Neodecanoic acid, iron salt

Endpoint summary

Description of key information

The test article was considered to be negative in the ARE-Nrf2 Luciferase Test (OECD 442D), but was considered to be positive in the test "In Vitro Skin Sensitisation (human Cell Line Activation Test)" (OECD 442E). However, due to concern of DMF activity causing an anomalous increase in fluorescence, a repeat experiment was conducted with isopropanol as the vehicle. An in vivo sensitisation assay was conducted to confirm the skin sensitisation potential of the test item due to the conflicting in vitro h-CLAT results. The guinea pig Buehler test (OECD 406) was selected as the Local Lymph Node Assay is not suitable for metal salts.

No skin reactions were seen for any of the animals after completion of the induction exposure and after the challenge exposure. Based on these results, the test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-09-05 to 2017-11-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

adopted February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The data will be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Specific details on test material used for the study:

- Storage: 2 to 8°C, protected from light, under nitrogen
- Batch Number: Ln11013894

Details on the study design:

The study was conducted to investigate the potential of the test item to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
The ARE-Nrf2 luciferase test method utilises an immortalised adherent cell line derived from HaCaT human keratinocytes. The cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 promoter fused with the ARE from a gene known to be up-regulated by contact sensitisers.
The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic substances.

Positive control results:

The EC1.5 values for the positive control were 20.81 and 9.54 µM in Repetitions 1 and 2, respectively. The average induction in the two replicates for the positive control at 64 µM was 3.09 and 6.40 in Repetitions 1 and 2, respectivley.

Key result

Parameter:

other: The four conditions specified in the prediction model were not met in either repetition. The test article was therefore considered to be negative in the ARE-Nrf2 Luciferase Test.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The four conditions specified in the prediction model were not met in either repetition. The test article was therefore considered to be negative in the ARE-Nrf2 Luciferase Test.

Calculation of Imax and EC1.5 Values

Luminescence readings and fold increases are given in the attached Table 10.1 and Table 10.2.

The maximal fold increases (Imax) were 0.94 and 3.79 for Repetitions 1 and 2, respectively.

There was no EC1.5 value for Repetition 1 as there were no statistically significant increases in induction.

The EC1.5 value for Repetition 2 was 0.78 mM.

Viability

MTT-absorbance readings are given in the attached Table 10.3 and Table 10.4.

This measurement was not applicable for Repetition 1 as there was no EC1.5 determining concentration.

Cell viability was 21.51% at the EC1.5 determining concentration in Repetition 2.

In Repetition 1, there was no apparent overall dose response for lucipherase and the dose response curve was not biphasic.

In Repetition 2 the dose response curve for luciferase induction was biphasic.

Assay Acceptance

Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 32 and 64 µM in Repetition 1 and at concentrations of 16 to 64 µM in Repetition 2.

The EC1.5 values for the positive control were 20.81 and 9.54 µM in Repetitions 1 and 2, respectively. The average induction in the two replicates for the positive control at 64 µM was 3.09 and 6.40 in Repetitions 1 and 2, respectivley.

The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 6.56% and 11.62% in repetitions 1 and 2, respectively.

Interpretation of results:

GHS criteria not met

Conclusions:

The four conditions specified in the prediction model were not met in either repetition. The test article, was therefore considered to be negative in the ARE-Nrf2 Luciferase Test.

Executive summary:

In a dermal sensitisation study conducted according to OECD 442D, the test article was dissolved in isopropanol to the final concentration (200 mM). Serial dilutions were then made using isopropanol to obtain 12 master concentrations of the test article (0.098, 0.196, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50, 100 and 200 mM). The master concentrations were then further diluted 25 fold into culture medium containing serum and finally used for treatment with a further 4 fold dilution factor so that the final concentrations ranged from 0.98 to 2000 µM.

Aliquots of 50 µL of each of the final concentrations were transferred to each of three luciferase plates and a single viability plate. Each plate was sealed using a plate sealer and then incubated at 37±1 °C, 5% (v/v) CO2 in air, in a humidified environment for 48±1 hours.

