Magnesium neodecanoate

Endpoint summary

Description of key information

Eye irritation: corrosive/severe irritant to the eyes (OECD 437, GLP)

Skin irritation: irritating to the skin (OECD 404, 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:

2018-02-05 to 2018-03-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2015-07-28

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-06-05

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry; < 30 °C

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, 97633 Sulzfeld, Germany
- Age at study initiation: approx 41 and 43 weeks old
- Weight at study initiation: 3.9 – 4.3 kg
- Housing: individually housed in ABS-plastic or Noryl rabbit cages, floor 4200 cm2
- Diet (ad libitum): autoclaved hay and Altromin 2123 maintenance diet for rabbits, rich in crude fibre
- Water (ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 ± 3 °C
- Relative humidity: 55 ± 10 %
- Air changes: at least 10 x / hour
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g of the test item
The test item was applied directly without moistening, due to the sticky and hydrophobic nature of the test item. The test item was evely distributed on the skin and a good test item contact with the skin was given.

Duration of treatment / exposure:

initial animal: 3 minutes, 1 hour and 4 hours
confirmatory animal: 4 hours

Observation period:

Initial animal: 14 days
Confirmatory animal: 21 days

Number of animals:

2 male rabbits

Details on study design:

TEST SITE
- Area of exposure/Type of wrap if used: approx. 24 hours before the test, the fur was removed from the dorsal area of the trunk by using an electric clipper. The test item was applied to a small area (approx. 6 cm²) of skin on one side of the dorsal area and covered with a gauze patch, which was held in place with a non-irritating tape. The untreated other side served as control. The test item was applied to the patch first and then applied to the skin. The patch was fixed with a semi-occlusive dressing. The limits of the application site were marked with an ink marker.

INITIAL AND CONFIRMATORY TESTING
Initially, a single animal test was employed. Up to three test patches were applied sequentially to the animal. The first patch was removed after three minutes. No serious skin reaction was observed, so a second patch was applied at a different site and removed after one hour. The observations at this stage indicated that exposure can humanely be allowed to extend to four hours, so a third patch was applied and removed after four hours, and the response was graded. No corrosive effect was observed after the last patch was removed, so the animal was observed for 14 days.

The results of the initial test indicate the test item is not corrosive to the skin using the procedure described. In order to confirm the irritant response, one additional animal was treated with one patch for an exposure period of 4 hours. According to OECD 404, section 17, treatment of a third animal can be omitted when animal no. 1 and 2 exhibit the same response. Furthermore, according to the classification directives the results of two animals were sufficient for classification of the test item. Moreover, as the test item showed no signs of corrosion in two animals, it is considered that adding a third animal would not change the outcome of the study

REMOVAL OF TEST SUBSTANCE
- Washing: at the end of the exposure period, the residual test item was not removed.

OBSERVATION TIME POINTS
- initial animal: immediatley and 1 hour, 24, 48 and 72 hours after patch removal
- confirmatory animal: 1 hour, 24, 48 and 72 hours after patch removal

SCORING SYSTEM: according to the Draize scale
In addition, all local effects such as hyperplasia, scaling, discolouration, fissures and scabs were also recorded.

FURTHER OBSERVATIONS
- body weights: prior to the administration and at the end of the observation period
- any systemic effects were also recorded.

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

- initial animal: the examination of the test site immediately and 1 hour after the patch removal revealed slight erythema (grade 1). Furthermore, moderate erythema (grade 2) was observed 24, 48 and 72 hours and up to day 7 after patch removal as well as on days 12 to 14 after patch removal. Slight erythema (grade 1) was observed on day 8 to day 11 after patch removal. Lastly, slight oedema (grade 1) was observed on 24 and 48 hours after patch removal.
No adverse changes apart from the reactions described above were observed at the skin sites.

- confirmatory animal: the examination of the test site 1 hour after the patch removal revealed slight erythema (grade 1). Furthermore, moderate erythema (grade 2) was observed 24, 48 and 72 hours and up to day 4 after patch removal. Lastly, slight erythema (grade 1) was observed on day 5 to day 21 after patch removal.
The animal showed scaling at the application site from day 9 to day 15.

Other effects:

- body weight: there were no significant body weight changes during the observation period
- clinical signs: no mortality was observed.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test item is irritating to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent amendments and corrections, the test item is irritating to the skin (Category 2; H315).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

2017-08-09

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)

Version / remarks:

2013-07-26

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-06-05

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry; < 30 °C

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: abattoir A. Moksel AG, Buchloe, Germany
- Characteristics of donor animals: cattle was between 16 and 60 months.
- Storage, temperature and transport conditions of ocular tissue: fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories
- Time interval prior to initiating testing: immediately after arrival of the eyes, cornea preparation was initiated and was used for BCOP testing on the same day.

