Isodecyl pivalate

Endpoint summary

Description of key information

Skin sensitisation (OECD 442C and 442E): negative

Skin sensitisation (OECD 406): not sensitising (based on read-across from CAS 110-27-0 and CAS 91031-48-0 and (Q)SAR modelling of the target substance in a weight of evidence approach)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

09 Mar - 30 Apr 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

no rationale for used concentrations given, limited etails on test substance purity

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

analytical purity of test substance not specified, no range finding for irritation, thus no explanation for used concentrations of 5% for intra- and epidermal induction, 25% for challenge

Principles of method if other than guideline:

Magnusson and Kligman method in guinea pigs

GLP compliance:

not specified

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A non-LLNA test is available that was performed prior to the current data requirements, stipulated in Regulation (EC) No 1907/2006. In accordance with the same Regulation, the data was included to avoid unnecessary testing.

Species:

guinea pig

Strain:

Pirbright-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Züchter Winkelmann, Borchen
- Weight at study initiation: test group mean: 382 g; control group mean: 337 g
- Housing: 5 per cage
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23
- Humidity (%): 50 - 56
- Air changes (per hr): 11
- Photoperiod (hrs dark / hrs light): 12 / 12

Route:

intradermal and epicutaneous

Vehicle:

other: Carboxymethylcellulose, Cremophor

Concentration / amount:

5% for injection, 5% for dermal induction

Route:

epicutaneous, occlusive

Vehicle:

other: Carboxymethylcellulose, Cremophor

Concentration / amount:

25%

No. of animals per dose:

15 for treatment, 19 as control

Details on study design:

RANGE FINDING TESTS: no data

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: injection on day 1, epidermal application on day 7
- Test groups: 3 injections: 0.1 mL Freund's adjuvant, 0.1 mL 5% test substance in 2% Carboxymethylcellulose and 0.5% Cremophor, 0.1 mL Freund's adjuvant with test substance 1:1, concentration 5%; epidermal application: 5% in Vaseline
- Control group: the same than test group without test substance
- Site: two rows with 3 injections right and left of the scapular midline, dermal application on the injections sites
- Frequency of applications: 1
- Duration: single injections and 48 h dermal exposure under occlusive conditions
- Concentrations: 5%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 14 days after dermal induction (day 22)
- Exposure period: 24 h
- Test groups: 0.1 mL 25% test substance in 2% Carboxymethylcellulose and 0.5% Cremophor
- Control group: 0.1 mL 2% Carboxymethylcellulose and 0.5% Cremophor
- Site: test substance on the right flank, vehicle on the left
- Concentrations: 25% and vehicle only
- Evaluation (hr after challenge): 48 and 72 h

Positive control substance(s):

no

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

15

Clinical observations:

none

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

19

Clinical observations:

none

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

15

Clinical observations:

none

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

19

Clinical observations:

none

Group:

positive control

Remarks on result:

not measured/tested

The test substance is not sensitising to the skin of guinea pigs, when used as 5% solution for induction and 25% solution for challenge.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

CLP: not classified

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

6 Aug - 30 Aug 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

analytical purity of test substance not specified, no positive control group

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted in 1981

Deviations:

yes

Remarks:

analytical purity of test substance not specified, no positive control group

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A non-LLNA test is available that was performed prior to the current data requirements, stipulated in Regulation (EC) No 1907/2006. In accordance with the same Regulation, the data was included to avoid unnecessary testing.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Lebeau breeding centre, Gambais, France
- Age at study initiation: approx. 5 weeks
- Weight at study initiation: male mean 436 g, female mean 423 g
- Housing: individual housing in polycarbonate cages
- Diet: guinea pigs sustenance ref. 106 (U.A.R., Villemoisson-sur-Orge, France), ad libitum
- Water: tap water, ad libitum
- Acclimation period: for a minimal period of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Photoperiod (hrs dark / hrs light): 12 / 12

Route:

intradermal and epicutaneous

Vehicle:

paraffin oil

Concentration / amount:

25% at intradermal induction, 100% at epidermal induction

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Concentration / amount:

50%

No. of animals per dose:

Treatment group: 20 (10 males and 10 females)
Control group: 10 (5 males and 5 females)

Details on study design:

RANGE FINDING TESTS:
Based on the results of a preliminary study a 25% dilution of the test substance in paraffin oil was used for intradermal induction and the undiluted (100%) substance was used for the epidermal induction exposure. The undiluted test substance was found to be slightly irritating upon dermal application.
A 50% test substance concentration was selected for the challenge phase as the maximum non-irritant dose upon dermal exposure for 24 h under occlusive dressing.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: Intradermal induction on day 1. On day 7 a local irritation was induced using 0.5 mL of a 10 % sodium laurylsulphate in vaseline. On day 8 a epidermal induction was performed with 0.5 mL vehicle or 0.5 mL test substance. The dermal applications were held in place for 48 hours under occlusive dressing.
- Test groups: 20 animals treated with test substance
- Control group: 10 animals treated with vehicle only
- Site: the scapular region of both sides
- Concentrations: 0.1 mL of 25% dilution of the test substance in paraffin oil in the presence of Freund' adjuvant was used for intradermal induction and 0.5 mL of the undiluted (100%) test substance was used for epidermal induction.
- Duration: 10 days

