4-O-α-D-glucopyranosyl-D-glucitol

Endpoint summary

Description of key information

Skin sensitisation: Not a skin sensitizer based on the results of the three in chemico / in vitro skin sensitisation tests (adressing the 3 key events of the skin sensitisation adverse outcome pathway (AOP)) performed with the registered substance (Reliability 1 key studies; GLP compliant; OECD 442 C, OECD 442 D, OECD 442 E guidelines; 2019) (CitoxLab France, 2019), together with the results of the in vivo skin sensitisation test with a relevant structural analogue (Reliability 1 key study; GLP compliant; OECD 429 guideline; 2010) (Seibersdorf Labor GmbH, 2010).

Respiratory sensitisation: no data available.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study in compliance with guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Animals: Mice, CBA/Ca, females, young adult nulliparous.

Justification for the selection of the species: Mice are recommended by the guidelines for LLNAs.

Age at the first administration: About 8 – 12 weeks.

Body mass range: At the initiation of the study a maximum deviation of 20 % of any individual body weight from the mean is accepted.

Hygiene: Optimal hygienic conditions.

Room No.: EI 1-13.

Room temperature: About 22 °C (continuous monitoring and recording).

Relative humidity: Between 30% and 70% (continuous monitoring and recording).

Lighting: Only artificial light from 6.00 a.m. to 6.00 p.m.

Cages: Single caging. Makrolon cages type II, (22 cm x 16,5 cm ground area, 15 cm high).

Feed: Maintenance diet for rats and mice R/M-H (item V1534-300), autoclavable, ad libitum.

Water: Tap water from Makrolon-bottles with stainless steel canules or from an automatic watering system, ad libitum.

Bedding material: Aspen wood chips, ABEDD®, LAB & VET Service GmbH, Hasnerstraße 84/6, 1160 Wien (item LTE E-001). Germ reduction by autoclaving; Changed 1/week.

Environmental Enrichment: Nibbling wood bricks and/or nesting material, same material and source as the bedding material, are offered once a week. A "mouse house" (red polycarbonate shelter, 9.8 x 8.8 cm ground area, 5.5 cm high) is offered per cage. Germ reduction by autoclaving.

Acclimatisation: At least 5 days.

Identification of the animals: By shearing a defined fur region on the back of the animal:
Group A: top left
Group B: top right
Group C: bottom left
Group P: top left and top right
Group K: unshorn
and by cage tag.

Vehicle:

dimethylformamide

Concentration:

Solubility testing of the test substance in the guideline-recommended vehicles showed that the highest concentrations suitable for application of the test substance can be achieved with DMF (14.9%, w/w).

No. of animals per dose:

Test substance concentrations:
Group A (low dose), 5 animals, 5% test substance in DMF (w/w)
Group B (mid dose), 5 animals, 10% test substance in DMF (w/w)
Group C (high dose), 5 animals, 14.9% test substance in DMF (w/w)

Control substance concentrations:
Group K (negative control), 5 animals, DMF
Group P (positive control), 5 animals, 25% HCA in AOO (v/v)

Details on study design:

The individual animals are allocated to their groups by random numbers (5 mice/group). The test substance is administered in 3 concentrations to the dorsal surfaces of the ears of the animals of the test substance groups. In a manner identical to that of animals in the treatment groups animals of one negative control group and one positive control group are treated with DMF and HCA respectively. The positive and negative control groups might be simultaneously used for other, concurrently performed studies. Each animal is treated for 3 consecutive days. 3 days after the last administration the proliferation of the lymphocytes of the draining lymph nodes is measured by the determination of the amounts of incorporated radioactive ³H-methyl thymidine (3HTdR). If the dpm data of the negative or positive control differ very strong from the historical data the main study will be repeated.

Time schedule
Day 1,2,3: Epicutaneous administration of the appropriate dilutions of the test or control substances to the dorsum of both ears.
Day 6: Administration of 20 μCi 3HTdR to all mice via a tail vein.
5 hours later, the draining auricular lymph nodes are excised and pooled for each experimental group.
Day 7: Measurement of 3HTdR incorporation by ß-scintillation counting.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The allocation of the animals to the groups is performed by randomisation.

Positive control results:

Positive control is valid. SI was higher than 3.

Parameter:

SI

Remarks on result:

other: The substance is regarded as non sensitizer; since the SIs of all tested concentrations were clearly lower than 3.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see below

Key result

Parameter:

SI

Value:

0.7

Test group / Remarks:

Low dose: 5% test substance in DMF (w/w)

Key result

Parameter:

SI

Value:

0.6

Test group / Remarks:

Mid dose: 10% test substance in DMF (w/w)

Key result

Parameter:

SI

Value:

0.7

Test group / Remarks:

High dose: 14.9% test substance in DMF (w/w)

Key result

Parameter:

SI

Value:

4.7

Test group / Remarks:

Positive control

	dpm	SI
group K(negative control)	494	1
group A(low dose)	360	0.7
group B(mid dose)	320	0.6
group C(high dose)	327	0.7
group P(positive control)	2315	4.7

Interpretation of results:

not sensitising

Conclusions:

Under the experimental conditions of the study, the tested substance was not found to be a skin sensitizer.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across data with original study of reliability 1

Justification for type of information:

See attached document for read across analogy rationale and justification.

