EPOFLOC L-1R

Endpoint summary

Description of key information

Not corrosive or irritating to the skin. Value used for CSA is taken from the 2000 study as explained in DNEL derivation.

As the pH is >11.5 the classification is; Eye Damage 1 H318: Causes serious eye damage.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25th July - 7th August 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd, Margate, Kent, England.
- Age at study initiation: Approximately 15 weeks of age (prior to treatment (Day 1)).
- Weight at study initiation: 3.3 to 3.6 kg.
- Housing: Individually in plastic cages with perforated floors.
- Diet (e.g. ad libitum): SDS Stanrab (P) Rabbit Diet ad libitum
- Water (e.g. ad libitum): Drinking water ad libitum
- Acclimation period: All rabbit were acclimatised to the experimental environment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 (approximately)
- Humidity (%): 30 - 70 (relative humidity)
- Air changes (per hr): 19 (approximately)
- Photoperiod (hrs dark / hrs light): 12 hours of artificial light (0700 - 1900 hours) in each 24 hours period.

IN-LIFE DATES: From: 25 July 1995 To: 7 August 1995.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

0.5 ml

Duration of treatment / exposure:

4 hours

Observation period:

- Environmental parameters: Recorded daily.
- Clinical signs: All animals were observed daily for signs of ill health or toxicity.
- Dermal responses: Examination of the treated skin was made on Day 1 (i.e. approximately 60 minutes after removal of the dressings) and on Days 2, 3 and 4 (equivalent to 24, 48 and 72 hours after exposure). Additional observations were made on Days 5 to 14.

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: Approximately 24 hours prior to application of the test substance, hair was removed with electric clippers from the dorso-lumbar region of each rabbit exposing an area of skin approximately 100 mm x 100 mm. A 0.5 ml amount of the test substance was applied under a 25 mm x 25 mm gauze pad to one intact skin site on each animal.

- Type of wrap if used: Each treatment site was covered with “Elastoplast” elastic adhesive dressing for four hours.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period, the semi-occlusive dressing and gauze pad were removed and the treatment site was washed with warm water (30° to 40°C) to remove any residual test substance. The treated area was blotted dry with absorbent paper.
- Time after start of exposure: 4 hours

SCORING SYSTEM: Local dermal irritation was assessed using the prescribed numerical system in the attached report. Any other lesion not covered by this scoring system, was described.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

3.3

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Remarks on result:

other: Necrosis.

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

3.3

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Remarks on result:

other: Necrosis.

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

3.3

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Remarks on result:

other: Necrosis.

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not fully reversible within: 14 days

Remarks on result:

other: Edges of necrotic area lifting.

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.3

Max. score:

2

Reversibility:

not fully reversible within: 14 days

Remarks on result:

other: Edges of necrotic area lifting.

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.3

Max. score:

2

Reversibility:

not fully reversible within: 14 days.

Remarks on result:

other: Edges of necrotic area lifting.

Irritant / corrosive response data:

- Dermal responses: The numerical values given to the dermal reactions elicited by New Eporva 800 are shown in Table 1 of the attached report. Well-defined dermal reactions were seen following removal of bandages. Severe dermal irritation with necrosis developed in all these animals on Day 2 and persisted throughout the observation period to Day 14 (day of termination).

Other effects:

- Clinical signs: There were no signs of toxicity or ill health in any rabbit during the observation period.

Table 1. Dermal reactions observed after application of New Eporva 800

Rabbit number and sex

E = Erythema O = Oedema

Day

1*

2

3

4

5

6

7

8

9

10

11

12

13

14

1250

E

a2

b4

b4

b4

b4

b4

b4

b4

b4

b4

b4

b4

b4

b4

O

2

2

2

1

1

1

1

c1

c1

c1

c1

c1

c1

c1

1251

E

a2

b4

b4

b4

b4

b4

b4

b4

b4

b4

b4

b4

b4

b4

O

2

1

1

1

1

1

1

c1

c1

c1

c1

c1

c1

c1

1259

E

a2

b4

b4

b4

b4

b4

b4

b4

b4

b4

b4

b4

b4

b4

O

2

1

1

1

1

1

1

1

c1

c1

c1

c1

c1

c1

* Approximately 60 minutes after removal of the dressing
a Brown staining from dose; b Necrosis; c Edges of necrotic area lifting

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Conclusions:

A single semi-occlusive application of New Eporva 800 to intact rabbit skin for four hours elicited severe persistent dermal irritation. The necrosis, severe erythema and the persistence until day 14 of the study after a 4 hour exposure period warrant a classification as category 1B (H314 causes severe burns and eye damage).

