hexane was not irritating to human skin under the conditions of this test.

Executive summary:

In a pre-test for a human maximization test, a 48 hour closed patch test with test material was conducted on the backs of 25 healthy, male and female volunteers. The skin irritation of Styrax honduras EX hexane was evaluated prior to a human maximization test and to determine whether sodium lauryl sulfate pretreatment was necessary. No skin irritation was observed. 8% Styrax honduras EX hexane was not irritating to human skin under the conditions of this test.

Reference 4

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Principles of method if other than guideline:

Kligman, A. M. (1966). "The identification of contact allergens by human assay. III. The maximization test: a procedure for screening and rating contact sensitizers". J. Invest. Dermatol. 47: 393–409.

GLP compliance:

no

Specific details on test material used for the study:

TEST MATERIAL
STYRAX TURKISH EX HEXANE 81-8-PROD-2

Species:

other: human

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

30 healthy male and female volunteers were screened.

Type of coverage:

occlusive

Preparation of test site:

not specified

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

8%

Duration of treatment / exposure:

48 hours

Number of animals:

27

Details on study design:

A 48 hour closed patch test on the backs of 27 volunteers. The skin irritation of Styrax turkish EX hexane was evaluated prior to a human maximization test and to determine whether sodium lauryl sulfate pretreatment was necessary.

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

48 h

Score:

<= 0

Reversibility:

other: not relevant

Remarks on result:

no indication of irritation

Remarks:

Styrax turkish EX hexane

Irritation parameter:

erythema score

Basis:

mean

Time point:

48 h

Score:

<= 0

Reversibility:

other: not relevant

Remarks on result:

no indication of irritation

Remarks:

Styrax turkish EX hexane

Irritant / corrosive response data:

No skin irritation was observed.

Interpretation of results:

GHS criteria not met

Conclusions:

8% Styrax turkish EX hexane was not irritating to human skin under the conditions of this test.

Executive summary:

A 48 hour closed patch test on the backs of 27 volunteers. The skin irritation of Styrax turkish EX hexane was evaluated prior to a human maximization test and to determine whether sodium lauryl sulfate pretreatment was necessary. No skin irritation was observed. 8% Styrax turkish EX hexane was not irritating to human skin under the conditions of this test.

Reference 5

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Principles of method if other than guideline:

Kligman, A. M. (1966). "The identification of contact allergens by human assay. III. The maximization test: a procedure for screening and rating contact sensitizers". J. Invest. Dermatol. 47: 393–409.

GLP compliance:

no

Specific details on test material used for the study:

TEST MATERIAL
S-TYRAX ABS. RESIN, 76-8-288

Species:

other: human

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

Thirty-five healthy inmate volunteers were screened and twenty-seven completed the study.

Type of coverage:

occlusive

Preparation of test site:

not specified

Vehicle:

other: petrolatum

Number of animals:

27

Details on study design:

In a pre-test for a human maximization study, a patch of the test material in petrolatum was applied to normal sites on the backs of 27 subjects for 48 hours under occlusion. Vehicle was petroleum. The skin irritation of Styrax absolute resin in a concentration of 8% was evaluated prior to a human maximization test and to determine whether sodium lauryl sulfate pretreatment was necessary.

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

48 h

Score:

<= 0

Reversibility:

other: not relevant

Remarks on result:

no indication of irritation

Remarks:

Styrax absolute resin in a concentration of 8%

Irritation parameter:

erythema score

Basis:

mean

Time point:

48 h

Score:

<= 0

Reversibility:

other: not relevant

Remarks on result:

no indication of irritation

Remarks:

Styrax absolute resin in a concentration of 8%

Irritant / corrosive response data:

No skin irritation was observed.

Interpretation of results:

GHS criteria not met

Conclusions:

8% Styrax absolute resin was not irritating to human skin under the conditions of this test.