Reason / purpose for cross-reference:

read-across source

Positive control results:

Positive control is valid. SI was higher than 3.

Key result

Parameter:

SI

Test group / Remarks:

Test item

Remarks on result:

other: The substance is regarded as non sensitizer; since the SIs of all tested concentrations were clearly lower than 3.

Key result

Parameter:

SI

Value:

0.7

Test group / Remarks:

Low dose: 5% test substance in DMF (w/w)

Key result

Parameter:

SI

Value:

0.6

Test group / Remarks:

Mid dose: 10% test substance in DMF (w/w)

Key result

Parameter:

SI

Value:

0.7

Test group / Remarks:

High dose: 14.9% test substance in DMF (w/w)

Key result

Parameter:

SI

Value:

4.7

Test group / Remarks:

Positive control

	dpm	SI
groupK(negative control)	494	1
groupA(low dose)	360	0.7
groupB(mid dose)	320	0.6
groupC(high dose)	327	0.7
groupP(positive control)	2315	4.7

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of the study, the source substance was not found to be a skin sensitizer. Based on the weight of evidence approach (used in combination with the negative results of the in chemico / in vitro skin sensitisation tests with Maltitol), the target substance was considered as not sensitizing to skin and was not classified according to CLP criteria.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From to

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor; Batch/Lot number: ELEL7
- Expiration date of the lot/batch: 07 October 2022

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: not applicable
- Specific activity: not applicable
- Locations of the label: not applicable
- Expiration date of radiochemical substance: not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: controlled room temperature
- Stability under test conditions: not applicable
- Solubility and stability of the test substance in the solvent/vehicle: The test item was prepared at 100 mM in milli-Q water using vortex until reaching a colorless solution. This formulation was used just after its preparation.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: see section "details on study design"
- Final preparation of a solid: none

FORM AS APPLIED IN THE TEST (if different from that of starting material): tested diluted in milli-Q water

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): not applicable

OTHER SPECIFICS: none

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design: The test item was tested in one validated run (no second run was required since no borderline results were obtained). The run was processed as described below:

PREPARATION OF THE SAMPLES
The following samples were prepared in triplicate except for the co-elution control samples for which only one sample was prepared per peptide buffer.
1) Co-elution control samples preparation
a) For the co-elution control with cysteine peptide:
50 µL of test item formulation was incubated with 750 µL of cysteine peptide dilution buffer (without cysteine peptide) and 200 µL of acetonitrile.
b) For the co-elution control with lysine peptide:
In parallel, 250 µL of test item formulation was incubated with 750 µL of lysine peptide dilution buffer (without lysine peptide).

2) Reference control samples preparation
a) Reference control A and B samples
In a vial, acetonitrile was added to a volume of peptide solution (cysteine or lysine) to achieve a nominal concentration of 0.500 mM.
b) Reference control C samples
Reference control C samples were prepared for each vehicle used to dissolve the test and positive control items. Since several test items were assayed concurrently and were dissolved using the same vehicle, the reference control C samples were shared.
- For the reference control C prepared with cysteine peptide:
50 µL of each vehicle used to dissolve the test item or the positive control (i.e. milli-Q water or acetonitrile) was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile.
- For the reference control C prepared with lysine peptide:
In parallel, 250 µL of each vehicle used to dissolve the test item or the positive control (i.e. milli-Q water or acetonitrile) was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate buffer at pH 10.2).

3) Cinnamaldehyde (positive control) depletion control samples preparation
a) For the reactivity of cinnamaldehyde with cysteine peptide: 50 µL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile.
b) for the reactivity of cinnamaldehyde with lysine peptide: In parallel, 250 µL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

4) Test item samples preparation:
a) For the reactivity of test item with cysteine peptide: 50 µL of test item formulation was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile.
b) For the reactivity of test item with lysine peptide: In parallel, 250 µL of test item formulation was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).

INCUBATION OF THE SAMPLES
All samples (co-elution controls, reference controls, test item and positive control samples) were then incubated during 24 (± 2) hours at 25°C and protected from light before injection into the HPLC/UV system. At the end of the incubation period, a visual inspection of the samples was performed prior to HPLC analysis to detect precipitate or phase separation. Samples presenting precipitate and/or phase separation (micelles) were centrifuged at 400g for 5 minutes at room temperature and only supernatants were then injected onto the HPLC/UV system. Otherwise, vials were directly transferred onto the HPLC/UV system.

PREPARATION OF CALIBRATION CURVE SAMPLES
One set of calibration standards was prepared with each analytical sequence by spiking each peptide (lysine and cysteine) in separate solutions of 20% acetonitrile:peptide dilution buffer to obtain at least six different concentration levels ranging from 0.0167 to 0.534 mM. A dilution buffer blank was also included in the standard calibration curve. The calibration curves were defined by the relationships between the peak area signal of the peptide versus the nominal concentration. These curves were obtained by using the appropriate mathematical model.

HPLC/UV ANALYSIS OF THE SAMPLES
-The study samples were assayed in batches using HPLC/UV analysis.
For each peptide, the analytical sequence included at least:
one blank sample (peptide dilution buffer),
one calibration curve injected at the beginning of the analytical batch,
three reference control A samples,
the co-elution control sample,
three reference control B samples,
reference control C sample (replicate 1),
positive control sample (replicate 1),
test item study sample (replicate 1),
The injection order of the reference control C, positive control and test item study samples were reproduced identically for replicate 2 and then replicate 3:
three reference control B samples.

