Calcium 3,5,5-trimethylhexanoate

Endpoint summary

Description of key information

Skin irritation: not irritating (OECD 404, GLP)
Eye irritation: irritating (OECD 405, GLP)

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:

2013-03-15 to 2013-03-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

adopted 2002-04-24

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-11-12

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Branch Löhndorf, 24601 Löhndorf/Post Wankendorf, Germany
- Age at study initiation: Approx. 7 months
- Weight at study initiation: 2.5 - 2.9 kg
- Housing: before and after the 4-hour exposure period, the animals were kept singly in cages measuring 380 mm x 425 mm x 600 mm (manufacturer: Dipl. Ing. W. EHRET GmbH, 16352 Schönwalde, Germany). During the exposure period, the animals were kept singly in restrainers which allowed free movement of the head but prevented a complete body turn. The cages excluded irritation of the skin by excrements and urine.
- Diet (ad libitum; before and after exposure period): commercial diet, ssniff® K-H V2333 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum; before and after exposure period): drinking water
- Acclimation period: at least 20 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature: 20°C ± 3°C (maximum range)
- Relative humidity: 30% - 70% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 500 mg of the test item was applied to the test site 1000 mg of the test item were mixed with 500 mg aqua ad iniectabilia (Batch no. 123868001, B. Braun Melsungen AG, 34212 Melsungen, Germany),
750 mg of this paste were applied per animal (^ 500 mg test item/animal)

Duration of treatment / exposure:

4 hours

Observation period:

Prior to the administration and 60 minutes, 24, 48 and 72 hours after the exposure period

Number of animals:

3 female rabbits

Details on study design:

TEST SITE
- Area of exposure: approximately 24 hours before the test, the fur was removed by closely clipping the dorsal area of the trunk of the animals. Care was taken to avoid abrading the skin. Only animals with healthy intact skin were used.
The test item was applied to the test site (area: approx. 6 cm²) and then covered with a gauze patch. The patch was held in contact with the skin with non-irritating tape for the duration of the exposure period. The surrounding untreated skin served as a control.

INITIAL TEST AND CONFIRMATORY TEST
As it was expected that the test item would not produce any severe irritancy or corrosion, the test was started using at first only one animal, receiving a single patch for an exposure period of 4 hours.
As neither a corrosive effect nor a severe irritant effect was observed after a four-hour exposure, the test was completed using two additional animals, each with one patch only, for an exposure period of 4 hours.

SCORING SYSTEM: according to the Draize scale

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

None of the three rabbits exposed for 4 hours to 500 mg Calcium-2-ethylhexanoate/animal showed any skin reactions

Other effects:

There were no systemic intolerance reactions.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is not classified as skin irritant.

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:

2013-01-02 to 2013-02-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted 2002-04-24

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2012-11-30

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS - New Zealand White (Hsdlf:NZW) strain rabbits
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK
- Age at study initiation: twelve to twenty weeks old
- Weight at study initiation: 2.24 or 2.75 kg
- Housing: the animals were individually housed in suspended cages.
- Diet (ad libitum): 2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK)
- Water (ad libitum): mains drinking water
- Acclimation period: at least five days

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 17 to 23°C
- Relative humidity: 30 to 70%
- Air exchange: at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): a volume of 0.1 mL of the test item, which was found to weigh approximately 76 mg was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes.

Duration of treatment / exposure:

not applicable

Observation period (in vivo):

Approximately 1 hour and 24, 48 and 72 hours as well as 7 and 14 days following treatment

Number of animals or in vitro replicates:

3 male rabbits

Details on study design:

Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.

INITIAL AND CONFIRMATORY TEST
Initially, a single rabbit was treated. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the six point scale (please refer to table 2 in the field "Any other information on materials and methods incl. tables" below).
After consideration of the ocular responses produced in the first treated animal, two additional animals were treated.

SCORING SYSTEM: according to the Draize scale
Any other ocular effects were also noted.
Any clinical signs of toxicity, if present, were also recorded.
Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.
If evidence of irreversible ocular damage is noted, the test item will be classified as corrosive to the eye.