After the 48-hour exposure period, the medium in the luciferase plates was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The plate was sealed and incubated for 4 hours at 37 ± 1 °C, 5% (v/v) CO2. The MTT medium was then removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator at 37 ± 1 °C, 5% (v/v) CO2 in air and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e.

After the 48-hour exposure period, the cells in the viability plate were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 to 22 minutes at 25 ± 2 °C, loaded into the luminescence plate reader and read.

The results are summarised as follows:

Condition	Rep 1	Rep 2
IMAX (fold increase)	0.94	3.79
Cell Viability at the EC1.5 (%)	N/A	21.51
EC1.5 (mg/mL)	N/A	0.78
Dose response for luciferase Induction	No	Yes

The four conditions specified in the prediction model were not met in either repetition. The test article, was therefore considered to be negative in the ARE-Nrf2 Luciferase Test.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-10-02 to 2018-03-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

The data will be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Specific details on test material used for the study:

Storage: 2 to 8°C under nitrogen, protected from light.
Batch Number: LN11013894
Retest Date: 31 July 2019
Purity: 100%

Details on the study design:

The study was conducted to investigate the potential of test item to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling. The human Cell Line Activation Test (h-CLAT) has been evaluated in a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)-coordinated validation study and subsequent independent peer review by the EURL ECVAM Scientific Advisory Committee (ESAC) and has been recommended to be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Positive control results:

In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI =150% and CD54 RFI =200%) and cell viability should be more than 50%.

Run / experiment:

other: CD54

Parameter:

other: %

Remarks:

DMF Vehicle

Value:

200

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: CD86

Parameter:

other: %

Remarks:

DMF vehicle

Value:

150

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: CD54

Parameter:

other: %

Remarks:

Isopropanol vehicle

Value:

200

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: CD86

Parameter:

other: %

Remarks:

Isopropanol vehicle

Value:

150

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The significance of these results is unclear because of the concern about the potential for activity of DMF itself causing an anomalous increase in fluorescence.

Dose Finding Assays

The cell viability results are given in the folowing tables:

Table 6.1 Dose Finding Assay (DMF): Cell Viability

	Viability (%) at Concentration (µg/mL)
Run	7.8	15.6	31.3	62.5	125.0	250
1	99.1	99.0	99.3	99.0	98.6	98.0
2	99.0	99.2	99.1	99.0	98.5	98.1

 

Table 6.2       Dose Finding Assay (Isopropanol): Cell Viability

	Viability (%) at Concentration (µg/mL)
Run	1.6	3.1	6.3	12.5	25.0	50.0	100.0	200.0
1	98.8	99.1	99.1	99.1	99.2	99.2	99.2	99.0
2	99.1	99.1	99.4	99.1	98.7	99.0	98.6	98.3

For treatments with DMF, precipitate was observed during the serial dilution steps at concentrations of 500 and 1000 µg/mL, therefore the maximum attainable concentration in DMF was 250 µg/mL.

The highest four concentrations using isopropanol (25 – 200 µg/mL) were noted to have formed a hazy, but stable suspension. The maximum attainable concentration in ispropanol was therefore 200 µg/mL.

No CV75 value was calculated for treatments with either DMF or ispropanol as the vehicle, as there was no effect on viability.

CD86/CD54 Expression Results with DMF

The data for Experiment 1 treatments with DMF as vehicle were obtained in a repeat experiment, as the RFI values for the vehicle control in the initial experiment did not meet the acceptance criteria criteria (data not reported).

The relative fluorescence intensity (RFI) values for the test article in DMF were calculated as follows:

Concentration (µg/mL)	RFI (CD86) Exp 1	Exp 2	RFI (CD54) Exp 1	Exp 2
69.7	89	167	126	129
83.7	110	169	142	136
100.4	116	192	147	152
120.5	127	187	155	158
144.6	152	190	162	141
173.6	162	149	151	165
208.3	158	193	154	155
25.0	163	165	148	150
Solvent cotrol	134	145	151	164
Positive control	306	217	860	684

No RFI values for CD54 were greater than 200 therefore the EC200 could not be calculated. The EC150 for CD86 calculated by linear regression was 142.4 µg/mL in Experiment 1. The EC150 value calculated by log-linear extrapolation in Experiment 2 was 22.5 µg/mL.

