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Description of key information

The skin irritant potential of propyl gallate (> 99.93% purity) was analysed according to OECD 439 using the EPISKIN-Standard Model™ (EPISKIN-SMTM). Hereby, the test item was applied topically. In this study under the given conditions the test item showed no irritant effects (70.3% mean relative tissue viability) after 15 min exposure and 42 h post-incubation.

 

The eye irritation potential of propy gallate (> 99.93% purity) was examined according to OECD 437 using the BCOP model. As this test gave indefinite results, an in vivo study according to OECD 405 was performed showing a severe irritant effect of the test item in the rabbit 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:
2016-11-10 to 2017-01-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM™ (SkinEthic)
- Tissue batch number(s): 16-EKIN-048

SkinEthic Kit:
- EPISKIN-SM™ plate containing 12 reconstructed epidermis units (area: 0.38 cm²); each reconstructed epidermis is attached to the base of a tissue culture insert with an O-ring set and maintained on nutritive agar for transport (Lot No.: 16-EKIN-048):
1x 12-well assay plate
1x flask of sterile maintenance medium (basic medium for incubations, Lot No.: 16-MAIN3-079)
1x flask of sterile assay medium (basic medium for use in MTT assays, Lot No.: 16-ESSC-051)

- Validity controls as provided by the supplier (SkinEthic):
Morphology:
Histology scoring (HES stained vertical paraffin sections, n = 6):
Specification ≥ 19.5
Result: 22.3 ± 0.3, CV = 1.2%
Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
Barrier function:
IC50 determination (SDS concentration, MTT test, n= 14):
Specification ≥ 1.5 mg/mL
Result: 1.7 mg/mL

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C, 5.0 % CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- washed with DPBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT solution
MTT stock solution: 3 mg/mL MTT (Sigma; Lot No.: MKBR6576V) in PBS (Gibco; Lot No.: 1722256)
MTT medium: MTT stock solution was diluted 1 + 9 with DMEM-based medium (final concentration 0.3 mg/mL)
- Acidic isopropanol
0.04 N HCl (AppliChem; Lot No.: 0000687102) in isopropanol (AppliChem; Lot No.: 0000694025)
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

NUMBER OF REPLICATE TISSUES: 3 tissues per dose group

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 h post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 h post-treatment incubation is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg + 5 µL aqua dest.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL DPBS (Gibco, Cat No. 14040-091, Lot No.: 1737107)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL Sodium dodecyl sulfat (SDS; AppliChem, Art.-No. A1112,0500, CAS No.: 151-21-3, Lot No.: 40015277)
- Concentration (if solution): 5% in aqua dest.
Duration of treatment / exposure:
15 min ± 0.5 miin
Duration of post-treatment incubation (if applicable):
42 ± 1 h
Number of replicates:
3 tissues per dose group
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of three tissues
Value:
70.3
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
For detailed results see "Any other information on results" Table 1

Results of thePre-Experiments

The mixture of 10 mg test item per 2 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/purple.

For quantitative correction of results the part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using killed tissues. Therefore, three killed tissues were treated with the test item (KT) and with the negative control DPBS (KU), respectively. NSMTT was calculated relative to the negative control of living tissues (NK) according to the following formula:

NSMTT [%] = [(ODKT- ODKU)/ODNK] * 100

Mean ODKT= 0.066

Mean ODKU= 0.032

Mean ODNK= 0.685

The calculated NSMTT was ≤ 30% (4.9%) relative to the negative control of livingepidermisand could therefore be used for determination of the true MTT metabolic conversion (TODTT) of the test item treated living tissues (TM) according to the following formula:

TODTT= ODTM- (ODKT- ODKU) (results see Table 1)

The mixtures of 10 mg of the test item per 90 µL aqua dest. and per 90 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSClivingequalled 0%. No additional controls were necessary.