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

24th January 2014

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

according to guideline

Guideline:

other: ECVAM´s “Performance standards for applying human skin models to in vitro skin irritation testing”.

Deviations:

not specified

Principles of method if other than guideline:

- Based on the normative references ISO-10993-1 (2009), Chapter 4.6 and ISO 10993-10 (2009)

GLP compliance:

not specified

Remarks:

No data

Species:

other: not applicable

Strain:

other: not applicable

Type of coverage:

open

Preparation of test site:

other: no preparation

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

50 µl

Duration of treatment / exposure:

20 minutes

Observation period:

42 hours post exposure incubation

Details on study design:

An in vitro reconstituted, human epidermal 3D-skin model, type epiCS, Lot. No. 100-AC2452-1 was used.

Prior to performing the test procedure the skin model cell culture inserts were incubated for 24 h at 37°C and 5% pCO2 in a cell culture incubator in fresh Maintenance Medium. After this preincubation time 50 μl of PBS was pipetted on the surface of each skin model. Then 50 μl of the product to be tested was applied on the skin surface. 50 μl Triton X 100 were used as a (skin irritating) positive control, 50 μl PBS were used as a (not skin irritating) negative control. All experiments were performed in duplicate. After 20 min incubation all inserts were rinsed thoroughly with sterile PBS, blotted on a paper towel to remove excess PBS and cultivated in Maintenance Medium for 42 h in a cell culture incubator at 37°C and 5% pCO2.

Measurement of LDH-release: After the 42h incubation period 2 x 100 μl Maintenance Medium Samples were taken from every skin model and the LDH-release was measured using a ‘’Cytotoxicity Detection Kit (LDH)’’ from Roche Diagnostics.

Measurement of cell vitality (MTT-test): The inserts were rinsed once in Assay Medium using a 24-well cell culture plate and transferred in a second 24-well cell culture plate with 300 μl Cellsystems Assay Medium containing 1mg/ml MTT (Sigma M5655). All inserts were incubated for additional 3h in a cell culture incubator at 37°C and 5% pCO2. Afterwards all inserts were blotted on a paper towel and the MTT-dye was extracted from the skin samples using 2 ml Isopropanol per insert. The extinction of each Isopropanol extract was measured in a photometer at 570 nm. With this data the relative vitality of the cells in the skin samples compared to the negative control (PBS) was calculated.

Irritation / corrosion parameter:

% tissue viability

Remarks:

42h

Value:

77.02

Measurement of cell vitality (MTT-test): In the presence of Triton X 100 on the skin culture inserts 1,2% of the cell vitality compared to the negative control was reached. This value is within the valid range of 15% cell vitality or less compared to the negative control. A product is considered as skin irritating, if it reduces the cell vitality of the skin samples to less than 50% compared to negative control skin samples. This is not the case in this experiment. The material didn´t show a skin irritating effect.

 

LDH-Release: Human skin inserts charged with the product didn´t display a significantly different LDH activity in the cell culture medium compared to PBS incubated skin inserts.

Interpretation of results:

GHS criteria not met

Conclusions:

Epofloc L1-R did not cause sufficient loss of viability to be considered as irritating.

Reference 3

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22nd January 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

GLP compliance:

not specified

Species:

other: not applicable

Strain:

other: not applicable

Type of coverage:

open

Preparation of test site:

other: no preparation

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

50 µl

Duration of treatment / exposure:

Cluster 1: 3 minutes
Cluster 2: 1 hour

Irritation / corrosion parameter:

other: Cell Vitality (MTT test)

Run / experiment:

3 minutes

Value:

98.78

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

other: Cell Vitality (MTT test)

Run / experiment:

1 hour

Value:

104.96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Measurement of cell vitality (MTT-test), 3 min incubation: The vitality data of the skin corrosive positive control (8 N KOH) of 16,36% falls within the valid range of considerably lower than 50% vitality compared to the PBS negative control for corrosive chemicals after 3 min incubation. Vitality data of chemicals after an incubation time of 3 min on the skin inserts which fall below the vitality data of the non-corrosive negative control PBS solution by more than 50% lead to the evaluation of the substance as “skin corrosive”. This was not the case in this test. The tested product didn´t show a skin corrosive effect after the 3 min incubation time.