The HPLC/UV method used for the samples analysis is described in the testing laboratory internal analytical method (summarized in the study report).

Positive control results:

Results for the positive control are presented in Tables 2 a and b) in the section "any other information incl. Tables".
The acceptance criteria for the positive controls were satisfied.

Key result

Run / experiment:

other: 1

Parameter:

other: mean of the percentage cysteine and percentage lysine depletions

Value:

0.82

Vehicle controls validity:

other: reference control samples

Negative controls validity:

other: co-elution controls valid

Positive controls validity:

valid

Remarks on result:

other: no or minimal peptide reactivity; DPRA prediction is considered as negative.

Other effects / acceptance of results:

SOLUBILITY RESULTS
Two vehicles were tested during the solubility assays. The test item was found not soluble at 100 mM in acetonitrile. The test item was found soluble 100 mM in milli-Q water using vortex until reaching a colorless solution. Therefore, the retained vehicle was milli-Q water.

EVALUATION OF THE PRESENCE OF PRECIPITATE AT THE END OF THE INCUBATION WITH PEPTIDES
At the end of the incubation period, a visual inspection of all samples (co-elution controls, reference controls, test item and positive control samples) was performed prior to HPLC analysis.
Phase separation was observed for the positive control samples incubated with lysine. These vials were therefore centrifuged at 400g for a period of 5 minutes at room temperature to force precipitate to the bottom of the vial. Then, only supernatants were injected onto the HPLC/UV system.
For the other samples, the vials were directly transferred into the HPLC/UV system.

EVALUATION OF THE RESULTS (Tables 1 to 6 in the section “any other information on results incl. Tables”)
Results for the test item and the positive control are presented in Tables 1 (a, b and C) and 2 (a and b), respectively. The peak areas for calibration curve samples are presented in Tables 3 and 4. The peptide concentrations and peak areas in reference control samples are presented in Tables 5 and 6.
Representative chromatograms of the co-elution, reference control C and test item samples were presented for each peptide in the study report (not reported in the current endpoint study record).

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.
Analysis of the chromatograms of the co-elution samples (not reported in the current endpoint study record) indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percentage depletion values were calculated for each peptide using the formula described previously:
• for the cysteine peptide, the mean depletion value was 0.58%,
• for the lysine peptide, the mean depletion value was 1.06%.
The mean of the percentage cysteine and percentage lysine depletions was equal to 0.82%.

Tables 1 a, b and c. Percentage peptide depletion for the test item samples

a. Determination of Cysteine peptide and Lysine peptide depletion in samples spiked with a solution at 100mMof Maltitol

Sample number	Cysteine peptide		Lysine peptide		Mean depletion rate (%) of Maltitol	Depletion classification
	Peak area (μV/sec)	% depletion	Peak area (μV/sec)	% depletion
1	1836823	1.75	1565589	1.28
2	1875781	0.00*	1565170	1.31
3	1879083	0.00*	1576466	0.60
Mean	-	0.58	-	1.06	0.82	No reactivity/ minimal reactivity
SD	-	1.01	-	0.40
% CV	-	173.2	-	38.0
Precipitate:	No		No
Micelle	No		No

*: Value set to 0 due to negative depletion

-: not applicable

 

b. Determination of Cysteine peptide and lysine peptide concentration in Reference Control C samples prepared in water

Sample number	Cysteine peptide			Lysine peptide
	Peak Area (μV/sec)	Concentration (mM)	%Dev	Peak Area (μV/sec)	Concentration (mM)	%Dev
1	1840973	0.488	(-2.5)	1588973	0.494	(-1.2)
2	1896063	0.502	(0.4)	1584844	0.493	(-1.5)
3	1871655	0.496	(-0.9)	1583921	0.492	(-1.5)
Mean	1869564	0.495	(-1.0)	1585913	0.493	(-1.4)
SD	-	0.007	-	-	0.001	-
% CV	-	1.5	-	-	0.2	-

-: not applicable

c. Determination of % interference due to co-elution of Maltitol with Cysteine or Lysine peptides

	Peak detected at the cysteine retention time		Peak detected at the lysine retention time
Sample number	Peak Area (μV/sec)	% Interference	Peak Area (μV/sec)	% Interference
1	0	(0.0)	0	(0.0)
Precipitate:	No		No
Micelle	No		No

 

Tables 2 a and b. Percentage peptide depletion for the positive control samples

a. Determination of Cysteine peptide and Lysine peptide depletion in samples spiked with a solution at 100mMofCinnamaldehyde

Sample number	Cysteine peptide		Lysine peptide		Mean depletion rate (%) ofCinnamaldehyde	Depletion classification
	Peak area (μV/sec)	% depletion	Peak area (μV/sec)	% depletion
1	516113	72.31	595631	62.78
2	532370	71.44	596574	62.72
3	519074	72.15	597988	62.63
Mean	-	71.97	-	1.06	67.34	High reactivity
SD	-	0.46	-	0.40
% CV	-	0.6	-	38.0

-: not applicable

 

b. Determination of Cysteine peptide and lysine peptide concentration in Reference Control C samples prepared inAcetonictrile