Results of the main experiment

Table1:  Result of the test Item

Name Negative Control Positive Control Test Item
Tissue 1 2 3 1 2 3 1 2 3
Absolute OD570 0.606
0.643
0.671
0.728
0.737
0.726
0.096
0.102
0.076
0.084
0.096
0.093
0.540
0.546
0.558
0.565
0.372
0.382
OD570 (Blank corrected 0.563
0.600
0.628
0.685
0.694
0.683
0.053
0.059
0.033
0.041
0.053
0.050
0.497
0.503
0.515
0.522
0.329
0.342
Mean OD570 of the Duplicates (Blank corrected)  0.582 0.656 0.689 0.056 0.037 0.052 0.500 0.519 0.336
Total Mean OD570 of 3 Replicate Tissues (Blank corrected) 0.642* 0.048 0.451
SD OD570 0.055 0.010 0.101
TODIT - - 0.417
Relative Tissue Viabilities [%] 90.6 102.2 107.2 8.7 5.8 8.0 77.8 80.7 52.3
Mean Relative Tissue Viabilities [%] 100.0 7.5** 70.3
SD Tissue Viabilities [%]*** 8.5 1.5 15.7
CV [% Viability] 8.5 20.5 22.3

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

**Mean relative tissue viability of the three positive control tissues is40%.

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

Table 2: Test Acceptance Criteria

 

Value

Cut off

pass/fail

Mean OD570 nmBlank

0.043

< 0.1

pass

Mean Absolute OD570 nmNK

0.685

0.6 ≤ NK ≤1.5

pass

Mean Relative Viability [%] PC

7.5

≤ 40%

pass

Max. SD of % Viability [%]

15.7

≤ 18%

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 therefore classified as “non-irritant” in accordance with UN GHS “No Category”.
Executive summary:

In the present study the skin irritant potential of Propyl gallate (> 99.93% purity) was analysed according to OECD 439 using the EPISKIN-Standard Model™ (EPISKIN-SMTM), a reconstituted three-dimensional human epidermis model to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 15 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls.

In this study under the given conditions the test item showed no irritant effects (70.3% mean relative tissue viability). As the relative mean tissue viability after 15 min of exposure and 42 h post-incubation was > 50%, the test item is therefore classified as “non-irritant” 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 vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-01-17 to 2017-04-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A Bovine Corneal Opacity and Permeability Assay (OECD Guideline number 437) with propyl gallate was performed (Eurofins Munich / BSL Munich Project No. 167610). Based on the results of this test further in vivo testing for assessing acute eye irritation/corrosion is necessary.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
2 October 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, 97633, Sulzfeld, Germany
- Age at study initiation: approximately 39 weeks old
- Weight at study initiation: >2 kg
- Housing: ABS-plastic or Noryl rabbit cages, floor 4200 cm2, in an air-conditioned room
- Diet (e.g. ad libitum): free access to autoclaved hay and to Altromin 2123 maintenance diet for rabbits, rich in crude fibre
- Water (e.g. ad libitum): free access to tap water (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: adequate acclimatisation period (at least 5 days) under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): at least 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g of test item was applied to the test site
Duration of treatment / exposure:
single treatment, 1h
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
1 rabbit
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): The treated eye was rinsed with physiological saline 0.9% NaCl 1 hour after the application.
- Time after start of exposure: 1 h

SCORING SYSTEM: Grading System for Ocular Lesion (for details see Table 1 in "Any other information on material and methods")

TOOL USED TO ASSESS SCORE: fluorescein
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
3
Reversibility:
not reversible
Remarks:
within 72 hours
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
not reversible
Remarks:
within 72 hours
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
not reversible
Remarks:
within 72 hours
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
not reversible
Remarks:
within 72 hours
Irritant / corrosive response data:
After the application into the eye of one male NZW rabbit the test item produced severe irritant occular effects (see Table 2 and Table 3 in "Any other information on results"). Local effects at the treated eye and clinical signs were observed (see Table 3). The inclined position of the head 72 hours after application is considered to be related to the local effects observed at the treated eye and not due to systemic effects of the test item. Before fluorescein examination at the end of the observation period of 72 hours the animal was euthanised for animal welfare reasons. Conjunctival redness, chemosis, hypersecretion, corneal effects and iris lesion were observed and described in Table 2.
Other effects:
- Lesions and clinical observations: after 1 hour slight hypersecretion, after 24 hours moderate hypersecretion, after 48 and 72 hours severe hypersecretion