Executive summary:

In a pre-test for a human maximization study, a patch of the test material in petrolatum was applied to normal sites on the backs of 27 subjects for 48 hours under occlusion. Vehicle was petroleum. The skin irritation of Styrax absolute resin in a concentration of 8% was evaluated prior to a human maximization test and to determine whether sodium lauryl sulfate pretreatment was necessary. No skin irritation was observed. 8% Styrax absolute resin was not irritating to human skin under the conditions of this test.

Reference 6

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1972

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline available

Principles of method if other than guideline:

Three primary irritation tests were performed with human volunteers.

GLP compliance:

no

Specific details on test material used for the study:

Styrax resin

Species:

other: Human

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

29 healthy volunteers participated in test A
30 healthy volunteers participated in test B
48 volunteers with dermatoses participated in test C

Type of coverage:

occlusive

Preparation of test site:

not specified

Vehicle:

other: In test A: vaselinum Aldum or Unguentum Hydrophilicum (Phermacopoerria Japonica) In test B: Urguentum Simplex* or Unguentum Hydrophilicum (Phermacopoerria Japonica) In test C: 99% Ethanol or non-irritative cream base

Remarks:

*Urguentum Simplex formula: Bees wax 14%, Liquid paraffine 49%, emulsifier 1%, sodium borate 1%, pure water 35%.

Amount / concentration applied:

Test A: 20%
Test B: 2%
Test C: 0.05%

Duration of treatment / exposure:

Test A: 48h
Test B: 24 - 72h
Test C: 24-48h

Number of animals:

Test A: 29
Test B: 30
Test C: 48

Details on study design:

Primary irritation tests were performed with a closed patch test on human volunteers.
The test conditions of test A ,B, and C were as follows:

Test A:
29 healthy Male/female volunteers
small application on the back
Exposure duration: 48 hours
Sample concentration: 20% styrax resin in vaselinum Aldum or Unguentum Hydrophilicum (Phermacopoerria Japonica)

Test B:
30 healthy Male/female volunteers
Application on upper inside of the arm
Exposure duration: 24-72 hours
Sample concentration: 2% styrax resin in Urguentum Simplex* or Unguentum Hydrophilicum (Phermacopoerria Japonica)

Test C:
48 male/female volunteers with dermatosis
Application on upper inside of the arm
Exposure duration: 24 – 48 hours
Sample concentration: 0.05% in 99% Ethanol or non-irritative cream base

Irritation parameter:

other: Erythema

Remarks:

20% test substance, test A

Basis:

other: one subject

Time point:

48 h

Score:

<= 1

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Remarks:

Test A with 20% styrax resin in vaselinum Aldum or Unguentum Hydrophilicum (Phermacopoerria Japonica); 1 out of 29 subjects a suspected positive reaction was observed

Irritation parameter:

other: erythema

Remarks:

20% test substance, test A

Basis:

other: one subject

Time point:

48 h

Score:

<= 1

Reversibility:

not specified

Remarks on result:

probability of weak irritation

Remarks:

Test A with 20% styrax resin in vaselinum Aldum or Unguentum Hydrophilicum (Phermacopoerria Japonica); 1 out of 29 subjects a suspected positive reaction was observed

Irritation parameter:

other:

Remarks:

20% test substance, test A

Basis:

other: 27 subjects

Time point:

48 h

Score:

<= 0

Reversibility:

other: not relevant

Remarks on result:

no indication of irritation

Remarks:

TEST A with 20% styrax resin in vaselinum Aldum or Unguentum Hydrophilicum (Phermacopoerria Japonica); in 27 out of 29 subjects no irritation reactions were observed.