Sample number	Cysteine peptide			Lysine peptide
	Peak Area (μV/sec)	Concentration (mM)	%Dev	Peak Area (μV/sec)	Concentration (mM)	%Dev
1	1848296	0.490	(-2.1)	1598351	0.497	(-0.6)
2	1896544	0.502	(0.5)	1600053	0.497	(-0.5)
3	1846762	0.489	(-2.2)	1602181	0.498	(-0.4)
Mean	1863867	0.494	(-1.3)	1600195	0.497	(-0.5)
SD	-	0.007	-		0.001	-
% CV	-	1.5	-		0.1	-

-: not applicable

Table 3. Peak areas of the calibration curve samples for Cysteine peptide (μV/sec)

Analyte:Cysteinepeptide                              Regressiontype :linear                   Unit:μV/sec

Weighingfactor :1/x

 

Concentration (mM)
Date	0.0167	0.0334	0.0667	0.134	0.267	0.534	r2	slope	intercept
25 October 2018	60774	122109	247834	504055	1006082	2016897	1.0000	3780000	3780000

 

Table 4. Peak areas of the calibration curve samples for Lysine peptide (μV/sec)

Analyte: Lysine peptide                  Regressiontype :linear                   Unit:μV/sec

Weighingfactor :1/x

 

Concentration (mM)
Date	0.0167	0.0334	0.0667	0.134	0.267	0.534	r2	slope	intercept
25 October 2018	54383	109157	213346	434992	862465	1711998	1.0000	3210000	1090

 

Tables 5 a and b. Concentrations and peak areas of Cysteine peptide in reference control samples

a. Determination of Cysteine peptide concentration in reference ControlAsamples

Date	Sample number	Cysteine peptide
		Area (μV/sec)	Concentration (mM)	%Dev
25 October 2018	Reference control A.1	1939183	0.514	(2.7)
	Reference control A.2	1847185	0.489	(-2.2)
	Reference control A.3	1866917	0.494	(-1.1)
Mean		-	0.499	(-0.2)
SD		-	0.013	-
% CV		-	2.6	-

-: not applicable

b. Cysteine peptide peak areas in Reference Control B and C samples prepared in Acetonitrile

Date	Sample number	Peak areas (μV/sec)
25 October 2018	Reference control B.1	1886085
	Reference control B.2	1866249
	Reference control B.3	1847569
	Reference control B.4	1848799
	Reference control B.5	1868449
	Reference control B.6	1875684
	Reference control C.1	1848296
	Reference control C.2	1896544
	Reference control C.3	1846762
Mean		1864937
SD		18524
% CV		1.0

 

Tables 6 a and b. Concentrations and peak areas of Lysine peptide in reference control samples

a. Determination of Lysine peptide concentration in reference ControlAsamples

Date	Sample number	Lysine peptide
		Area (μV/sec)	Concentration (mM)	%Dev
23 October 2018	Reference control A.1	1595595	0.496	(-0.8)
	Reference control A.2	1600581	0.498	(-0.5)
	Reference control A.3	1614079	0.502	(0.3)
Mean		-	0.498	(-0.3)
SD		-	0.003	-
% CV		-	0.6	-

-: not applicable

b. Lysine peptide peak areas in Reference Control B and C samples prepared in Acetonitrile

Date	Sample number	Peak areas (μV/sec)
23 October 2018	Reference control B.1	1603463
	Reference control B.2	1599317
	Reference control B.3	1599906
	Reference control B.4	1595433
	Reference control B.5	1606503
	Reference control B.6	1601619
	Reference control C.1	1598351
	Reference control C.2	1600053
	Reference control C.3	1602181
Mean		1600758
SD		3173
% CV		0.2

 

Interpretation of results:

other: this test is a part of a tiered strategy for the evaluation of skin sensitisation potential in the context of an integrated approach to testing and assessment.

Conclusions:

The mean of the percentage cysteine and percentage lysine depletions was equal to 0.82%. Under the experimental conditions of this study, the DPRA prediction is considered as negative and the test item MALTITOL, was considered to have no or minimal peptide reactivity.

Executive summary:

This GLP compliant study was performed to evaluate the reactivity of the test item MALTITOL to synthetic cysteine and lysine peptides (protein reactivity). This in chemico study was performed according to the OECD Guideline 442C: in chemico skin sentitization: Direct Peptide Reactivity Assay (DPRA), adopted on 04 February 2015. This test addresses the molecular initiating event of the skin sensitisation adverse outcome pathway (AOP) and is part of a tiered strategy for skin sensitization assessment.

Material and methods

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours, protected from light. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm.

Peptide reactivity was reported as percentage depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate vehicle).

Results

The test item was dissolved at 100 mM in milli-Q water.

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percentage depletion values were calculated for each peptide using the formula described in § Data analysis and calculation:

- for the cysteine peptide, the mean depletion value was 0.58%,

- for the lysine peptide, the mean depletion value was 1.06%.

The mean of the percentage cysteine and percentage lysine depletions was equal to 0.82%. Accordingly, the test item was considered to have no or minimal peptide reactivity. Therefore, the DPRA prediction is considered as negative.