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 12 days after the last induction treatment
- Exposure period: 24 h under occlusive dressing
- Test groups: 20 animals treated with test substance
- Control group: 10 animals treated analogous to the test groups
- Site: the right flank was treated with the test substance; the left flank was treated with vehicle.
- Concentrations: 0.5 mL of a 50% solution in paraffin oil
- Evaluation (hr after challenge): 24 and 48 h after removal of the dressing

Positive control substance(s):

no

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25% (intradermal induction, 50% (challenge)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25% (intradermal induction, 50% (challenge)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25% (intradermal induction, 50% (challenge)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25% (intradermal induction, 50% (challenge)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Group:

positive control

Remarks on result:

not measured/tested

Table 1: Challenge readings

Group	Sex	Animals	24 h scoring period				48 h scoring period
			Erythema		Edema		Erythema		Edema
			LF	RF	LF	RF	LF	RF	LF	RF
Control	Males	1	0	0	0	0	0	0	0		0
		2	0	0	0	0	0	0	0		0
		3	0	0	0	0	0	0	0		0
		4	0	0	0	0	0	0	0		0
		5	0	0	0	0	0	0	0		0
	Females	16	0	0	0	0	0	0	0		0
		17	0	0	0	0	0	0	0		0
		18	0	0	0	0	0	0	0		0
		19	0	0	0	0	0	0	0		0
		20	0	0	0	0	0	0	0		0
Treated	Males	6	0	0	0	0	0	0	0		0
		7	0	0	0	0	0	0	0		0
		8	0	0	0	0	0	0	0		0
		9	0	0	0	0	0	0	0		0
		10	0	0	0	0	0	0	0		0
		11	0	0	0	0	0	0	0		0
		12	0	0	0	0	0	0	0		0
		13	0	0	0	0	0	0	0		0
		14	0	0	0	0	0	0	0		0
		15	0	0	0	0	0	0	0		0
	Females	21	0	0	0	0	0	0	0		0
		22	0	0	0	0	0	0	0		0
		23	0	0	0	0	0	0	0		0
		24	0	0	0	0	0	0	0		0
		25	0	0	0	0	0	0	0		0
		26	0	0	0	0	0	0	0		0
		27	0	0	0	0	0	0	0		0
		28	0	0	0	0	0	0	0		0
		29	0	0	0	0	0	0	0		0
		30	0	0	0	0	0	0	0		0

No deaths occured. No significant differences in the gain of body weight was observed between treatment and control group.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

CLP: not classified

Executive summary:

A Guinea pig maximization test was performed according to OECD Guideline 406. 20 test and 10 control animals (Dunkin-Hartley guinea pigs) were induced with 25% substance solution and challenged with a 50% test substance solutionl. 24 and 48 hours after termination of challenge exposure skin readings revealed no indications for a skin sensitising potential of Fatty acids, C16-18, 2-ethylhexyl esters.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

Adopted 4 February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 03.07.2020


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Details on the study design:

Skin sensitisation (In chemico test system)
- Details on study design:
The direct peptide reactivity test is proposed to estimate the skin sensitisation potential of a test item by quantifying its reactivity towards proteins using 2 model synthetic peptides containing either cysteine or lysine. The peptide reactivity of the test item is expressed as cysteine and lysine peptide percent depletion values following 24 hours incubation with the test item. Relative peptide concentration is measured by spectrofluorimetric assay methods.
- Model synthetic peptides: synthetic cysteine and lysine peptides of purity higher than 85% and preferably in the range of 90-95%:
CYSTEINE PEPTIDE (Cys-Pep) from JPT Peptide Technologies (AcRFAACAA-OH, Ref. 30793_1)
LYSINE PEPTIDE (Lys-Pep) from JPT Peptide Technologies (AcRFAAKAA-OH, Ref. 30793_2)
The peptide reactivity of the test item is evaluated by measuring the remaining concentration of model peptides (Cys-Pep and /or Lys-Pep) in the reaction medium after being reacted with the test item during 24 h.

- CYS-PEP assay:
- Solutions preparation:
Cys-Pep reaction solution was prepared by diluting to 400 µM with 100 mM sodium phoshate buffer (pH 7.5), cysteine peptide stock-solution (10mM) prepared in dimethyl sulfoxide (DMSO). Test item reaction solution was prepared by diluting test item in 100 mM sodium phosphate buffer (pH 7.5) in order to obtain final concentration of test item equal to 5X (in w/v or mM) final concentration of Cyst-Pep reaction solution.
- Cyst-Pep reaction:
90 µL of Cyst-Pep reaction solution were distributed in a polystyrene transparent 96-well tissue culture treated microplate (Costar®). Then, 90 µL of test item reaction solution were allotted to the wells of reaction plate. After shaking (approx. 5 sec) and sealing the plate with adhesive film, the reaction solution was left in the dark at room temperature (RT) for 24 (±2) h before running the spectrofluorimetric analysis of cysteine peptide.
- Cysteine peptide analysis:
After incubation, 10 µL of reaction mixture were transferred to black polystyrene 96-well plate (Greiner®), and 90 µL of thiol detection reagent in Sensolyte® 520 Thiol Quantitation Assay Fluorimetric kit (AnaSpec Inc) labeled with a quencher (QXL 520) and a fluorophore 5-FAM (carboxyfluorescein) was added to the wells of the black microplate. After shaking (approx. 5 sec), black microplate was left at RT in the dark for 30 (±5) min to achieve complete reaction between unreacted cysteine peptide and thiol detection QXL 520/5-FAM reagent. After reaction with free peptide-SH, 5-FAM was released and fluorescence intensity (FI) signal proportional to the unreacted free cysteine peptide-SH concentration in the analyte was measured at λExc/λEm 485/535 nm using a microplate spectrofluorimeter.
3 reference controls were included in Cyst-Pep assay: test item control: wells containing only test item (wo Cys-Pep), peptide control: wells containing only Cys-Pep (wo test item), solvent control: wells containing only the solvents (solvent(Cys-Pep)) + (solvent(test item))