CD86/CD54 Expression Results with isopropanol

The relative fluorescence intensity (RFI) values for the test article in isopropanol were calculated as follows:

Concentration (µg/mL)	RFI (CD86) Exp 1	Exp 2	RFI (CD54) Exp 1	Exp 2
55.8	122	123	175	175
67.0	129	123	185	181
80.4	137	147	181	205
96.5	182	173	150	225
115.7	197	173	244	232
138.9	197	189	224	280
166.7	216	195	271	332
200.0	222	186	271	284
Solvent cotrol	93	117	74	93
Positive control	353	365	512	284

 

The EC150 values for the two runs were 84.97 and 82.14 µg/mL for CD86 and the EC200 values for the two runs were 106.74 and 77.70 µg/mL for CD54.

In accordance with the OECD Test Guideline, when only two runs have been conducted the highest EC150 or EC200 value is adopted.

Assay Acceptance Criteria Results

All assay acceptance criteria were met.

The cell viabilities of medium and solvent control were higher than 90% in each independant run.

In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI =150% and CD54 RFI =200%).

For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control was >105% on all occasions.

For the positive control, RFI values were =150%forCD86 and =200% for CD54 in all independent runs. Cell viability was >50% in each independent run.

For the test article, the cell viability was more than 50% in all tested concentrations in each independent run.

Interpretation of results:

study cannot be used for classification

Conclusions:

The RFI values for CD54 were <200% in both independent runs using DMF as the vehicle. However, the RFI values for CD86 were >150%. The test article was therefore considered to be postitive in the human Cell Line Activation Test using DMF as the vehicle. However, due to concern of DMF activity causing an anomalous increase in fluorescence, a repeat experiment was conducted with isopropanol as the vehicle.
The RFI values for CD54 were >200% in both independent runs and the RFI values for CD86 were >150% using isopropanol as the vehicle. The test article was therefore considered to be positive in the human Cell Line Activation Test using ispropanol as the vehicle. In conclusion, the test item induced the expression of CD86 and CD54 cell surface markers when isopropanol was used as the vehicle and was considered to be positive in the human Cell Line Activation Test. An induction of the CD86 cell surface marker was also observed when using DMF as the vehicle, but the significance of these results is unclear because of the concern about the potential for activity of DMF itself.

Executive summary:

The study was conducted according to OECD 442E to investigate the potential of test item to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

The test article was initially dissolved in dimethylformamide (DMF) and a dose finding assay was conducted to determine a concentration showing 75% THP-1 cell survival (CV75). No CV75 value was calculated as there was no effect on viability at the maximum attainable concentration in DMF (250 µg/mL).

Eight stock solutions were prepared by 1.2-fold serial dilutions using DMF to give eight doses ranging from 69.7 to 250 µg/mL. These stock solutions were then diluted 250-fold into the culture medium (working solutions).

Aliquots of 500 µL of each of the working solutions were mixed 1:1 with cell suspensions at 1 x 106 cells per well. Each plate was sealed using a plate sealer and then incubated at 37 ± 1 °C, 5% (v/v) CO2 in air, in a humidified environment for 24±0.5 hours.

After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation, washed with FACS buffer and then blocked with 600 µL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) at 4 °C for 15 minutes.

After blocking, the cells were split into three aliquots of 180 µL into a 96-well plate, centrifuged and then stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies at 4 °C for 30 minutes.

The stained cells were washed with FACS buffer and re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

The RFI values for CD54 were <200% in both independent runs. However, the RFI values for CD86 were >150%. The test article was therefore considered to be positive in the human Cell Line Activation Test using DMF as the vehicle. No RFI values for CD54 were greater than 200 therefore the EC200 could not be calculated. The EC150 for CD86 calculated by linear regression was 142.4 µg/mL in Experiment 1. The EC150 value calculated by log-linear extrapolation in Experiment 2 was 22.5 µg/mL.