Conclusion

Under the experimental conditions of this study, the DPRA prediction is considered as negative and the test item MALTITOL, was considered to have no or minimal peptide reactivity.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 13 September 2018 to 04 January 2019

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:

The OECD guideline 442D mentions that test items prepared in sterile water or culture medium have to be filtered after preparation for sterilization prior treatment. Filtration of such preparations can be possible when a test item formulation is a solution. However, in case the test item formulation is a suspension, filtration process can retain test item particles in the filter and considerably decrease test.
item concentration in the filtered formulation. Therefore, in view of the above and considering that
pre-treatment filtration procedure is not of common practice in other in vitro cell-based assays, and
that water and culture media used in the assay are already sterile, test item formulations were not
filtered prior treatment.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor; Batch/Lot number: ELEL7
- Expiration date of the lot/batch: 07 October 2022

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: not applicable
- Specific activity: not applicable
- Locations of the label: not applicable
- Expiration date of radiochemical substance: not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: controlled room temperature
- Stability under test conditions: not applicable
- Solubility and stability of the test substance in the solvent/vehicle: A solubility assay was performed prior the first treatment in order to select the vehicle. The vehicles tested simultaneously, were DMSO, water or culture medium (at 200 mM and at 100 mM).
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: none

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: On the basis of solubility results, the test item was dissolved in DMSO at 200 mM. One formulation was prepared for each run, this formulation was homogenized by vortex at least 12 minutes before use and then diluted in DMSO by serial dilutions, using a dilution factor of 2 to obtain a total of 12 concentrations in a 96-well plate; this 96-well plate was called "Master plate 100x". Subsequently, each formulation of the Master plate 100x was 25-fold diluted in treatment medium in another 96-well plate called "Master plate 4x" taking care to adjust all wells to the same DMSO level. All formulations were prepared within 4 hours before use, and kept at room temperature and protected from light until use.
- Final preparation of a solid: none

FORM AS APPLIED IN THE TEST (if different from that of starting material): diluted in the appropriate solvent.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): not applicable

OTHER SPECIFICS: none

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design: The test item was tested in two independent runs using cells from a different passage number

TEST SYSTEM:
- KeratinoSens cells (batch D1; supplied by Givaudan): the cell line KeratinoSens is stably transfected with a modified plasmid. This plasmid contains an ARE sequence from the AKR1C2 gene and a SV40 promotor which are inserted upstream of a luciferase gene. The resulting plasmid was transfected into HaCaT keratinocytes and
clones with a stable insertion selected in the presence of Geneticin / G-418. Induction of luciferase gene is the end-point evaluated and reflects the activation by the test item of the Nrf2 transcription factor in this test.

SOLUBILITY ASSAY
- A solubility assay was performed prior the first treatment in order to select the vehicle. The vehicles tested simultaneously, were DMSO, water or culture medium (at 200 mM and at 100 mM). Since the test item was found soluble in DMSO at 200 mM, this stock formulation was diluted in treatment culture medium to the final concentration of 2000 μM. Then, a visual inspection of the sample was performed immediately as well as after an overnight period of incubation at 37°C to evaluate the presence or absence of precipitate/emulsion.

VEHICLE AND NEGATIVE CONTROL
Based on solubility results, the vehicle was DMSO. This vehicle was used as the negative control, and was applied to cells at a concentration of 1% in culture
medium.

POSITIVE CONTROL
- Cinnamic Aldehyde (CA): for each run, the positive control item was dissolved in DMSO to a final concentration of 200 mM. This solution was then further diluted to a final concentration of 6.4 mM. It was diluted in DMSO by serial dilutions in the Master plate 100x, using a dilution factor of 2, to obtain a total of 5 concentrations. Subsequently, each formulation of the Master plate 100x was diluted 25-fold in treatment medium in another 96-well plate called "Master plate 4x". The final tested concentrations ranged from 4 to 64 μM. All these formulations were prepared within 4 hours before use, then kept at room temperature and protected from light until use.

METHOD FOR A RUN OF KERATINOSENS ASSAY
- Cell seeding for testing: Cells were grown using general culture procedures up to 80-90% confluence. The day prior to treatment, cells were washed twice with D-PBS containing 0.05% EDTA, harvested, re-suspended in Maintenance medium and counted using Trypan Blue dye. Cell concentration was adjusted to a density of 8 x 104 cells/mL. Cells were then distributed into four 96-well plates (three white plates and one transparent plate), by adding 125 μL (representing 1 x 104 cells) per well taking care to avoid sedimentation of the cells during seeding. After seeding, the cells were grown for 24 (± 1) hours in the 96-well microtiter plates prior to test item addition.
- Treatment: After the 24-hour growing period, the medium was removed by aspiration and replaced by 150 μL of treatment medium. From the Master plate 4x, a volume of 50 μL was added to each well of the three white assay plates and 50 μL to the transparent plate for the cytotoxicity evaluation. All plates were covered by a sealing membrane to avoid evaporation of volatile test items and to avoid cross-contamination between wells. The plates were then incubated for 48 (± 2) hours at 37°C, 5% CO2, 90% humidity.
- End-point measurements:
--> Microscopic observation to evaluate the presence or absence of precipitate - transparent plate: After the 48 (± 2) hours incubation period, the presence or absence of precipitate/emulsion was determined in each well by microscopic inspection.
--> Luminescence flash signal to evaluate induction signal - white plates: After incubation, the supernatants from the white assay plates were discarded. The cells were washed once with D-PBS. A volume of 20 μL of passive lysis buffer was added to each well and the cells were incubated for 20 (± 2) minutes at room temperature and under orbital shaking. The plates containing the passive lysis buffer were then placed in the luminometer for reading using the following program:
- 50 μL of the luciferase substrate was added to each well,
- 1 second after this addition, the luciferase signal was integrated for 2 seconds.
--> Absorbance signal to evaluate the cytotoxicity - transparent plate: For the cell viability assay plate, the medium was replaced by 200 μL of treatment medium. A volume of 27 μL of a MTT solution at 5 mg/mL in D-PBS was then added to each well of the transparent 96-well plate. The plates were covered with a sealing membrane and returned at 37°C in the incubator in humidified atmosphere for 4 hours (± 10 minutes). At the end of the incubation period, the medium was removed and a volume of 200 μL of a 10% SDS solution was added to each well. The plates were covered with a sealing membrane and placed at 37°C in the incubator in humidified atmosphere for an overnight period to extract the formazan from cells. After the overnight incubation, the absorption of each well was determined at 600 nm using the plate reader.