Irritation parameter:

other:

Remarks:

2% test substance, Test B

Basis:

other: all subjects

Time point:

other: 24 - 72h

Score:

<= 0

Reversibility:

other: not relevant

Remarks on result:

no indication of irritation

Remarks:

Test B with 2% styrax resin in Urguentum Simplex* or Unguentum Hydrophilicum (Phermacopoerria Japonica); no irritation was observed in any of the subjects participating in test B

Irritation parameter:

other:

Remarks:

0,05% test substance, test C

Basis:

other: all subjects

Time point:

other: 24 - 48h

Score:

<= 0

Reversibility:

other: not relevant

Remarks on result:

no indication of irritation

Remarks:

Test C with 0.05% in 99% Ethanol or non-irritative cream base; no irritation was observed in any of the participants of test C

Irritant / corrosive response data:

In test A, Erythema and slight erythema were observed in two subjects. No reactions were observed in the remaining 27 subjects.
No irritation reactions were observed in the subjects participating in test B and C

Results

Test	Number of subjects	-	±	+	++
A	29	27	1	1	0
B	30	30	0	0	0
C	48	48	0	0	0

Signs for recording of patch test reaction

-	No reaction	Negative reaction
±	Slight erythema	Suspected positive reaction
+	erythema	Positive reaction
++	Erythema and dropsical swelling
+++	Erythema, papules, and vesicles

Interpretation of results:

GHS criteria not met

Conclusions:

Erythema and slight erythema was observed in two healthy subjects exposed to 20% test subtance. No reactions were observed in the remaining 27 subjects exposed to 20% test substance. No irritation reactions were observed in the subjects exposed to 2 and 0.05% test substance. Under the conditions of the test it can be concluded that styrax resin is not irritating.

Executive summary:

Three primary irritation tests were performed with a closed patch test on human volunteers. The first test was performed with 29 healthy Male/female volunteers, who were exposed to 20% test substance for  48h with a small application on the back. The test substance was applied in vaselinum Aldum or Unguentum Hydrophilicum (Phermacopoerria Japonica). The second test was performed with 30 healthy Male/female volunteers, who where exposed to 2% test substance for 24 – 72h with an application on upper inside of the arm. The test substance was applied in Urguentum Simplex or  Unguentum Hydrophilicum (Phermacopoerria Japonica). The final test was performed in 48 male/female volunteers with dermatosis, who were exposed to 0.05% test subance for 24- 48h on the upper inside of the arm. The test substance was applied in Ethanol or non-irritative cream base.

Erythema and slight erythema was observed in two healthy subjects exposed to 20% test subtance. No reactions were observed in the remaining 27 subjects exposed to 20% test substance. No irritation reactions were observed in the subjects exposed to 2 and 0.05% test substance. Under the conditions of the test it can be concluded that styrax resin is not irritating.

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:

25 Apr 2017 - 25 Apr 2017

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes

Specific details on test material used for the study:

Identification: Styrax resinoid oil
Appearance: Orange viscous liquid

Test item storage: At room temperature
Stable under storage conditions until: 02 February 2019

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Bovine eyes were used as soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration: neat

Duration of treatment / exposure:

10 ± 1 minutes

Duration of post- treatment incubation (in vitro):

120 ± 10 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C. Three corneas were selected at random for each treatment group.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
physiological saline

POSITIVE CONTROL USED
Ethanol

APPLICATION DOSE AND EXPOSURE TIME
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. The opacity was measured with the OP-KIT device.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

- Corneal permeability: Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.

After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μl of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: According to guideline.

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

-2.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

- 0.7

Positive controls validity:

valid

Remarks:

17.2

Remarks on result:

other: values ranging from -3.7 to -2.0

Irritation parameter:

fluorescein leakage

Run / experiment:

mean

Value:

0.004

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.004

Positive controls validity:

valid

Remarks:

0.937

Remarks on result:

other: values ranging from -0.003 to 0.018.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

-2.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

-0.6

Positive controls validity:

valid

Remarks:

31.3

Remarks on result:

other: ranging from -3.4 to -2.0

Other effects / acceptance of results:

The individual in vitro irritancy scores for the negative controls ranged from -1.8 to 1.1. The individual positive control in vitro irritancy scores ranged from 24 to 40. The corneas treated with the positive control item were turbid after the 10 minutes of treatment. The corneas treated with Styrax resinoid oil showed opacity values ranging from -3.7 to -2.0 and permeability values ranging from -0.003 to 0.018. The corneas were clear after the 10 minutes of treatment with Styrax resinoid oil. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -3.4 to -2.0 after 10 minutes of treatment with Styrax resinoid oil.