 

Result data	n=2, (%)
(rel. cell vitality)	8 N KOH	PBS	Test material
Mean	16.36%	100%	98.78%
Standard deviation	0.93%	5.76%	5.84%

 

Measurement of cell vitality (MTT-test), 1 h incubation: The vitality data of the skin corrosive positive control (8 N KOH) of 0,43% falls within the valid range of considerably lower than 15% vitality compared to the PBS negative control for corrosive chemicals after 1 h incubation. Vitality data of chemicals after an incubation time of 1 h on the skin inserts which fall below the vitality data of the non-corrosive negative control PBS solution by more than 85% lead to the evaluation of the substance as “skin corrosive”. This was not the case in this test. The tested product didn’t show a skin corrosive effect after the 1 h incubation time.

 

Result data	n=2, (%)
(rel. cell vitality)	8 N KOH	PBS	Test material
Mean	0.43%	100%	104.96%
Standard deviation	0.35%	1.86%	3.44%

 

Explanatory notes: According to OECD 431-TG the skin inserts incubated with the substance to be tested need to show at least a cell vitality of 50% after 3 min incubation and at least a cell vitality of 15% after 1 h incubation. These essential requirements were met after both tested incubation times. Therefore the tested substance was classified as not skin corrosive according to OECD 431-TG.

Interpretation of results:

GHS criteria not met

Conclusions:

The EPOFLOC L1-R, used for precipitation of heavy metal ions out of aqueous solutions, didn’t cause a skin corrosive effect.

Reference 4

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

9th October to 17th October 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Higate Farm, Market Rasen, Lincolnshire, England.
- Age at study initiation: Approximately 9 weeks of age (prior to treatment (Day 1)).
- Weight at study initiation: 2.6-3.1 kg
- Housing: Individually in plastic cages with perforated floors
- Diet (e.g. ad libitum): SDS Stanrab (P) Rabbit Diet ad libitum
- Water (e.g. ad libitum): Drinking water ad libitum
- Acclimation period: All rabbit were acclimatised to the experimental environment for a minimum of 6 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19(±2)
- Humidity (%): 30-70 (relative humidity)
- Air changes (per hr): No Data
- Photoperiod (hrs dark / hrs light): 12 hours of artificial light (0600 - 1800 hours) in each 24-hour period.
IN-LIFE DATES: From: 9th October to 17th October 2000.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

0.5 mL

Duration of treatment / exposure:

4 hours

Observation period:

- Environmental parameters: Recorded daily.
- Clinical signs: Observed daily for signs of ill health or toxicity.
- Dermal responses: Examination of the treated skin was made on Day 1 (i.e. immediately after removal of the dressings following 3 minute, 60 minute and 4 hour ) and on Days 2, 3 and 4 (equivalent to 24, 48 and 72 hours after exposure). Additional observations were made on Days 5 to 8.

Number of animals:

3 male

Details on study design:

TEST SITE
- Area of exposure: Approximately 24 hours prior to test substance application, hair was removed with electric clippers from the dorso-lumbar region of each rabbit exposing an area of skin approximately 100 mm X 100 mm. 0.5 mL of the test substance was applied under a 25 mm x 25 mm gauze pad to each intact skin site on each animal.

- Type of wrap if used: Each treatment site was covered with “Elastoplast” elastic adhesive dressing for the exposure period.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure periods, the semi-occlusive dressing and gauze pad were removed and the treatment site washed with warm water (35° to 37°C) to remove any residual test substance. The treated area was blotted with absorbent paper.
- Time after start of exposure: 4 hours

SCORING SYSTEM: Local dermal irritation was assessed using the prescribed numerical system in the attached report. Any lesion not covered by this scoring system was described.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

other: 8 days

Score:

0

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

other: 8 days

Score:

0

Max. score:

1

Reversibility:

fully reversible

Remarks on result:

other: Desquamation noted on day 6 and 7.