Subsequently, it was discovered that there is published data indicating that DMF may activate the CD86 receptor. Thus, there was some concern that DMF caused an anomalous increase in fluorescence when exposed to the test article in the presence of DMF as the vehicle, therefore, a repeat experiment was performed using isopropanol as the vehicle. Isopropanol is a known non-sensitizer and has been used in-house in human Cell Line Activation Tests with no impact on the performance or reliability of results and was therefore considered to be an appropriate solvent for testing in this assay type.

A dose finding assay was conducted using isopropanol to determine the CV75. No CV75 value was calculated as there was no effect on viability at the maximum attainable concentration of the test article in isopropanol (200 µg/mL).

Eight stock solutions were prepared by 1.2-fold serial dilutions using isopropanol to give eight doses ranging from 55.82 to 200 µg/mL. These stock solutions were then diluted 250-fold into the culture medium (working solutions). CD86/CD54 expression measurements and assessment of cell viability were then performed as described above for treatments with DMF.

The RFI values for CD54 were >200% in both independent runs and the RFI values for CD86 were >150%. The test article was therefore considered to be positive in the human Cell Line Activation Test using ispropanol as the vehicle. The EC150 for CD86 and the EC200 for CD54 were calculated to be 84.97 µg/mL and 106.7 µg/mL, respectively.

In conclusion, the test article, test item, induced the expression of CD86 and CD54 cell surface markers when isopropanol was used as the vehicle and was considered to be positive in the human cell line activation test. an induction of the CD86 cell surface marker was also observed when using DMF as the vehicle, but the significance of these results is unclear because of the concern about the potential for activity of DMF itself.

Reference 3

Endpoint:

skin sensitisation: in vitro

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-05-16 to 2018-09-12

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

The guinea-pig Bueher test was selected as the OECD guideline for skin sensitisation suggests that the Local Lymph Node Assay is not suitable for metal salts.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: E01133-065
- Expiration date of the lot/batch: 31 July 2019
- Purity: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator container flushed with nitrogen

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L'Arbresle, France; Charles River Deutschland, Kisslegg, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 4-7 weeks old
- Weight at study initiation: 200-550 g
- Housing: Noryl cages containing sterilized sawdust as bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70%
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12 hours light/ 12 hours dark
- IN-LIFE DATES: From: 5th June 2018 To: 27 July 2018

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Concentration / amount:

100% (0.5 mL)

Day(s)/duration:

Induction: Days 1, 8 and 15 for 6 hours exposure

Adequacy of induction:

highest technically applicable concentration used

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Concentration / amount:

100% (0.1 mL)

Day(s)/duration:

Day 29-31; after 6 hours the dressing was removed and challenge reactions were assessed 24 and 48 hours after removal of the last dressing

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Experimental group: 20 females
Control group: 10 females

Details on study design:

Preliminary Irritation study:
A preliminary irritation study was conducted in order to select test item concentrations to be used in the main study. The selection of concentrations was based on the following criteria:
- The concentrations are well-tolerated systemically by the animals.
- For the induction exposures: the highest possible concentration that produced mild irritation (grade 2).
- For challenge exposure: the maximum non-irritant concentration.
Series of test item concentrations were tested. Practical feasibility of administration determined the highest starting-concentration for each route. The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 20%, 10%, 5%, 2%, 1%. The test system and procedures were identical to those used during the main study, unless otherwise specified. The four animals selected were 4 weeks old. No body weights were determined.

Epidermal application:
A series of four test item concentrations (100%, 50%, 20% and 10%) was tested, the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each or an equivalent amount when dosed with a spatula) per animal to the clipped flank, using Metalline patches (2x3 cm) mounted on Medical tape which were held in place with Micropore tape and subsequently Coban elastic
bandage. After 6 hours, the dressing was removed and the skin cleaned of residual test item using corn
oil. The treated skin areas were assessed for irritation 24 and 48 hours after removal of the dressings.