RESULTS ANALYSIS
- For the MTT and the luciferase data, the background value recorded in the empty well without cells (blank) was subtracted.
- For the MTT data, the % viability was calculated for each well in the test plate in relation to average of the six negative control wells.
- For the luciferase data, the average value of the six negative control wells was set to 1, and for each well in the plate, the fold induction was calculated in relation to this value.
- For wells in which a statistically significant gene-induction (using a student test, also called T-test) over the 1.5 threshold was found, the following parameters were calculated from the processed raw data:
- Imax: maximal induction factor of luciferase activity compared to the negative control over the complete dose-response range measured,
- EC1.5: concentration at which a 1.5-fold luciferase gene induction is obtained,
-IC50 and IC30: concentrations effecting a reduction of cellular viability by 50% and 30%,
- Indication whether significant 1.5-fold gene induction occurred below the IC30.
- The data were plotted in graphs and the Imax and the EC1.5 values were visually checked since uneven dose-response curves or large variation may lead to wrong extrapolations. Also, the individual and overall geometric means IC50 and IC30 were calculated, when applicable.

ACCEPTANCE AND EVALUATION CRITERIA

- Acceptance criteria: Each run was considered valid if the following criteria were met:
- at least 2 consecutive concentrations should have a viability ≥ 70%,
- the positive control results should be positive, thus the gene induction should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations,
- the average EC1.5 value for the positive control should be within two standard deviations of the historical mean. In addition, the average induction (Imax) in the three replicate plates for the positive control at 64 μM should be between 2 and 8. If the latter criterion was not fulfilled, the dose-response of cinnamic aldehyde are carefully checked, and the run is accepted if there was a clear dose-response with increasing luciferase activity at increasing concentrations for the positive control,
- the average coefficient of variation of the luminescence reading in the negative control wells of the triplicate plates should be < 20%.

- Evaluation criteria of the test item: The results of each run are analyzed individually and if the test item is classified as positive in two runs, the final outcome is considered positive. If the test item is classified as negative in two runs, the final outcomeis negative.
The test item is considered as positive if the following four conditions are all met in two of two or in two of three runs, otherwise the KeratinoSens prediction is considered as negative:
- the Imax is > 1.5-fold and statistically significantly different as compared to the negative control (as determined by a two-tailed, unpaired Student’s T-test),
- at the lowest concentration with a gene induction > 1.5-fold (i.e. at the EC1.5 determining value), the cell viability is > 70%,
- the EC1.5 value is < 1000 μM (or < 200 μg/mL for test item without MW),
- there is an apparent overall dose-response for luciferase induction (or a reproducible biphasic response).

Positive control results:

Viability (%), induction values, Imax, IC30, IC50 and EC1.5 obtained with the positive control are presented in Tables 4 and 5 in the section "any other information incl. tables".

Key result

Run / experiment:

other: 1

Parameter:

other: Imax

Remarks:

at 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM in culture medium containing 1% DMSO.

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: no precipitate/emulsion observed in any wells at the end of the 48-hour treatment period; cell viability > 70%; no statistically significant gene-fold induction above the threshold of 1.5 (compared to the negative control) at any tested concentration.

Key result

Run / experiment:

other: 2

Parameter:

other: at 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM in culture medium containing 1% DMSO.

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: no precipitate/emulsion observed in any wells at the end of the 48-hour treatment period; cell viability > 70%; no statistically significant gene-fold induction above the threshold of 1.5 (compared to the negative control) at any tested concentration.

Other effects / acceptance of results:

SOLUBILITY RESULTS:
- In the solubility test, the test item was found soluble in DMSO, water and treatment culture medium at 200 mM and 100 mM, as well as once all these formulations were 100-fold diluted in culture medium. DMSO was therefore retained as the vehicle to be used for the preparation of the test item stock formulations.

KERATINOSENS RUN:
The two runs were performed using the following concentrations: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM in culture medium containing 1% DMSO. At these tested concentrations and for both runs, the following results were obtained:
- no precipitate/emulsion were observed in any wells at the end of the 48-hour treatment period,
- no noteworthy decrease in cell viability was noted (i.e. cell viability > 70%),
- no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Therefore the Imax values were < 1.5.