Vehicle:

physiological saline

Remarks:

0.9 % NaCl

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL of the test substance mixture
- Concentration (if solution): 10 % w/v concentration
The test item was mixed with the vehicle by using ultrasonic technique. The sonicated suspension was incubated at 32 °C for 1 hour.
Prior to application, the mixture was vortexed with an ultraturrax for 2 - 3 minutes to achieve a better saturation.

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

Number of bovine corneae per dose:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- eyes were examined for defects and any defective eyes were discarded. Eyes with scratches or any kind of opacity were not used.
- tissue surrounding the eyeball was pulled away and the cornea was excised.
- isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (BASF, Duratec) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with pre-warmed RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI).
- corneas were incubated for one hour at 32 ± 1 °C for equilibration in an air incubator.

QUALITY CHECK OF THE ISOLATED CORNEAS
- after the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI.
- an initial measurement was performed on each of the corneas using the opacitometer.
- three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas.
- the illuminance of each cornea was read and recorded.
- only corneas that had an initial illuminance reading I > I0/1.1651 lux (an equivalent to the opacity threshold of 7 as listed in OECD 437) were used for the assay.

APPLICATION DOSE AND EXPOSURE TIME
- medium was removed from the anterior chamber and replaced with the test item or control.
- 750 μL of the test substance mixture was applied directly onto the cornea by removing the window-locking ring and glass window prior to treatment.
- 750 µL of the control substance was introduced into the anterior chamber (closed-chamber method).
- after 10 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed.

REMOVAL OF TEST SUBSTANCE/CONTROL SUBSTANCES
- epithelium was washed at least three times with MEM (containing phenol red).
- once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red).

METHODS FOR MEASURED ENDPOINTS:
- anterior chamber was refilled with complete RPMI and an illuminance measurement was performed after 2 hours incubation at 32 ± 1 °C.
- each cornea was observed visually and pertinent observations were recorded.
- corneas were visually examined for tissue peeling, residual test chemical and non-uniform opacity patterns and observation were recorded.
- after the illuminance measurement was performed, the medium was removed from both chambers of the holder.
- posterior chamber was refilled with fresh complete RPMI.
- 1 mL of a 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C.
- then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

Evaluation of the opacity:
- the following formula was used to calculate the opacity, whereas the values a and b are equipment-specific variables empirically determined by the manufacturer:
Opacity = ((I0/I) - b)/ a
with a = 0.025 and b = 0.9894
- value I0 is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is reevaluated annually. This I0 value is than calculated to the respective data of the opacitometer and the data according to guideline (opacity < 7). So the initial illuminance can be calculated and corneas below this value were discarded.
- change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.

Evaluation of the permeability:
- mean OD490 for the blank cuvettes was calculated.
- mean blank OD490 was subtracted from the OD490 of each cuvette (corrected OD490).
- any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor.
- final-corrected OD490 of the test article and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490
- mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula was used to determine the in vitro irritation score (IVIS):
IVIS = mean opacity value + (15 x mean permeability OD490 value)
For the IVIS cut-off values for identifying test substances as inducing serious eye damage and test substances not including eye irritation or serious eye damage please refer to table 1 in the field "Any other information onmaterial and methods incl. tables" below.

ACCEPTABILITY CRITERIA
- the BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.
- the negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Irritation parameter:

in vitro irritation score

Remarks:

(mean)

Value:

84.92

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

All 3 corneas treated with magnesium neodecanoate showed severe opacity of the tissue.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.
- Acceptance criteria met for positive control: the in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

The evaluation of acceptability criteria was performed by using the following historical data:
- for evaluation of the validity of the positive control, the historical mean IVIS score as obtained from 2015 until 2017 was used (please refer to the tables in the field "Any other information on results incl. tables").
- for the evaluation of the validity of the negative control, the historical upper limits of the opacity and permeability values as obtained in 2017 were used (please refer to the tables in the field "Any other information on results incl. tables" below)

Please also refer for results to the field "Any other information on results incl. tables" below

Table 1: Opacity

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1	Negative Control	1.65	1.08	-0.57
2		1.65	1.54	-0.11
3		1.58	1.04	-0.53
MV		1.63	1.22	-0.40
4	Positive Control	3.11	40.60	37.49	37.90
5		2.88	30.21	27.34	27.74
6		3.03	33.25	30.22	30.62
MV		3.01	34.69	31.68	32.09
7	Test Item	1.26	31.06	29.80	30.20
8		-0.21	29.70	29.91	30.32
9		0.97	31.70	30.73	31.13
MV		0.67	30.82	30.15	30.55

MV = mean value

Table 2: Permeability

Cornea No.	Test Item	OD490	Corrected OD490 Value
1	Negative Control	0.009
2		0.013
3		0.004
MV		0.009
4	Positive Control	1.013	1.004
5		1.147	1.138
6		2.120	2.111
MV		1.427	1.418
7	Test Item	2.985	2.976
8		3.175	3.166
9		4.740	4.731
MV		3.633	3.625