Styrax resinoid oil did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -2.8 after 10 minutes of treatment.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 31 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, since Styrax resinoid oil induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

To evaluate the eye hazard potential of Styrax resinoid oil an Bovine Corneal Opacity and Permeability test (BCOP test) was performed according to OECDTG 437. The eye damage of Styrax resinoid oil was tested through topical application for 10 minutes.The test item was applied as it is (750 μl) directly on top of the corneas. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 31 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.Styrax resinoid oil did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of –2.8 after 10 minutes of treatment. In conclusion, since Styrax resinoid oil induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.and according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin:

Four human pre-maximisation tests are available and performed according to the Kligman, 1966 procedure. A 48 hour closed patch test with 8% test substance was performed under occlusive conditions. 8% Styrax absolute resin, Styrax alcohol soluble Turkish in DEP, Styrax honduras EX hexane, and Styrax turkish EX hexane did not result in irritation under the conditions of the test.

To evaluate Styrax resinoid oil for its ability to induce skin irritation, a human three dimensional epidermal model (EPISKIN Small model (EPISKINSMTM)) was used according to OECDTG 439. Styrax resinoid oil was applied undiluted (25μl), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Styrax resinoid oil did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by Styrax resinoid oil was 7.8% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues. The positive control had a mean cell viability of 20% after 15 ± 0.5 minutes exposure. Theabsolute mean OD570(optical density at 570 nm) of the negative control tissues was within thelaboratory historical control data range.The standard deviation value of the percentageviability of three tissues treated identically was less than 11%, indicating that the test systemfunctioned properly.Skin irritation is expressed as the remaining cell viability after exposure to the test item. Therelative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Styrax resinoidoil compared to the negative control tissues was 80%. In conclusion, Styrax resinoid oil is non-irritant in the in vitro skin irritation test under theexperimental conditions described and should not be classified according to theGlobally Harmonized System of Classification and Labeling of Chemicals (GHS) of theUnited Nations.and according to the criteria outlined in Annex I of the CLP Regulation(1272/2008/EC).

Three primary irritation tests were performed with a closed patch test on human volunteers (Fujii et al., 1972). The first test was performed with 29 healthy Male/female volunteers, who were exposed to 20% test substance for  48h with a small application on the back. The test substance was applied in vaselinum Aldum or Unguentum Hydrophilicum (Phermacopoerria Japonica). The second test was performed with 30 healthy Male/female volunteers, who where exposed to 2% test substance for 24 – 72h with an application on upper inside of the arm. The test substance was applied in Urguentum Simplex or  Unguentum Hydrophilicum (Phermacopoerria Japonica). The final test was performed in 48 male/female volunteers with dermatosis, who were exposed to 0.05% test subance for 24- 48h on the upper inside of the arm. The test substance was applied in Ethanol or non-irritative cream base. Erythema and slight erythema was observed in two healthy subjects exposed to 20% test subtance. No reactions were observed in the remaining 27 subjects exposed to 20% test substance. No irritation reactions were observed in the subjects exposed to 2 and 0.05% test substance. Under the conditions of the test it can be concluded that styrax resin is not irritating.

Eye:

To evaluate the eye hazard potential of Styrax resinoid oil an Bovine Corneal Opacity and Permeability test (BCOP test) was performed according to OECDTG 437. The eye damage of Styrax resinoid oil was tested through topical application for 10 minutes.The test item was applied as it is (750 μl) directly on top of the corneas. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 31 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.Styrax resinoid oil did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of –2.8 after 10 minutes of treatment. In conclusion, since Styrax resinoid oil induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.and according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Justification for classification or non-classification

In conclusion, Styrax resinoid oil is non-irritant to the skin and eyes and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations and according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).