RSS-4, DRY SUBSTANCE

Endpoint summary

Description of key information

The skin and eye irritation potential of the substance have both been investigated in vitro as well as in vivo. Based on the results obtained in these 4 studies, the substance does not need to be classified for skin and eye irritation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 April - 04 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

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

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000, including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

- Source: Charles River Deutschland, Kisslegg, Germany
- Age at study initiation: Animals used withihn the study were at least 6 weeks old (two animals 8-9 weeks, and one animal 10-12 weeks)
- Weight at study initiation: Body weights were at least 1.0 kg (1507 grams or 1882 grams; weighing/recording error for other animal).
- Housing: Animals were housed individually in cages with perforated floors.
- Diet: Free access to pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad, Italy). Hay and wooden sticks were abailable during the study period.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle.

IN-LIFE DATES: From: 17 April - 04 May 2012

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

other: The test substance was moistened with water.

Controls:

other: Adjacent areas of the untreated skin of each animal served as controls.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 grams.

VEHICLE
The test substance was moistened with 0.3 mL of the vehicle.

Duration of treatment / exposure:

Single application.

Observation period:

14 days.

Number of animals:

3 males.

Details on study design:

STUDY DESIGN
The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner 14 days later, after considering the degree of skin irritation observed in the first animal.

TEST SUBSTANCE PREPARATION
The powdery test substance was moistened with water (Elix, Millipore S.A.S., Molsheim, France), immediately before application, to ensure close contact with the animal's skin.

TEST SITE
Approximately 24 hours before treatment, the dorsal fur was clipped with electric clippers, exposing an area of approximately 150 square centimeters (10x15 cm). To facilitate scoring, treated skin areas were re-clipped at least 3 hours before the observations.

The test substance was applied to the skin of one flank, using a patch of 2x3 cm.

REMOVAL OF TEST SUBSTANCE
Four hours after the application, the dressing was removed and the skin cleaned of residual test substance using tap water.

OBSERVATIONS
- Mortality/Viability: Twice daily.
- Toxicity: At least once daily.
- Body Weight: Day of treatment (prior to application) and after the final observation.
- Necropsy:No necropsy was performed according to protocol.
- Irritation:
The skin reactions were assessed at approximately 1, 24, 48 and 72 hours after the removal of the dressings and test substance. The irritation scores and a description of all other (local) effects were recorded.

SCORING SYSTEM:
The irritation was assessed according to the numerical scoring system according to OECD 404.

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 24, 48 and 72 hours.

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 24, 48 and 72 hours.

Score:

0

Max. score:

4

Irritant / corrosive response data:

No skin irritation was caused by 4 hours exposure to Magnesium bis (2-ethylbutanoate).

Other effects:

Coloration/remnants:
No staining of the treated skin by the test substance was observed and no test substance remnants were seen.

Toxicity/mortality:
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on these results, Magnesium bis (2-ethylbutanoate) does not have to be classified and has no obligatory labelling requirement for skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.

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:

23 April - 21 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000, including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

- Source: Charles River Deutschland, Kisslegg, Germany
- Age at study initiation: Animals used withihn the study were at least 6 weeks old (one animal 7-8 weeks, and two animals8-9 weeks).
- Weight at study initiation: Body weights were at least 1.0 kg (1700, 1755 and 1833 grams, respectively).
- Housing: Individually housed in cages with perforated floors.
- Diet: Free access to pelleted diet for rabbits (Global Diet 2030 Harlan Teklad, Italy). Hay and wooden sticks were available during the study period.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle.

IN-LIFE DATES: From: 23 April - 21 May 2012

Vehicle:

unchanged (no vehicle)

Controls:

other: One eye of each animal remained untreated and served as the reference control.

Amount / concentration applied:

TEST MATERIAL- Amount(s) applied:
53.8 mg (range 53.4 – 54.0 mg)

Duration of treatment / exposure:

Single instillation on Day 1.

Observation period (in vivo):

The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7 and/or 14 days after instillation of the test substance

Number of animals or in vitro replicates:

3 males

Details on study design:

STUDY DESIGN
The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner 3 weeks later, after considering the degree of eye irritation observed in the first animal.

TREATMENT
Animals were treated by instillation of, on average, 53.8 mg (range 53.4 – 54.0 mg) of the test substance (a volume of approximately 0.1 mL), in the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test substance. The other eye remained untreated and served as the reference control.
Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt, Germany) in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area. After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).

REMOVAL OF TEST SUBSTANCE
-Washing: No

OBSERVATIONS
- Mortality/Viability: Twice daily.
- Toxicity: At least once daily.
- Body Weight: Day of treatment (prior to instillation) and after the final observation. For one animal also on Day 8 next to measurement after the final observation on Day 15. Evaluation: Provides additional information..
- Necropsy: No necropsy was performed according to protocol.
- Irritation:
The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7 and/or 14 days after instillation of the test substance. The irritation scores and a description of all other (local) effects were recorded.
The irritation scores and a description of all other (local) effects were recorded.The irritation was assessed according to OECD 405.