Main study;
The concentrations and induction method were selected based on the results of the preliminary irritation study.
INDUCTION - Experimental animals
Days 1, 8 and 15:
The left side of the scapular region was clipped and epidermally treated with an amount equivalent to 0.5 mL (dosed with a spatula) of the undiluted test item, using a Metalline patch (2x3 cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage. After 6 hours, the dressings were removed and the skin cleaned of residual test item using water. Immediately after removal of the last induction application on Day 15, the treated area was assessed for irritation.
INDUCTION - Control animals
The control animals were treated as described for the experimental animals, except that, instead of the test item, water (Elix) was administered, since a 100% concentration has been selected for both the induction and challenge phase.
CHALLENGE - All animals
Day 29-31:
Epidermal application of the undiluted test item and water (Elix) (0.1 mL each, equivalent amount when dosed with spatula) to the clipped, right flank of all animals, using Patch Test Plasters (Curatest®, Lohmann, Almere, The Netherlands). The patches were held in place with Micropore tape and subsequently Coban elastic bandage. After 6 hours, the dressings were removed and the skin cleaned of residual test item using water. The treated sites were assessed for challenge reactions 24 hours and 48 hours after removal of the last dressing.

Challenge controls:

water (Elix)

Positive control substance(s):

yes

Remarks:

A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques. Female Dunkin Hartley guinea pigs were checked for the sensitivity to Alpha- Hexylcinnamaldehyde.

Positive control results:

A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by Charles River Den Bosch. In this study, performed in Dec 2017, females of the Dunkin Hartley guinea pig (from Charles River France, L’Arbresle, France) were checked for the sensitivity to Alpha- Hexylcinnamaldehyde, technical grade. The females were approximately 4 weeks old at commencement of the study. The study was based on the OECD Guideline No. 406, EC No 440/2008 Part B.6 and on the method described in "Allergic Contact Dermatitis in the Guinea-Pig: Identification of Contact Allergens" Magnusson and Kligman, 1970. Alpha- Hexylcinnamaldehyde, technical grade (CAS no. 101-86-0) was fabricated under lot no. MKBX3483V (Sigma- Aldrich, Steinheim, Germany).

Skin Reactions In The Challenge Phase (Number of animals with positive skin reactions)
Vehicle Alpha- Hexylcinnamaldehyde, technical grade
Corn oil 50%
24/48* 24/48*
Experimental group, 10 females
Score 2 0/0 8/0
Score 1 0/0 2/2
Score 0 10/10 0/8
Control group, 5 females
Score 1 0/0 5/0
Score 0 5/5 0/5
*. time (hours) after the challenge exposure.

The skin reactions observed in eight experimental animals in response to the 50% test item concentration in the challenge phase were considered indicative of sensitisation, based on the absence of any response of a similar grade in the control animals. These results lead to a sensitisation rate of 80 percent to the 50% concentration.
From these results, it was concluded that the female guinea pig of the Dunkin Hartley strain is an appropriate animal model for the performance of studies designed to evaluate the sensitizing potential of a test item in a Buehler type of test.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No skin reactions

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reactions

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No skin reactions

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reactions

Remarks on result:

no indication of skin sensitisation

Reading:

other: Challenge phase

Hours after challenge:

24

Group:

positive control

Dose level:

50%

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Remarks:

See the positive control results description for more information

Table 1          Preliminary Irritation Study -Skin reactions after epidermal exposure

Animal Number	Test item Concentration %	24 Hours after exposure		48 Hours after exposure
		Erythema	Oedema	Erythema	Oedema
1	100	0t	0t	0t	0t
	50	0t	0t	0t	0t
2	100	0t	0t	0t	0t
	50	0t	0t	0t	0t
3	20	0	0	0	0
	10	0	0	0	0
4	20	0	0	0	0
	10	0	0	0	0

t. Brown staining of the skin due to test item remnants, which did not impair scoring for irritation.