- LYS-PEP assay:
- Solutions preparation:
Lys-Pep reaction solution was prepared by diluting to 200 µM with 100 mM sodium phoshate buffer (pH 10.0), lysine peptide stock-solution (10 mM) in sodium phosphate buffer (pH 10.0). Test item reaction solution was prepared by diluting test
item in isopropanol in order to obtain final concentration of test item equal to 10X (in w/v or mM) final concentration of Lys-Pep reaction solution.
- Lys-Pep reaction:
100 µL of Lys-Pep reaction solution and 100 µL of test item reaction solution were allotted to a 96-well polystyrene tissue culture treated microplate (Costar®). After shaking (approx. 5 sec), the microplate was sealed with adhesive film and then incubated in the dark at room temperature (RT) for 24 (±2) h before spectrofluorimetric analysis of Lys-Pep.
- Lysine peptide analysis:
After incubation, 180 µL of reaction mixture were transferred to black polystyrene 96-well plate (Greiner®), and 20 µL of 20 mM fluorescamine solution in DMSO were added to the wells. After shaking (approx. 5 sec), the plate was left at RT in the dark for 5 minutes, to achieve complete reaction between unreacted lysine peptide and fluorescamine. After reaction of fluorescamine with free peptide-NH2, to form a fluorescent pyrrolinone moiety, fluorescence intensity (FI) signal proportional to the unreacted free lysine peptide-NH2 concentration in the analyte was determined at λExc/λEm 360/465 nm using a fluorescence plate reader.
3 reference controls were included in Lys-Pep assay: test item control: wells containing only test item (wo Lys-Pep), peptide control: wells containing only Lys-Pep (wo test item), solvent control: wells containing only the solvents (solvent(Lys-Pep)) + (solvent(test item))

2 positive controls were tested in each experiment: positive control #1]: p-phenylenediamine (PPD, 2mM), positive control #2: cinnamaldehyde (CinAld, 2mM)

Each test item and each positive control were analyzed in triplicate for both peptides.

A standard calibration curve was generated for both peptides. Peptide standards were prepared in phosphate buffer (pH 7.5) for Cys-Pep and phosphate buffer (pH 10.0) for Lys-Pep. Using serial dilution standards of the peptide stock solution (400 µM for Cys-Pep, 200 µM for Lys-Pep, 6 calibration solutions (St 1-6) were prepared to cover the ranges from 400 to 12.5 µM for Cys-Pep and from 200 to 6.25 µM for Lys-Pep. A blank with the dilution buffer (St 0) was also included in the standard calibration curves.

- Peptide depletion expression:
Peptide reactivity of a test item is expressed as the peptide depletion ratio after incubation of peptide with the test item. For each sample, the mean fluorescence intensity (FI) of replicates (n=3) measured in the presence of peptide (+) is corrected by subtracting the mean FI value of replicates (n=3) measured in the absence of peptide (-): IFcorr = IF(+) - IF(-)
From IFcorr values, the concentration of peptide is calculated by extrapolation from the linear calibration curve derived from the with peptide standards. The percent peptide depletion (PD%) is calculated for each model peptide as follows:
(PD%) = 1 - [(FIcorr mean[treated] / FIcorr mean[Control]) x 100]

- Interpretation of results:
The peptide reactivity potential of the test item is categorized based on its percent peptide depletion (PD%) in both cysteine peptide- and lysine peptide-models. Positive: Cysteine PD ≥ 10%, Lyseine PD ≥ 20%. Negative depletion is considered as 0%. Overall qualification of the peptide reactivity of the test item: Negative: negative peptide depletion both for cysteine and lysine; positive: positive peptide depletion for either cysteine or lysine and both for cysteine and lysine.

- Assay validation:
The assay is validated if:
- the peptide depletion ratio of the positive control # 1 (PPD) is superior or equal to the cut-off value for both cysteine and lysine peptides;
- the peptide depletion ratio of the positive control #2 (CinAld) is superior or equal to the cut-off value for both cysteine and lysine peptides;
- the linearity of the calibration curve with cysteine and lysine peptides is established (r2 > 0.958);
- the mean percent peptide depletion value of the three replicates for the positive control #1 (PPD) and for the positive control #2 (CinAld) should be within ±2.5 standard deviations (SD) of the historical mean established by the laboratory for the cysteine peptide and for the lysine peptide;
- the mean peptide concentration of the three replicates for the negative control should be 200 ± 24 µM for the cysteine peptide, and 100 ± 12 µM for the lysine peptide.