Table 2: Eye Irritation Scores - Animal No. 1

Observation

Eye Irritation Scores Post-Application After

1(1h)

2(24h)

3(48h)

4(72h)

 

T

C

T

C

T

C

T

C

Average Score (24, 48 and 72 hours)

Redness

0

0

1#

0

1

0

1

0

1.00

Chemosis

2

0

2

0

2

0

2

0

2.00

Iris

n.a.*

0

2**

0

2**

0

2

0

2.00

cornea

3

0

2

0

2

0

2

0

2.00

t= test item; c= control; #= upper rim of conjunctiva and nicticating membrane discoloured white; *=due to complete opacity of the cornea the iris was not visible and thus could not be evaluated; **= no reaction to light

Table 3: Clinical Signs - Animal No. 1 

Time Post-Application Local Findings / Comments Systemic Findings
Test Item Control Test Item
1 h slight hypersecretion nsf nsf
24 h moderate hypersecretion nsf nsf
48 h severe hypersecretion nsf nsf
72 h severe hypersecretion nsf head inclined §

nsf= no specific findings; h= hour(s); §= euthanised for ethical reasons

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
In an in vivo acute eye irritation/corrosion study in one rabbit accroding to OECD 405, Propyl gallate produced severely irritant effects, which are not expected to reverse.
Executive summary:

In a primary eye irritation study according to OECD 405, 0.1 g of propyl gallate (99.93 % purity) was applied into the conjunctival sac of one eye of 1 male, 39 weeks old, New Zealand White rabbit. The test substance remained in the eye for 1 hour and was rinsed afterwards with physiological saline (0.9 % NaCl). The animal then was observed for 72 hours. Irritation was scored by the method of Draize after 1, 24, 48 and 72 h.

Under the conditions of the present study, a single ocular application of the test item Propyl gallate to one rabbit at a dose of 0.1 g produced severely irritant effects including conjunctival redness, chemosis, hypersecretion, corneal effects and iris lesion (iridial response grade 2 for 72 hours), which are not expected to reverse.

In this study, propyl gallate is a severe eye irritant based on CLP criteria.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The irritancy potential of propyl gallate to the skin was addressed by using a reconstituted three-dimensional human epidermis model according to OECD 439.

In the in vitro skin irritation test, the target substance (> 99.93% purity) showed no irritant effects (70.3% mean relative tissue viability) after 15 min exposure and 42 h post-incubation, classifying propyl gallate as non-irritant to the skin.

The eye irritation potential of propyl gallate was investigated using a BCOP test according to OECD 437 followed by an in vivo study according to OECD 405.

For the bovine corneal opacity and permeability assay (OECD 437), the test item was suspended with physiological saline to give a concentration of 20%. A mean in vitro irritation score of 29.65 was determined. The positive control; 20% imidazol in phsiological saline, induced the appropriate responses, indicating the validity of the assay. According to the UN GHS criteria, this mean in vitro irritation score does not allow to make any prediction regarding classification of propyl gallate. For the purpose of classification, an in vivo study according to OECD 405 was performed.

In a primary eye irritation study according to OECD 405, 0.1 g of propyl gallate (99.93 % purity) was applied into the conjunctival sac of one eye of one male New Zealand White rabbit. The test substance remained in the eye for 1 hour and was rinsed afterwards with physiological saline (0.9 % NaCl). The animal then was observed for 72 hours. Irritation was scored by the method of Draize after 1, 24, 48 and 72 h.

Under the conditions of the present study, a single ocular application of the test item propyl gallate to one rabbit at a dose of 0.1 g produced severely irritant effects including conjunctival redness, chemosis, hypersecretion, corneal effects and iris lesion (iridial response grade 2 for 72 hours), which are not expected to reverse.

In this study, propyl gallate is a severe eye irritant based on CLP criteria.

Justification for classification or non-classification

A GLP guideline study (OECD 439) was conducted to evaluate the skin irritation potential of propyl gallate with negative results. Therefore, the test item does not warrant classification for skin irritation.

Based on the results of the primary eye irritation study according to OECD 405, propyl gallate does warrant classification for eye irritation category 1 (Eye Dam. 1, H318) according to CLP criteria.