 

Grading erythema:

0 = No erythema

Table 2          Induction and Challenge Readings - Control Animals

Animal number	Induction		Challenge				Comments
	Day 15		Day 30		Day 31
	Erythema	Oedema	Test item concentration 100%	Vehicle	Test item concentration 100%	Vehicle
Control
6	0	0	0t	0	0t	0
7	0	0	0t	0	0t	0
8	0	0	0t	0	0t	0
9	0	0	0t	0	0t	0
10	0	0	0t	0	0t	0
11	0	0	0t	0	0t	0
12	0	0	0t	0	0t	0
13	0	0	0t	0	0t	0
14	0	0	0t	0	0t	0
15	0	0	0t	0	0t	0

t. Brown staining of the skin due to test item remnants, which did not impair scoring for irritation.

 

Grading induction reactions:

Erythema:

0 = No erythema

Oedema:

0 = No oedema

 

Grading challenge reactions:

0 = No visible change

Table 3          Induction and Challenge Readings – Experimental Animals

Animal number	Induction		Challenge				Comments
	Day 15		Day 30		Day 31
	Erythema	Oedema	Test item concentration 100%	Vehicle	Test item concentration 100%	Vehicle
Experimental
16	0	0	0t	0	0t	0	not sensitized
17	0	0	0t	0	0t	0	not sensitized
18	0	0	0t	0	0t	0	not sensitized
19	0	0	0t	0	0t	0	not sensitized
20	0	0	0t	0	0t	0	not sensitized
21	0	0	0t	0	0t	0	not sensitized
22	0	0	0t	0	0t	0	not sensitized
23	0	0	0t	0	0t	0	not sensitized
24	0	0	0t	0	0t	0	not sensitized
25	0	0	0t	0	0t	0	not sensitized
26	0	0	0t	0	0t	0	not sensitized
27	0	0	0t	0	0t	0	not sensitized
28	0	0	0t	0	0t	0	not sensitized
29	0	0	0t	0	0t	0	not sensitized
30	0	0	0t	0	0t	0	not sensitized
31	0	0	0t	0	0t	0	not sensitized
32	0	0	0t	0	0t	0	not sensitized
33	0	0	0t	0	0t	0	not sensitized
34	0	0	0t	0	0t	0	not sensitized
35	0	0	0t	0	0t	0	not sensitized

t. Brown staining of the skin due to test item remnants, which did not impair scoring for irritation.

 

Grading induction reactions:

Erythema:

0 = No erythema

Oedema:

0 = No oedema

 

Grading challenge reactions:

0 = No visible change

Grading Oedema:

0 = No oedema

Interpretation of results:

GHS criteria not met

Conclusions:

In a dermal sensitisation test (Buehler test, OECD 406), there was no evidence that the test item had caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in response to a 100% test item concentration in the challenge phase. This result indicates a sensitization rate of 0 per cent.

Executive summary:

A dermal sensitisation study was conducted according to OECD testing guideline 406 to evaluate whether the test item induces contact hypersensitivity in guinea pigs after three epidermal exposures under the conditions described in this report. Test item concentrations selected for the main study were based on the results of a preliminary study. In the main study, twenty experimental animals were epidermally treated on three occasions (Days 1, 8 and 15) with a 100% concentration of the test item and ten control animals were similarly treated, but with the vehicle alone (Elix water). Two weeks after the last induction exposure, all animals were challenged with a 100% concentration and Elix water. No skin reactions were seen for any of the animals after completion of the induction exposure and after the challenge exposure.

The reliability check with Alpha-hexylcinnamaldehyde, performed within the same time frame as this study, showed that the Buehler Assay as performed is an appropriate model for testing for contact hypersensitivity. There was no evidence that the test item had caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in response to a 100% test item concentration in the challenge phase. This result indicates a sensitization rate of 0 per cent.