TOOL USED TO ASSESS SCORE: examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: Initial pain reaction: practically no initial pain was observed. Area cornea involved: one quarter (or less) but not zero; Moderate conjunctival redness & slight conjunctival redness were observed 1 h & 7 d after test item administration, respectively.

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: Slight iridial inflammation was observed 1 hour after test substance administration.

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: Slight to moderate discharge was observed 1 hour to 7 days after test substance administration.

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: Moderate chemosis was observed 1 hour after test substance administration. Slight chemosis was observed 7 days after test substance administration.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: Initial pain reaction: practically no initial pain was observed. Slight discharge was observed 1 hour and 24 hours after test substance administration.

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

other: Slight iridial inflammation was observed 1 hour after test substance administration.

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Moderate conjunctival redness was observed 1 hour after test item administration.

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Slight chemosis was observed 1 hour after test substance administration.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: Initial pain reaction: practically no initial pain was observed. Slight to moderate discharge was observed 1 hour to 72 hours after test substance administration.

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0.67

Max. score:

2

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Slight iridial inflammation was observed 1 hour after test substance administration.

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: Moderate conjunctival redness & slight conjunctival redness were observed 1 h & 7 d after test item administration, respectively.

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: Moderate chemosis was observed 1 hour after test substance administration. Slight chemosis was observed 7 days after test substance administration.

Irritant / corrosive response data:

Diffuse corneal opacity was noted in one treated eye at the 24, 48 and 72-Hour observations.
Iridial inflammation was noted in all treated eyes one hour after treatment, in two treated eyes at the 24 and 48-Hour observations and in one treated eye at the 72-Hour observation.
Moderate conjunctival irritation was noted in all treated eyes one and 24 hours after treatment. Moderate conjunctival irritation was noted in two treated eyes and minimal conjunctival irritation was noted in one treated eye at the 48-Hour observation. Moderate conjunctival irritation was noted in two treated eyes at the 72-Hour observation with minimal conjunctival irritation at the 7-Day observation.
One treated eye appeared normal at the 72-Hour observation and two treated eyes appeared normal at the 14-Day observation.

Other effects:

- Body weight: all animals showed expected gain in body weight during the study.

Interpretation of results:

Category II

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test material is irritating to the eyes.
According to 67/548/EC and subsequent regulations, the substance is not classified as an eye irritant.
According to the EC Regulation No. 1272/2008 and subsequent regulations, the substance is classified as Category 2.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin irritation

One reliable in vivo study described by Hansen (2013)(OECD 404; GLP compliant) is considered to be reliable without restrictions. The substance was determined not to be irritating to the skin.

 

The data requirements as laid down in the REACH regulation Annex VII and VIII, foresee a sequential testing strategy for the endpoint skin irritation. According to Annex VII, an in vitro study for skin corrosion and skin irritation shall be performed, before further in vivo tests according to Annex VIII are conducted. For the conduct of the in vitro skin corrosion and skin irritation properties, the human skin model system is recommended for the assessment of both endpoints (OECD 431 and 439 respectively).

 

During the conduct of the sequential testing for skin corrosion and skin irritation by another registrant also using metal substances (salts with organic and inorganic anion/ligand of high water solubility, Cobalt, borate neodecanoate complexes, Cobalt di(acetate) and Cobalt dichloride), it became obvious that a high number of false positive test results were received. Six supporting study records for three substances are attached to the waiving statement for in vitro skin irritation in section 7.3.1 of the IUCLID file. The studies represent three in vitro studies with the human skin model test system and a subsequently conducted in vivo skin irritation study in rabbits. These results raised doubt on the suitability of the in vitro skin model test systems for metal substances.