Positive control results:

% PD OF POSITIVE CONTROLS (PC):
- PC #1: PD CYST = 76.5%, PD LYS = 94.3%
- PC #2: PD CYST = 57.0%, PD LYS = 45.5%

Key result

Run / experiment:

other: Mean of 3 measurements

Parameter:

other: Cysteine Peptide Reactivity

Remarks:

(%)

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Mean of 3 measurements

Parameter:

other: Lysine Peptide Reactivity

Remarks:

(%)

Value:

14.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Parameter:

other: Peptide Reactivity Potential

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Accordingly to the criteria defined, the DP% values obtained DP CYST <10% and DP LYS <20% allowed to qualify the peptide reactivity of the test item as negative.

Table 1: In Vitro Skin Sensitisation Potential - PSIV-MRP test method (Cysteine peptide assay)

	Negative control		Positive Control 1		Positive Control 2		Test substance
Cysteine peptide assay	(-) peptide	(+)peptide	(-) peptide	(+)peptide	(-) peptide	(+)peptide	(-) peptide	(+)peptide
FI1	1986	30366	1834	8911	1920	14385	1938	31623
FI2	1907	31841	1768	8795	1887	14542	1941	31668
FI3	1933	31067	1775	8255	1899	14339	1914	31254
Mean	1942	31091	1792	8654	1902	14422	1931	31515
St. Dev.	40	738	36	350	17	106	15	227
FIcorr. = FI(+) – FI(-)	-	29149	-	6861	-	12520	-	29584
Peptide reactivity (%)	-	-	-	76.5	-	57.0	-	0.0

 

Table 2: In Vitro Skin Sensitisation Potential - PSIV-MRP test method (Lysine peptide assay)

	Negative control		Positive Control 1		Positive Control 2		Test substance
Lysine peptide assay	(-) peptide	(+)peptide	(-) peptide	(+)peptide	(-) peptide	(+)peptide	(-) peptide	(+)peptide
FI1	1498	31178	1273	2824	1588	17892	1484	26696
FI2	1478	31720	1300	3067	1578	17816	1487	27242
FI3	1500	31403	1280	3039	1546	17989	1470	27074
Mean	1492	31434	1284	2977	1571	17899	1480	27004
St. Dev.	12	272	14	133	22	87	9	280
FIcorr. = FI(+) – FI(-)	-	29149	-	1692	-	16328	-	25524
Peptide reactivity (%)	-	-	-	94.3	-	45.5	-	14.8

 

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Conclusions:

CLP: not classified

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 442E

Version / remarks:

adopted 25 June 2018

Deviations:

no

GLP compliance:

yes

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch:
03.07.2020


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
room temperature

Details on the study design:

Skin sensitisation (In vitro test system)
- Details on study design:
The purpose of the human THP-1 cell line activation test (PSIV-MAC) is to estimate skin sensitisation potential of a test item by quantifying changes in the expression of the cell surface markers CD86 and CD54 associated with the process of activation of dendritic cells (DC), in the human leukemia cell line THP- L. The THP-1 cell line activation potential of the test item is expressed as the changes of CD86 and CD54 cell surface markers expression following exposure to the test substance at sub-cytotoxic concentrations. The changes of surface marker expression are measured by immuno-cytochemical analysis conducted concurrently to cytotoxicity measurement.

- Test system:
Human acute monocytic leukemia cell line, THP-1, obtained from ATCC cultured in RPMI-1640 medium with GlutaMAX-1 and HEPES, and supplemented with fetal calf serum (10% FCS), ß-mercaptoethanol (0.05mM) and antibiotics/antimycotic cocktail (penicillin, streptomycin, amphotericin B) (complete RPMI medium), maintained in suspension in 75-cm2 culture flasks with 0.2 µm filtered cap under the following incubation conditions:
- temperature: 37°C (±1°C)
- humidified 5% (±1%) CO2 atmosphere
Subculture every 2-3 days via centrifugation at 260 g for 5 minutes with medium renewal.
THP-1 cell suspension maintained at density inferior to 1x 10^6 cells/mL.
Absence of mycoplasma contamination: controled systematically at every thawing of cells and /or routinely during cell culture period (at least: 3 controls/year)

- Test procedure:
CELL MAINTENANCE: THP-1 cells are routinely passaged by centrifugation with medium renewal every 2-3 days, maintaining cell density ≤ 1 x 10^6 cells/mL.
REACTIVITY CHECK: The reactivity check of the THP-1 cell batch used in PSIVMAC assay is performed using 3 control substances: DNCB (2,4-dinitrochlorobenzene) and NiSO4 (Nickel sulphate) used as positive controls, LA (lactic acid) used as negative control.
After exposure to the control substances, the endpoints cell viability and CD86/CD54 expression are assessed according.
The test meets the quality criteria if:
- both DNCB and NiSO4 produced a positive response of both CD86 and CD54;
- LA (lactic acid) gives negative response of both CD86 and CD54;
Only the cells which passed the reactivity check are to be used for the assay. THP-1 cells can be propagated up to 2-3 months after thawing, but not in excess of 30 passages.