Based on these results the test item was not considered to be a skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

For the skin sensitisation endpoint, the ECHA preferred method of a battery of three in vitro tests was selected. The three tests investigate three steps in the adverse outcome pathway (AOP) that has been defined for skin sensitisation, i.e., peptide/protein binding, keratinocyte response, and monocytic/dendritic cell response. The standard assays that may be selected to investigate the three steps of the AOP are the Direct Peptide Reactivity Assay (DPRA) (OECD 442C), the KeratinoSensTM (OECD 442D) and the human Cell Line Activation Test (h-CLAT) (OECD 442E), and these were considered for use with the registration substance. The ECHA guidance on information requirements R7a (July 2017) indicates on page 281, that “In case several EU/OECD adopted test methods are available for a key event, the registrant should select the most appropriate test method available for their substance based on the applicability of the test method” and “The test methods have each been validated independently, with a limited scope. This means that each has its limitations and cannot be used as a stand-alone test method; however, the limitations may in many cases be compensated when used together with additional information e.g. by using information from similar substances within a Weight-of-Evidence approach.”

Peptide/Protein Binding

The OECD Guideline 442C states in paragraph 11 that “This Test Guideline is not applicable for the testing of metal compounds since they are known to react with proteins with mechanisms other than covalent binding.” The registration substance is a metal salt and consequently the DPRA assay was excluded from the test battery because the substance is outside of its domain of applicability. There are no other OECD test methods applicable to this key event and so it was not possible to investigate peptide/protein binding.

Keratinocyte Response

The OECD Guideline 442D does not exclude the possibility to test metal salts and consequently this assay was selected for the investigation of the second key event for the skin sensitisation potential of the registration substance; the results are given in the relevant study endpoint summaries of this dossier. Based on the results, the test article was therefore considered to be negative in the ARE-Nrf2 Luciferase Test.

Monocytic/Dendritic Cell response

The OECD Guideline 442E does not exclude the possibility to test metal salts and consequently this assay was selected for the investigation of the third key event for skin sensitisation potential. The study was conducted to investigate the potential of the registration substance to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54).

The h-CLAT study, Covance Study Number 8372237 is summarised in the relevant endpoint summary of this dossier. The standard vehicles for use in this assay are water or DMSO, but neither was found to be suitable for use with the registration substance. However, test substance formulations could be prepared in dimethyl formamide (DMF), and this was selected by the Study Director for use in the study. The results of two experiments showed no change in the expression of cell surface marker CD54, whereas for the cell marker CD86 there were increases in expression above the threshold for a positive response; an EC150 of 142.4 µg/mL and 22.5µg/mL was calculated for Experiment 1 and Experiment 2 respectively. The conclusion of this test was that “The test article was therefore considered to be positive in the human Cell Line Activation Test.” However, it was subsequently discovered, that DMF has not been validated by Covance for this assay and that there was a moderate stimulation of CD86 expression by DMF alone. Furthermore, MB Research Labs have published their vehicle validation data (see references below) and have shown that DMF stimulates CD86 expression, but not CD54 expression. It was concluded that DMF was not a suitable vehicle for use in this assay and there was a strong possibility that the result was a false positive.

It was decided to use isopropanol as the vehicle for a repeat test. Isopropanol has not been formally validated for use in the h-CLAT assay, but it had been used by Covance in previous studies without issue. Using isopropanol, in the expression assays, RFI values for CD86 and CD54 were >150% and >200% respectively in both independent runs. The registration substance was therefore predicted as positive in this assay. The EC150 for CD86 was calculated to be 84.97 µg/mL and the EC200 for CD54 was 106.7 µg/mL. The data of the test using isopropanol did not correlate well with those of the test using DMF because the expression of both cell markers increased whereas it was only CD86 expression that was increased with DMF.

The European Commission Joint Research Centre, ECVAM Scientific Advisory Committee (ESAC) performed a peer review of the transferability and reliability of the human Cell Line Activation Test (h-CLAT) for skin sensitisation testing and adopted their consensus report on the 11 March 2014. They concluded on page 8 “Empirically the applicability domain seems to exclude pre-/pro-haptens, autofluorescent compounds, and chemicals with limited solubility or stability in water, metal salts and volatile compounds.” and “The tested metal salts gave an inconsistent response.”