The Imax, IC30, IC50, EC1.5 and viability values obtained for cells treated with test item in each run as well as the mean and SD values are presented in Tables 1, 2 and 3 in the section "any other information incl. Tables".
The viability values (%), induction values, Imax, IC30, IC50 and EC1.5 values obtained with the positive control are presented in Tables 4 and 5 in the section "any other information incl. Tables". In addition the luminescence values of all negative control wells and the %CV between these values for each run are also presented in Table 6 in the section "any other information incl. Tables".

OTHER EFFECTS:
- Visible damage on test system: no effect.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Cinnamic aldehyde, used as positive control, showed positive response to the Keratinosens test as expected.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: All acceptance criteria were fulfilled for negative controls.
- Acceptance criteria met for positive control: All acceptance criteria were fulfilled for the positive controls.
- Acceptance criteria met for variability between replicate measurements: yes.
- Range of historical values is prensented in Table 7 in the section "any other information incl. Tables".

Table 1. Evaluation of the viability (%) of cultures treated with the test item for each run:

 

	Concentrations (µM)
MALTITOL	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Viability (%) in Run	102	102	105	107	103	101	104	102	101	107	106	104
Viability (%) in Run 2	124	124	109	122	131	113	135	110	109	102	102	104
Meanviability(%)	113	113	107	114	117	107	119	106	105	104	104	104
GeometricMean(%)	112	112	107	114	116	107	118	106	105	104	104	104
SD	15	15	3	11	19	9	22	5	5	3	3	1

 

Table 2.Gene induction values, mean and SD values obtained after treatment with the test item in each run:

 

	Concentrations (µM)
MALTITOL	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Induction values in Run 1	1.0	1.0	1.1	1.1	1.2	1.2	1.2	1.1	1.3	1.1	1.3	0.9
Induction values in Run 2	1.0	1.0	1.1	1.1	1.0	1.2	1.1	1.0	1.1	1.0	1.1	1.0
Meaninduction	1.0	1.0	1.1	1.1	1.1	1.2	1.2	1.1	1.2	1.1	1.2	1.0
SD	0.0	0.0	0.0	0.0	0.1	0.0	0.1	0.1	0.2	0.0	0.2	0.1

 

Table 3.Imax, IC30, IC50and EC1.5results after treatment with the test item in each run:

 

MALTITOL	Imax	EC1.5 (µM)	IC50 (µM)	IC30 (µM)
Run 1	1.33	-	-	-
Run 2	1.24	-	-	-
Mean	1.28	n.r.	n.r.	n.r.
GeometricMean	n.r.	-	-	-
SD	0.07	-	-	-

- : no data available

n.r.: not requested by the OECD Guideline

 

 

Table 4. Evaluation of the viability (%) of cultures treated with the positive control for each run:

 

	Concentrations (µM)
Cinnamicaldehyde	4	8	16	32	64
Viability (%) in Run	101	104	109	113	96
Viability (%) in Run 2	110	108	107	114	96
Meanviability(%)	106	106	108	113	96
GeometricMean(%)	106	106	108	113	96
SD	6	3	1	1	0

 

Table 5.Gene induction values,Imax, IC30, IC50 and EC1.5 values obtained with the positive control for each run:

 

	Concentrations (µM)
Cinnamic aldehyde	4	8	16	32	64	Imax	EC1.5 (μM)	IC50 (μM)	IC30 (μM)
Run 1	1.1	1.6	1.9	2.2	4.7	4.69	7.28	-	-
Run 2	1.5	1.2	2.1	2.7	6.2	6.23	10.70	-	-
Mean	1.3	1.4	2.0	2.5	5.5	5.46	n.r.	n.r.	n.r.
GeometricMean	n.r.	n.r.	n.r.	n.r.	n.r.	n.r.	8.82	-	-
SD	0.3	0.3	0.1	0.4	1.1	1.09	2.42	-	-

- : no data available

n.r.: not requested by the OECD Guideline                       

 

Table 6.Luminescence values for the negative control wells and the %CV between these values for each run:

 

Negative control	Luminescence reading							Mean	% CV
Run 1	Replicate 1	520931	528199	539373	543423	552912	575423	533688	9.16
	Replicate 2	504490	452077	482144	535292	623383	601672
	Replicate 3	582955	574600	487239	490317	452705	559244
Run 2	Replicate 1	1062450	1219460	988741	921754	1026200	1042570	1004715	16.39
	Replicate 2	834206	875791	856488	673151	1029270	806489
	Replicate 3	1106230	1005110	1067530	1016410	1169610	1383410

 

Table 7. Historical control data (from 14 October 2016 to 03 October 2017):

 

Control items	Negative control		Positive control
Parameter	Mean RLU	%CV	EC1.5	Imax
n	32	32	32	32
Mean	456682.9	14.9	8.4	8.2
SD	209768.4	4.1	2.8	5.4
Lower CL 95%	381053.3	13.4	7.4	6.2
Upper CL95%	532312.5	16.3	9.4	10.1
5^thPercentile	181303.0	8.4	3.9	2.8
Median	411488.5	14.8	7.9	5.9
95^thPercentile	845930.0	22.5	13.7	17.1
Min	171403.0	8.3	3.8	2.7
Max	983733.0	25.7	14.8	22.7
Mean – 2SD	37146.2	6.8	2.8	/
Mean + 2SD	876219.7	23.0	13.9	18.9

CL: Confidence limit

CV: Coefficient of Variation

EC_1.5: Extrapolated concentration for a 1.5 fold luciferase gene indication

I_max: Maximal induction factor of luciferase activity compared to the negative control over the complete dose response range measured

Min: Minimal value

N: number of values.