Irritation parameter:

cornea opacity score

Remarks:

(opacity)

Basis:

mean

Time point:

other: 24, 48 and 72 hrs.

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 24, 48 and 72 hrs.

Score:

0.6

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

mean

Time point:

other: 24, 48 and 72 hrs.

Score:

1.7

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 24, 48 and 72 hrs.

Score:

0.8

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

Instillation of approximately 54 mg of Magnesium bis (2-ethylbutanoate) (a volume of approximately 0.1 mL) into one eye of each of three rabbits resulted in effects on the cornea, iris and conjunctivae.
The corneal injury consisted of slight dulling of the normal luster on Day 1 for all animals. No corneal opacity was observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage. Iridial irritation (grade 1 or 2) was observed for two animals on Day 1 and for one animal at 1, 24, 48 and 72 hours after instillation. One of the animals showed absence of reactivity to light and reduced size of the pupil at 24, 48 and 72 hours after instillation. The irritation of the conjunctivae consisted of redness, chemosis and discharge and had completely resolved within 7 days. Bald skin around the eye was noted for one animal between 24 hours and 7 days after instillation.

Other effects:

Colouration/Remnants:
No staining of (peri) ocular tissues by the test substance was observed and no test substance remnants were seen.

Toxicity/Mortality:
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

other: Irritating to eyes, but insufficient for classification.

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Based on these results, Magnesium bis (2-ethylbutanoate) does not have to be classified and has no obligatory labelling requirement for eye irritation according to the:
- Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011)
- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

In an in vitro skin irritation test similar to OECD 439 (2010) using a human skin model ( EPISKIN Standard Model), the influence of the substance on the viability of human skin was tested. After 15 minutes, the substance was removed and cells were cultured for 42 hours. Survival of unexposed skin was set at 100%, the positive control had a relative mean cell viability of 6% whereas the substance showed a relative mean cell viability of 105%. Since the mean relative tissue viability after exposure to the substance was above 50%, it is concluded that the substance is not irritating in the in vitro skin irritation test.

In addition an in vivo skin irritation test according to OECD 404 (2002) and GLP principles was performed with the substance. The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner 14 days later, after considering the degree of skin irritation observed in the first animal. No skin irritation effects and no symptoms of systemic toxicity were observed during the test period and no mortality occurred.

Eye irritation

The eye irritation potential of the substance was investigated in vitro by using the SkinEthic Reconstituted Human Corneal Epithelium model. Triplicate tissues were treated with 30 mg of the test material for 10 minutes at 37°C. Survival of unexposed skin was set at 100%, the positive control had a relative mean cell viability of 37.6% whereas the substance showed a relative mean cell viability of 73.9%. Since the mean relative tissue viability after exposure to the substance was above 60%, it can be concluded that the substance is not irritating in the in vitro eye irritation test.

 

In addition an in vivo eye irritation test according to OECD 405 (2002) and GLP principles was performed with the substance. The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner 3 weeks later. Instillation of approximately 54 mg of the substance (a volume of approximately 0.1 mL) into one eye of each of three rabbits resulted in effects on the cornea, iris and conjunctivae. The corneal injury consisted of slight dulling of the normal luster on Day 1 for all animals. No corneal opacity was observed. Iridial irritation (grade 1 or 2) was observed for two animals on Day 1 and for one animal at 1, 24, 48 and 72 hours after instillation. One of the animals showed absence of reactivity to light and reduced size of the pupil at 24, 48 and 72 hours after instillation. The irritation of the conjunctivae consisted of redness, chemosis and discharge and had completely resolved within 7 days. The substance produced in 3 animals a response of conjunctival redness of 1.7, in 1 animal a response of iridial irritation of 2.0, in 1 animal a response of conjunctival oedema of 1.0 and in 2 animals a response of conjunctival oedema of 0.7, calculated as the mean scores following grading at 24, 48 and 72 hours after instillation of the test material.

Justification for selection of skin irritation / corrosion endpoint:
The available in vivo study

Justification for selection of eye irritation endpoint:
The available in vivo study

Justification for classification or non-classification

Skin irritation

Based on the results obtained in an vitro skin irritation test similar to OECD 439 (2010) using a human skin model ( EPISKIN Standard Model) and in an in vivo skin irritation test according to OECD 404 (2002), the substance does not need to be classified for skin irritation in accordance with the CLP Regulation.

 

Eye irritation

Based on the results obtained in an vitro eye irritation test using the SkinEthic Reconstituted Human Corneal Epithelium model and in an in vivo eye irritation test according to OECD 405 (2002), the substance does not need to be classified for eye irritation in accordance with the CLP Regulation. Ocular effects were observed in the in vivo study, but not sufficient for classification.