Other Considerations – Read-Across

The registration substance is the iron salt of neodecanoic acid and in an aqueous environment it disassociates into Fe2+, Fe3+, and neodecanoic acid. These substances have been demonstrated not to be skin sensitisers. Iron, CAS number 7439-89-6, is not classified as a skin sensitiser when tested as various iron oxides in the guinea pig optimisation test (Maurer T., 1979). Neodecanoic acid, CAS number 26896-20-8, is not classified for skin sensitisation and was tested in a guinea pig maximisation test (1997) according to OECD guideline 406.

 

Conclusion

The three-test in vitro battery proposed by ECHA for the evaluation of skin sensitisation potential was applied to the registration substance, however, only one step was adequately concluded. The DPRA test was not performed because the substance is outside the applicability domain of the assay. The KeratinoSensTM assay was performed and demonstrated a clear negative result. The h-CLAT assay was also performed but gave a positive result. The data of the h-CLAT assay are considered to be unreliable predominantly because of the observations of the ESAC demonstrating that the applicability domain for the assay excludes metal salts, but also due to the uncertainty of the impact of the vehicle interfering with the cell marker expression.

The OECD guidance indicates that the limitations of the three assays may in many cases be compensated for when used together with additional information e.g. by using information from similar substances within a Weight-of-Evidence approach. This has been done for the registration substance to some extent in that there is data available indicating that the two components of the salt are not classified for skin sensitisation. Moreover, these dissociated substances were tested in guinea pig assays which evaluate the entire AOP for skin sensitisation, whereas the Local Lymph Node Assay (LLNA) only evaluates the induction phase of the AOP.

The valid data on the substance (negative for keratinocyte response) and the read-across data of the dissociated components of the substance (Iron and neodecanoic acid), when taken together, suggest that the registration substance may not be a skin sensitizer. However, due to the uncertainty of the results, and only one step in the AOP being adequately concluded (Keratinosens), the skin sensitisation potential of the registration substance remains inconclusive.

To establish the sensitisation potential of the registration substance, an in vivo study has been organised. The LLNA is usually the first-choice method for in vivo testing, however, with justification, other tests can be used. The Buehler assay (OECD 406) is the recommended choice for this substance due to the chemistry; it states in the OECD 429 guideline that “Despite the advantages of the LLNA over TG 406, it should be recognised that there are certain limitations that may necessitate the use of TG 406 (13) (e.g. false negative findings in the LLNA with certain metals, false positive findings with certain skin irritants [such as some surfactant type chemicals] (19) (20), or solubility of the test substance). In addition, test substance classes or substances containing functional groups shown to act as potential confounders (21) may necessitate the use of guinea pig tests (i.e. TG 406).” (OECD, 2010). The registration substance encompasses all these limitations - it contains an Iron metal which may cause false negative results, and it is a Category 2 skin irritant with the same chemical properties associated with surfactants which may cause a false positive result. Therefore, it was suggested that a Buehler assay is performed to limit any potential for a false positive and/or false negative result.

 

In vivo Buehler assay

 

The study was carried out in compliance with the guideline OECD 406. In the Main study, twenty experimental animals were epidermally treated on three occasions (Days 1, 8 and 15) with a 100% concentration of the test item and ten control animals were similarly treated, but with the vehicle alone (Elix water). Two weeks after the last induction exposure, all animals were challenged with a 100% concentration and Elix water.

No skin reactions were seen for any of the animals after completion of the induction exposure and after the challenge exposure.

The reliability check with Alpha-hexylcinnamaldehyde, performed within the same time frame as this study, showed that the Buehler Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

There was no evidence that the test item had caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in response to a 100% test item concentration in the challenge phase. This result indicates a sensitization rate of 0 per cent.

Based on these results the test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, no classification for skin sensitisation is warranted in accordance with CLP Regulation 1272/2008.