- CV75 DOSE FINDING ASSAY:
TEST SOLUTION PREPARATION:
The test item solutions are prepared on a weight/volume basis using complete RPMI medium for hydrophilic test substances, and with the solvent (DMSO, EtOH) used to dissolve hydrophobic products.
APPLICATION OF TEST ITEM:
100 µL of 2X working solutions of the test item are mixed 1:1 (v /v) with the THP1 cell suspension in a 96-well flat-bottom plate. The treated plate is then incubated for 24 (±2) h at 37 (±1)°C under 5% C02.
CELL VIABILITY MEASUREMENT:
After 24 h of exposure to the test item, 10 µL per well of CellTiter-Blue® Reagent (Promega) is added. After shaking, the 96-well plate is incubated at 37 (±1)°C under 5% CO2, for 90 (±5) minutes to allow reduction reactions of the detection reagent (resazurin) by metabolically active cells to fluorescent resorufin. The fluorescence produced, monitored at Ex:Em = 535/590nm is proportional to the number of viable cells.
ESTIMATION OF CV75 VALUE:
The fluorescence intensities Fl mes of measured wells are corrected by subtracting the mean FI NSB value of non specific fluorescence blank (n=4). The geometric mean fluorescence intensity is calculated: Fl Mcor (n=4).
The percentage of cell viability Viab.% is calculated for each concentration tested, according to: Viab.% = [FI Mcor (Treated wells) / FI Mcor (Control wells)] x 100
The concentration of the test item showing 75% of THP-1 cell survival (25% cytotoxicity) is calculated from the dose response curve using a linear probit-log regression model after conversion of the % cell death into probit and by plotting the probit value against logarithm of the concentration.

- INTERFERENCES TEST ITEM / RESA TEST:
DIRECT RESAZURIN REDUCTION:
10 µL of CellTiter-Blue® Reagent (Promega) are added into 200µL of solution of test item at maximal test concentration (1 mg/mL) and the reaction mixture is incubated for 90 minutes (± 5 min) at standard culture conditions. If the mixture demonstrates a fluorescent signal at excitation and emission wavelengths of 535 nm and 590 nm respectively, the test item is identified as a direct resazurin reducer. A non-specific resazurin reduction (NSRESA) control is performed by adding test item to replicates of resazurin solution with no cells, which undergo the entire testing procedure.
FLUORESCENCE INTERFERENCE:
200 µL of test material solution at the maximal test concentration (1 mg/mL) are incubated for 90 (±5) min at standard culture conditions. If the mixture shows a fluorescent signal at excitation and emission wavelengths of 535 nm and 590 nm respectively,
the test item is identified as interfering with the standard fluorescence measurement of resorufin. In this case, a nonspecific fluorescence in living THP-1 cells (NSF THP1) control is performed by application of test item on viable THP-1 cells which undergo the entire testing procedure but are incubated with medium without resazurin during the CellTiter-Blue® Reagent incubation step.