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

other: 8 days

Score:

0

Max. score:

1

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: 8 days

Score:

0

Max. score:

0

Irritation parameter:

edema score

Basis:

animal #2

Time point:

other: 8 days

Score:

0

Max. score:

0

Irritation parameter:

edema score

Basis:

animal #3

Time point:

other: 8 days

Score:

0

Max. score:

0

Irritant / corrosive response data:

- Dermal responses:
No dermal reaction observed following 3 minute application. Well defined erythema with slight oedema observed following 1 hour exposure with blanching after (days 4-6) and desquamation (days 7-8). Well defined erythema was observed in a single animal following 4 hour exposure with very slight erythema observed in remaining two animals. Desquamation was observed on (days 6 and 7) in a single animal. Reactions had resolved by days 4,5 or 8.

Other effects:

- Clinical signs: There were no signs of toxicity or ill health in any rabbit during the observation period.

Interpretation of results:

slightly irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

A single semi-occlusive application of New Eporva 800 to intact rabbit skin for four hours elicited very slight to well-defined dermal irritation.

New Eporva 800 does not meet the classification criteria for skin corrosion or irritation in accordance with Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, Version 4.0, November 2013. No classification required.

Executive summary:

A study was performed to assess the skin irritation potential of New Eporva 800 to the rabbit. Three rabbits were each administered a single dermal dose of 0.5 mL of the test substance and observed for 8 days. A single semi-occlusive application of New Eporva 800 to intact rabbit skin for four hours elicited very slight to well-defined dermal irritation.  New Eporva 800 does not meet the classification criteria for skin corrosion or irritation in accordance with Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, Version 4.0, November 2013. No classification required.

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

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is a strong acid (pH <= 2.0) or base (pH => 11.5) and the available information indicates that it should be classified as serious eye damage (Category 1)

Endpoint conclusion

Endpoint conclusion:

no study available

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The 1995 study (OECD 404) was classified a key study based on its rationale for reliability (Klimisch 1, reliable without restriction). As severe persistent dermal irritation with necrosis and severe erythema persisting until day 14 was observed, Epofloc L-1R warranted classification as category 1C (H314 causes severe burns and eye damage). The 2000 study of the same guideline was also classified a key study (Klimisch 1). In this study, Epofloc L-1R caused only very slight to well-defined dermal irritation, and did not meet the classification criteria for skin corrosion or irritation in accordance with Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, Version 4.0, November 2013. Although both are key studies, the 2000 study is the pertinent study following minor test material changes between tests, a result of the manufacturing process being optimized in 1995 and 2000. The current batch used in the 2000 study and thereafter has been confirmed as that currently used in the EU. Furthermore, data from two supporting in vitro studies performed in 2014 using the reconstituted, human epidermal 3D-skin model (Klimisch 3, not reliable), and skin corrosion test (OECD 431) (Klimisch 2, reliable with restrictions) both confirmed Epofloc L-1R was not a skin irritant or caused a skin corrosive effect. This substance has a pH of 12.1, therefore, on grounds of animal welfare, no eye irritation studies will be undertaken.

Justification for selection of skin irritation / corrosion endpoint:
The 1995 study (OECD 404) was classified a key study based on its rationale for reliability (Klimisch 1, reliable without restriction). As severe persistent dermal irritation with necrosis and severe erythema persisting until day 14 was observed, Epofloc L-1R warranted classification as category 1C (H314 causes severe burns and eye damage). The 2000 study of the same guideline was also classified a key study (Klimisch 1). In this study, Epofloc L-1R caused only very slight to well-defined dermal irritation, and did not meet the classification criteria for skin corrosion or irritation in accordance with Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, Version 4.0, November 2013. Although both are key studies, the 2000 study is the pertinent study following minor test material changes between tests, a result of the manufacturing process being optimized in 1995 and 2000. The current batch used in the 2000 study and thereafter has been confirmed as that currently used in the EU. Furthermore, data from two supporting in vitro studies performed in 2014 using the reconstituted, human epidermal 3D-skin model (Klimisch 3, not reliable), and skin corrosion test (OECD 431) (Klimisch 2, reliable with restrictions) both confirmed Epofloc L-1R was not a skin irritant or caused a skin corrosive effect.

Effects on skin irritation/corrosion: not corrosive or irritant

Justification for classification or non-classification

This substance is not classified as corrosive or irritant to the skin (s. additional information). In accordance with CLP guidance, as the pH is >11.5 the classification is; Eye Damage 1 H318: Causes serious eye damage.