MV = mean value

Table 3: In vitro irritation score

Cornea No.	Test Item	Corrected Opacity	Corrected OD490 Value	IVIS
1	Negative Control	-0.57	0.009
2		-0.11	0.013
3		-0.53	0.004
MV		-0.40	0.009	-0.27
4	Positive Control	37.90	1.004
5		27.74	1.138
6		30.62	2.111
MV		32.09	1.418	53.36
7	Test Item	30.20	2.976
8		30.32	3.166
9		31.13	4.731
MV		30.55	3.625	84.92

MV = mean value

Table 4: Historical mean in vitro irritation score of the positive control from February 2015 until August 2017

	IVIS Positive Control – Ethanol 100%
Mean Value (MV)	48.38
Standard Deviation (SD)	9.98
MV-2xSD	28.41
MV+2xSD	68.34
Number of Replicates providing Historical Mean: 44

Positive controls are updated after every single experiment or at least every 3 months

Table 5: Historical data on opacity and permeability of the positive control (Ethanol 100 %) from August 2017 until October 2017

Incubation: 10 min
Number of Replicates providing Historical Mean	Cornea No.	Opacity		Permeability		IVIS
		Change of Opacity Value	Corrected Opacity Value	OD490 Value	Corrected OD490 Value
1	4	37.495	37.899	1.013	1.004
	5	27.335	27.739	1.147	1.138	53.36
	6	30.218	30.622	2.120	2.111
2	4	24.492	23.921	1.442	1.436
	5	31.754	31.182	1.108	1.102	49.70
	6	30.259	29.688	1.755	1.749
Mean Value (MV)		30.259	30.175	1.431	1.424	51.530
Standard Deviation (SD)		4.391	4.608	0.433	0.434	2.588
MV-2xSD		21.477	20.960	0.564	0.557	46.354
MV+2xSD		39.040	39.391	2.298	2.291	56.706

Table 6: Historical mean in vitro irritation score of the negative control from February 2015 until September 2017

	IVIS Negative Control – NaCl 0.9 %
Mean Value (MV)	0.81
Standard Deviation (SD)	0.66
MV-2xSD	-0.50
MV+2xSD	2.12
Number of Replicates providing Historical Mean: 44

Negative controls are updated every single experiment or at least every 3 months

Table 7: Historical data on opacity and permeability of the negative control (NaCl 0.9 %) from August 2017 until October 2017

Number of Replicates providing Historical Mean	Cornea No.	Opacity	Permeability	IVIS
		Change of Opacity Value	OD490 Value
1	1	0.234	0.008	1.49
	2	1.738	0.008
	3	0.800	0.098
2	1	0.978	0.019	2.52
	2	3.920	0.022
	3	1.617	0.028
3	1	-0.149	0.009	-0.50
	2	-0.415	0.015
	3	-1.427	0.009
1	1	-0.57	0.009	-0.27
	2	-0.11	0.013
	3	-0.53	0.004
2	1	-0.25	0.004	0.66
	2	0.80	0.005
	3	1.17	0.008
Mean Value (MV)		0.520	0.017	0.780
Standard Deviation (SD)		1.296	0.023	1.254
MV-2xSD		-2.072	-0.029	-1.727
MV+2xSD		3.112	0.064	3.287

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

According to the bovine corneal opacity and permeability assay, since the IVIS was > 55 (IVIS score: 84.92), the test item magnesium neodecanoate can be considered as requiring classification for eye irritation or serious eye damage.
According to the Regulation (EC) No 1272/2008 and subsequent adaptions, the substance is classified for serious eye damage (Category 1; H318).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Eye irritation:

The substance was observed to be corrosive/severe irritanting to the eyes in a reliable in vitro eye irritation study according to OECD 437.

Skin irritation:

The substance was observed to be either corrosive or irritating to the skin in a reliable in vitro skin irritation/corrosion study according to OECD 439. As the results of an OECD 439 test are not suitable to differentiate between skin categories 1 and 2, the substance was tested in an in vitro skin corrosion test.

Magnesium neodecanoate was tested in an in vitro skin corrosion test according to OECD 435, but the substance was incompatible with the CORROSITEX™ Assay, as assessed in the qualification step. Consequently, the study cannot be used for classification.

Finally, the substance was observed to be irritating to the skin in a reliable in vivo acute dermal irritation/corrosion study according to OECD 404.

Justification for classification or non-classification

Skin irritation:

Magnesium neodecanoate induced serious reversible skin irritation in an in vivo test according to OECD Guideline 404 and is therefore classified as skin irritant category 2 with labelling H315: Causes skin irritation in accordance with Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation:

Magnesium neodecanoate induced a mean in vitro irritation score (IVIS) > 55 in a bovine corneal opacity and permeability assay according to OECD Guideline 437 indicating on irreversible effects on the eye and is therefore classified in category 1 with labelling H318: Causes serious eye damage in accordance with Regulation (EC) No 1272/2008 and its subsequent adaptations.

Respiratory irritation:

Magnesium neodecanoate is not classified for respiratory irritation (STOT SE category 3) because of lacking data.