 

It was therefore decided by the registrant to deviate from the suggested in vitro human skin model test for skin irritation and skin corrosion testing, with reference to the,

1. ECHA position paper in on skin irritation/corrosion (ECHA, 2014), in which it is stated that “It is important to note, that it is the responsibility of the Registrant to ensure that the chosen test method is suitable for the substance in order to obtain adequate information from the in vitro studies.”


2. the information on an increased number of false positive findings using metal substances (Cobalt, borate neodecanoate complexes, Cobalt di(acetate) and Cobalt dichloride) in the human skin model test (supporting information attached to the waiving statement for in vitro skin irritation in section 7.3.1 of the IUCLID file).

Not to cause unnecessary suffering in experimental animals by exposing them to corrosive substances, an alternative testing strategy to assess the skin corrosive properties for calcium 3,5,5-trimethylhexanoate was developed. The alternative testing strategy was tailored to the properties of calcium 3,5,5-trimethylhexanoate, comprising the following steps:

 

1. Collect information on pH shifts during water solubility testing (according to OECD 105), a pH value of <3 or >11.5 is indicative for skin corrosive properties. 


2. Assess the potential of oxidising properties of the anion or the cation individually. Agents with oxidising properties are likely to react with biological membranes and exert irritating or corrosive properties.


3. Assess corrosive effects on the bovine cornea via BCOP test (according to OECD 437). 


4. In case pH shifts, oxidising properties or the BCOP test results indicate a potential for skin corrosive/irritating properties, tiered testing should be performed:
   a)    conduct a membrane barrier test (according to OECD 435), in case negative
   b)   conduct an in vivo skin irritation test
   


5. In case pH shifts, oxidising properties or the BCOP test results indicate a lack of skin corrosive/irritating properties, in vivo skin irritation testing (according to OECD 404) should be performed.

 

The in vivo skin irritation testing does not breach the last resort principle, since the data requirements of regulation (EC) 1907/2006 at the time of dossier preparation (before 1st June 2013) foresaw the conduct of an in vivo skin irritation study (Annex VIII, Section 8.1.1 Column 1). Since the substance calcium 3,5,5-trimethylhexanoate had to fulfil the data requirements inter alia laid down in Annex VIII, the conduct of an in vivo skin irritation study was statutory.

 

Further details on the testing strategy for skin irritation/corrosion testing and the findings with calcium 3,5,5-trimethylhexanoate are presented in the report in attached to the waiving statement for in vitro skin irritation in section 7.3.1 of the IUCLID file.

 

 

Eye irritation

One reliable in vivo study described by Pooles (2013) (OECD 405; GLP compliant) is considered to be reliable without restrictions. The substance was determined to be irritating to the eyes.

Furthermore, a reliable in vitro study described by Heppenheimer (2013) (OECD 437; GLP compliant) is considered to be reliable without restrictions. The substance was determined to be neither corrosive nor severely irritating to the eye.

Justification for classification or non-classification

Skin irritation

Reference Hansen (2013) is considered as the key study for in vivo skin irritation and will be used for classification. The skin irritation was scored according to the Draize scale. The mean score (24, 48, 72 h) for erythema and oedema for all three animals were as follows:

 

Erythema: 0 for all animals

Oedema: 0 for all animals

 

Thus, according to Regulation (EC) 1272/2008 and subsequent amendments the substance will not be classified as irritating to the skin. Furthermore, the substance has not to be classified as skin irritant according to Directive 67/548 EC.

 

Eye irritation

Reference Pooles (2013) is considered as the key study for in vivo eye irritation and will be used for classification. During the study the test item was applied to one eye of three animals each and the eye irritation was scored according to the Draize scale. The following mean scores (24, 48 and 72 hours) were obtained for the three animals:

 

cornea: 1, 0 and 0

iris: 1, 0 and 0.67

conjunctival redness: 2, 1 and 2

chemosis: 1.67, 0.67 and 1.67

 

All effects were reversible within 14 days. Thus, according to Regulation (EC) 1272/2008 and subsequent amendments the substance will be classified as Category 2. Furthermore, the substance does not have to be classified as irritating to the eyes according to Directive 67/548 EC.