- CD86 /CD54 EXPRESSION MEASUREMENT:
TEST SOLUTION PREPARATION:
The test item solutions are prepared on a weight/volume basis using complete RPMI medium for hydrophilic test substances, and with the solvent (DMSO, EtOH) used to dissolve hydrophobic products.
6 concentrations are tested in each experiment:
- C6 = 1.2 x C5
- C5 = 1.2^0 x C5 = CV75 or DNC THP1*
- C4 = 1.2^-1 x C5
- C3 = 1.2^-2 x C5
- C2 = 1.2^-3 x C5
- C1 = 1.2^-4 x C5
with DNC THP1 *: non toxic dose in THP1-CV75 assay.
Concurrently to test sample assay, 1 positive control was used in the experiment: DNCB: 2,4-dinitrochlorobenzene (5 µg /mL).
TREATMENT OF CELLS:
200 µL of 2X working-solution of test item are added to appropriate wells (n=3) of titrated THP- 1 cell suspension in a 48-well flat-bottom plate. The plate is then incubated for 24 (±2) h at 37 (± 1)°C under 5 (± 1)% CO2.
CELL IMMUNOSTAINING AND ANALYSIS:
After 24 h of exposure, aliquots (100 µL) of cell suspension are transferred into 96-well flat bottom plate coated with Corning® Cell-Tak cell and tissue adhesive. After centrifugation, immobilized THP-1 cells are fixed with 100 µL of fixative solution (8% formaldehyde). After centrifugation, cells are maintained in fixative solution for 15 min at room temperature (RT) to complete support adsorption. After attachment, cells are washed with staining buffer (PBS+BSA(0.1%)) and blocked with blocking solution
(PBS+BSA(1%)) for 15 minutes at RT before immuno-staining. After washing with staining buffer, cells are stained with antihuman CD86, anti-CD54 antibodies diluted in staining buffer for 3 h (± 10 min) at RT. After washing, an alcaline phosphatase-conjugated anti-IgG is added and incubated with cells for 2 h (± 5 min) at RT. After washing (3x), 150 µL of p-nitrophenyl phosphate substrate is added and incubated for 30 (± 5) min at RT. The absorbance of the samples is measured at 405 nm. Concurrently, a non specific binding control (NSB) is performed by incubating cells with staining reagents using the detection mouse isotype IgG antibody (mouse anti-IgG) but without the primary antibodies CD86 and CD54.
CELL VIABILITY MEASUREMENT:
After exposure to the test item,100 µL of cell suspension are transferred into a black 96-well plate. 10 µL per well of CellTiter-Blue® Reagent (Promega) are added. After shaking, the plate is placed at 37 (± 1)°C under 5% CO2 for 90 (±5) min. The fluorescence intensities are quantified (λExc: 535 nm, λEm: 590 nm) and expressed in fluorescence relative unit (FRU).
CELL VIABILITY:
The fluorescence intensities (FImes) of measured wells are corrected by subtracting the mean FI NSB value of non specific fluorescence blank (n=3). The geometric mean fluorescence intensity is calculated: FlMcor (n=3). The percentage of ceil viability (Viab.%) is calculated for each concentration tested, according to: Viab.% = (MFIcor[Treated] / MFIcor[Control]) x 100
CD86/CD54 EXPRESSION:
For each ceil marker of interest, the optical densities [OD CD86/CD54] of wells recorded in CD86 and CD54 assays are corrected by subtracting the mean OD value of NSB (no specific binding) blank (n=3). The geometric mean [ODcorr] is calculated in each assay (n=3). The relative expression level of CD86 and CD54 is calculated in each experimental condition, as follows: CD86/CD54 (5) = (ODcorr mean [CD86/CD54 assay] / Viab.%) x 100
The test satisfies the qualified testing requirement if cell viability of test-item doses is 50. When the ceil viability is less than 50%, the [CD86/CD54 (%)] value can not be calculated. The standard fluorescence intensity (Resazurin test) measurement is appropriate to assess direct Resazurinreducers and fluorescence interfering test item when the uncorrected percent cell viability obtained with the test item is already 50% and <= 100%. For test items which are not compatible with the standard
fluorescence measurement due to too strong interference with the Resazurin assay, the XTT assay may be used to assess cell viability of THP-1 cells.

- INTERPRETATION OF RESULTS:
Based on CD86/CD54 expression measurement, the PSIV-MAC assay is considered POSITIVE if at least one of the following conditions is met, otherwise the PSIV-MAC assay is considered NEGATIVE:
- the CD86 (%) value is equal to or greater than 150 at any tested concentration (with cell viability >= 50%)
- the CD54(%) value is equal to or greater than 200 at any tested concentration (with cell viability >= 50%)

- ASSAY VALIDATION:
The PSIV-MAC assay is validated if:
- the cell viabilities of medium and solvent (DMSO) controls are more than 90%;
- both CD86 and CD54 expression in the positive control (DNCB) are over the positive criteria (CD86 (%) >= 150 and CD54 (%) >= 200) and cell viability is more than 50%;
- the CD86 (%) and CD54 (%) in the DMSO solvent control does not exceed the positive criteria (CD86 (%) >= 150 and CD54 (%) >= 200);
- the cell viability of test item at more than four tested doses is 50%.
Negative results are acceptable only for test item exhibiting a cell viability of less than 90% at 1.2xCV75 (or highest concentration). Negative results with cell viability of 90% or higher are discarded. In such a case, it is recommended to try to refine the dose selection by repeating the CV75 determination.
It should be noted that when 5000 µg/mL in medium, 1000 µg /mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test item, a negative result is acceptable even if the cell viability is above 90%.

- ASSAY PROCEDURE:
A stock solution of the test item was prepared in ethanol (500 mg/mL or 600 mg/mL). The different test solutions were obtained by dilution of the stock solution in complete RPMI medium (EtOH final concentration: 0.2%).
INTERFERENCE TEST ITEM / RESA:
Direct Resazurin reduction: no
Fluorescence interference: no
TEST ITEM CYTOTOXICITY:
Broad range: 9 concentrations between 0.0001 and 1 mg/mL (n=3)
Narrow range: 9 concentrations between 0.3 and 3 mg/mL (n=4)
CD86/CD54 EXPRESSION:
6 test-solutions:
C6 = 1.2 mg/mL
C5 = [DNC*] = 1 mg/mL
C4 = 0.83 mg/mL
C3 = 0.69 mg/mL
C2 = 0.58 mg/mL
C1 = 0.48 mg/mL
with [DNC* ]: maximal non toxic dose (Resa test)

Positive control results:

DNCB: CD86 (%) = 400 >= 150
- DNCB: CD54 (%) = 340 > 200
- DNCB: Viab.(%) = 85.22% > 50%

Key result

Run / experiment:

mean

Parameter:

other: CD86 (%)

Remarks:

C1

Value:

97

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

mean

Parameter:

other: CD86 (%)

Remarks:

C2

Value:

103

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

mean

Parameter:

other: CD86 (%)

Remarks:

C3

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

mean

Parameter:

other: CD86 (%)

Remarks:

C4

Value:

96

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

mean

Parameter:

other: CD86 (%)

Remarks:

C5

Value:

94

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

mean

Parameter:

other: CD86 (%)

Remarks:

C6

Value:

102

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

mean

Parameter:

other: CD54 (%)

Remarks:

C1

Value:

96

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

mean

Parameter:

other: CD54 (%)

Remarks:

C2

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

mean

Parameter:

other: CD54 (%)

Remarks:

C3

Value:

95

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

mean

Parameter:

other: CD54 (%)

Remarks:

C4

Value:

92

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

mean

Parameter:

other: CD54 (%)

Remarks:

C5

Value:

83

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

mean

Parameter:

other: CD54 (%)

Remarks:

C6

Value:

88

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Control of reactivity of THP-1 cell line:
- Positive controls:
DNCB 5 µg/mL: positive result for CD86 and CD54
NiSO4 150 µg/mL: positive result for CD86 and CD54
- Negative control:
LA 1800 µg /mL: negative result for CD86 and CD54
Cells satisfyed the reactivity control.

Broad range :
No significant cytotoxicity (cell death <= 10%) was observed in the range of tested concentrations.

Narrow range :
A very slight dose-dependent cytotoxicity was observed in the range of the tested concentrations.

The CV75 value has been calculated using the probit-log[concentration] regression model:
Probit (% death) = 0.812 x log(conc.) + 3.605 r = 0.987
CV75 = 7.80 mg/mL

Highest tested concentration (C5) to be applied in CD86/CD54 expression assay:
C5 = DNC THP1 = 1 mg/mL

Accordingly to the criteria defined, the CD86(%) values obtained (<150 with the 6 tested concentrations) and the CD54(%) values obtained <200 with the 6 tested concentrations allowed to qualify the dendritic cell activation potential (DCAP) of the test item as negative.

Test item viability:
Cell viability > 50%: 6 concentrations

Table 1: In Vitro Skin Sensitisation Potential - PSIV-MACtest method (CD86 assay)

	Control	Control EtOH	Positive Control	Test substance (mg/mL)
CD86 assay	-	EtOH (0.2%)	DNCB 5 µg/mL)	0.48	0.58	0.69	0.83	1.00	1.20
ODmes.1	0.284	0.278	0.767	0.269	0.282	0.279	0.268	0.261	0.287
ODmes.2	0.275	0.277	0.708	0.272	0.273	0.277	0.274	0.275	0.280
ODmes.3	0.299	0.278	0.703	0.277	0.295	0.266	0.278	0.267	0.275
Mean	0.286	0.277	0.726	0.273	0.283	0.274	0.273	0.267	0.281
St. Dev.	0.012	0.001	0.035	0.004	0.011	0.007	0.005	0.007	0.006
ODcor.Mean	0.731	0.697	2.491	0.679	0.721	0.683	0.681	0.657	0.710
ODcor.St. Dev.	0.049	0.002	0.141	0.015	0.044	0.028	0.021	0.028	0.025
Resazurin assay	-	EtOH (0.2%)	DNCB 5 µg/mL)	0.48	0.58	0.69	0.83	1.00	1.20
FImes.1	40823	40462	35564	41305	40718	39807	40660	40647	40521
FImes.2	41224	40533	35289	40117	40386	40060	40731	40378	40667
FImes.3	40803	40708	35143	41378	41152	39850	41759	41176	40621
Mean	40950	40568	35332	40933	40752	39906	41050	40734	40603
St. Dev.	238	127	214	708	384	135	615	406	75
Viability (%) Mean	100	100	85	101	100	98	101	100	100
Viability (%) St. Dev.	0.34	0.34	0.56	1.88	1.02	0.36	1.64	1.08	0.20
CD86 (%) Mean	100	100	400	97	103	100	96	94	102
CD86 (%) St. Dev.	0,43	0.43	20.11	2.58	5.27	3.95	1.95	4.58	3.71

 

Table 2: In Vitro Skin Sensitisation Potential - PSIV-MACtest method (CD54 assay)

	Control	Control EtOH	Positive Control	Test substance (mg/mL)
CD54 assay	-	EtOH (0.2%)	DNCB 5 µg/mL)	0.48	0.58	0.69	0.83	1.00	1.20
ODmes.1	0.329	0.313	0.762	0.310	0.338	0.314	0.307	0.293	0.292
ODmes.2	0.321	0.328	0.729	0.320	0.315	0.309	0.311	0.280	0.299
ODmes.3	0.306	0.327	0.666	0.319	0.321	0.304	0.307	0.289	0.298
Mean	0.316	0.323	0.719	0.316	0.325	0.309	0.308	0.287	0.296
St. Dev.	0.012	0.008	0.049	0.006	0.012	0.005	0.002	0.006	0.004
ODcor.Mean	0.845	0.862	2.447	0.836	0.869	0.807	0.804	0.720	0.756
ODcor.St. Dev.	0.048	0.033	0.194	0.022	0.048	0.020	0.009	0.026	0.017
Resazurin assay	-	EtOH (0.2%)	DNCB 5 µg/mL)	0.48	0.58	0.69	0.83	1.00	1.20
FImes.1	40823	40462	35564	41305	40718	39807	40660	40647	40521
FImes.2	41224	40533	35289	40117	40386	40060	40731	40378	40667
FImes.3	40803	40708	35143	41378	41152	39850	41759	41176	40621
Mean	40950	40568	35332	40933	40752	39906	41050	40734	40603
St. Dev.	238	127	214	708	384	135	615	406	75
Viability (%) Mean	100	100	85	101	100	98	101	100	100
Viability (%) St. Dev.	0.63	0.34	0.56	1.88	1.02	0.36	1.64	1.08	0.20
CD54 (%) Mean	100	100	333	96	100	95	92	83	88
CD54 (%) St. Dev.	5.59	3.66	24.41	3.89	5.39	2.50	2.24	2.68	1.75

 

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Conclusions:

CLP: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin Sensitisation, in chemico / in vitro:

There is an in vitro and an in chemico skin sensitisation test with isodecyl pivalate (CAS 60209-82-7).

In order to assess the in chemico skin sensitising potential of isodecyl pivalate (CAS 60209-82-7) its capacity to activate the human leukemia cell line THP-1 was investigated in a study equivalent to OECD 442C and under GLP (WoE, Stearinerie, 2018). The reactivity of the test item to protein was evaluated by measuring the concentration of model peptides (Cys-Pep and Lys-Pep) in the reaction medium after being reacted with the test item (2 mM in phosphate buffer pH7.5 for the Cys-Pep assay and in isopropanol for the Lys-Pep assay) in triplicate during 24 h via spectrofluorimetric analysis. P-phenylenediamine and cinnamaldehyde were inserted as positive controls. Positive and negative controls showed valid results and all acceptability criteria were fulfiled, proving that the test design was valid. Under the experimental conditions described, the cysteine and lysine peptide values were 0% and 14.8%, respectively. And therefore the reactivity of the test item to protein is concluded to be negative.

In order to assess the in vitro skin sensitising potential of isodecyl pivalate (CAS 60209-82-7) its ability to activate human dendritic cells was investigated in a study equivalent to OECD 442E and under GLP (WoE, Stearinerie, 2018). The PSIV-MAC assay is based on the quantification of CD86 and CD54 surface marker expression via immunocytochemistry in THP-1 cells after 24 h contact with the test compound at six concentrations (0.48 – 1.2 mg/mL in ethanol) in triplicate. 2,4-dinitrochlorobenzene was used as the positive control. Positive and negative controls showed valid results and all acceptability criteria were fulfiled, proving that the test design was valid. Under the experimental conditions described, the CD86 (%) value less than 150 and the CD54 (%) value less than 200 at the 6 tested concentrations means that the DC activation potential of the test item is negative.

Skin sensitisation, in vivo:

In addition to the in chemico/in vitro data on isodecyl pivalate (CAS 60209-82-7), data from in vivo studies of structurally related substances are available, which support the negative outcome of the in chemico / in vitro skin sensitisation studies with isodecyl pivalate (CAS 60209-82-7).

CAS 110-27-0

A Guinea pig maximisation test was performed with isopropyl myristate (CAS 110-27-0) according to a protocol similar to the OECD Guideline 406 (WoE, BASF, 1984). 15 female Pirbright-Hartley guinea pigs were induced with a 5% solution of the test substance via intradermal injection on Day 1 and via epidermal application on Day 7. 20 animals served as negative controls, of which one died during the study period of causes unrelated to the treatment. No positive control group was included in the study and no information was given on periodical testing of strain sensitivity. 14 days after the epidermal induction, the challenge was performed with a 25% solution of test material in 2% carboxymethylcellulose and 0.5% cremophor. 48 and 72 hours after challenging skin examination revealed no irritation the test group or in the control group. The readings 24 and 48 hours after the challenge exposure ended showed no indications of skin sensitising potential of the test substance in any of the animals.

CAS 91031-48-0

Fatty acids, C16-18, 2-ethylhexyl esters (CAS 91031-48-0) was tested for its skin sensitisation potential in a Guinea pig maximization test according to OECD Guideline 406 (WoE, Oleon, 1991). 20 test and 10 control animals (Dunkin-Hartley guinea pigs) were induced intradermally with the test substance in a 25% dilution in paraffin oil. 7 days later the epidermal induction was performed with the undiluted test substance, as this concentration (100%) was found to be slightly irritating in the range-finding test. 12 days after the last induction treatment (Day 19) the animals were challenged with a 50% test substance solution in paraffin oil. The readings 24 and 48 hours after the challenge exposure ended showed no indications of skin sensitising potential of the test substance in any of the animals.

Conclusions for skin sensitisation:

An in vitro and an in chemico skin sensitisation test with isodecyl pivalate (CAS 60209-82-7) and two Guinea Pig Maximisation Tests performed with the structurally related substances isopropyl myristate (CAS 110-27-0) and Fatty acids, C16-18, 2-ethylhexyl esters (CAS 91031-48-0) are available and no skin sensitisation was evaluated. Taking all the available data into consideration isodecyl pivalate (CAS 60209-82-7) is considered not to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on skin sensitisation do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.

There are no data available on respiratory sensitisation.