RLU: Relative Luminescence Unit

SD: Standarddeviation

/ :notapplicable,negativecalculatedvalue

 

 

 

 

 

 

Interpretation of results:

other: This test is a part of a tiered strategy for the evaluation of skin sensitisation potential in the context of an integrated approach to testing and assessment.

Conclusions:

Under the experimental conditions of this study, the test item, MALTITOL, was negative in the KeratinoSens assay.

Executive summary:

This GLP compliant study was performed to evaluate the potential of the test item, MALTITOL, to activate the Nrf2 transcription factor in the KeratinoSens cell line. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase. This in vitro study was performed according to the OECD Guideline 442D: In Vitro Skin Sensitisation assays addressing the AOP key event on keratinocyte activation, adopted on June 2018.

This test is a part of a tiered strategy for the evaluation of skin sensitisation potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

Material and methods

The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed.

Results

All acceptance criteria were fulfilled for the positive and negative controls. These runs were therefore considered to be valid.

For each run, the test item was dissolved in DMSO at 200 mM.

Both runs were performed using the following concentrations: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM in culture medium containing 1% DMSO. At these tested concentrations and for both runs, the following results were obtained:

- No precipitate/emulsion were observed in any wells at the end of the 48-hour treatment period,

- No noteworthy decrease in cell viability was noted (i.e. cell viability > 70%),

- No statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Therefore the Imax values were < 1.5.

Conclusion

Under the experimental conditions of this study, the test item, MALTITOL, was negative in the KeratinoSens assay.

Reference 5

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 13 September 2018 to 01 February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD guideline No. 442E: In vitro skin sensitization assays addressing the Key Event on activation of dendritic cells on the adverse outcome pathway for Skin sensitization adopted 25 June 2018.

Version / remarks:

With the following exceptions:
- Reactivity check was performed for each ATCC batch of cells and each working cell bank, and not each time frozen cells are thawed. Validation of cells reactivity was guaranteed in each run by running concurrently both positive controls (NiSO4 and DNCB) instead of only one (DNCB),
- According to the OECD guideline No. 442E, the first dose-range finding (DRF) assay should be performed at the maximum concentration of 1000 µg/mL before running another assay with a maximum concentration of 5000 µg/mL if no cytotoxicity is noted in the first assay. In the present study design, the first DRF assay was directly performed at concentrations greater than 1000 µg/mL if allowed by solubility. Depending on the viability values obtained in the first DRF, the concentration range used for the second DRF may have been adapted accordingly,
- When preparing cells for treatment, they were seeded between 0.1 and 0.2 x 106 cells/mL before incubating them for 48h to 72 hours (as noted in the DB-ALM protocol), instead of at 0.2 x 106 cells/mL for 48h incubation, or 0.1 x 106 cells/mL for 72h as noted in the OECD guideline.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor; Batch/Lot number: ELEL7
- Expiration date of the lot/batch: 07 October 2022

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: not applicable
- Specific activity: not applicable
- Locations of the label: not applicable
- Expiration date of radiochemical substance: not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: controlled room temperature
- Stability under test conditions: No chemical analysis of the test item formulations was performed as part of this study. For this short-term study, the formulations were prepared within 4 hours before treatments.
- Solubility and stability of the test substance in the solvent/vehicle: based on the results of the solubility test, 0.9%NaCl was selected as vehicle
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: see the "Solubility assessment" and "TEST ITEM AND CONTROLS PREPARATION" sections in "Details on study design"
- Final preparation of a solid: see the "Solubility assessment" and "TEST ITEM AND CONTROLS PREPARATION" sections in "Details on study design"

FORM AS APPLIED IN THE TEST (if different from that of starting material): diluted in 0.9%NaCl.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): not applicable

OTHER SPECIFICS: none

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

SOLUBILITY ASSESSMENT
The solubility of the test item was assessed visually for each preparation (particles, drops, cloudiness, non-miscible phases, etc) and recorded in the study files. A preparation was deemed appropriate for cell treatment as long as it was qualified as a solution or stable dispersion (homogenous emulsion/suspension). Saline (0.9% NaCl) and DMSO are the only vehicles allowed in the assay. The vehicle was chosen between these two in the order of preference, and in accordance with the steps described below:
- First, the test item was dissolved in saline at 100 mg/mL. If the test item was soluble or gave a stable dispersion (homogenous emulsion/suspension) saline was used as a vehicle and the Highest Soluble Concentration (HSC) was determined by testing greater concentrations as follows: 300 mg/mL -> 500 mg/mL.
- If the test item was not soluble at the concentration of 100 mg/mL, it was dissolved at 500 mg/mL in DMSO.
- In case it was not soluble at 500 mg/mL in DMSO, the HSC was determined by testing lower concentrations in a common ratio of two (250 mg/mL ->125 mg/mL -> continued if needed). Minimal possible concentration was 1 mg/mL in DMSO.
- When a solution or a stable dispersion was obtained, no solubility evaluation was performed after a further 100-fold dilution in RPMI-1640 culture medium.
- Neither heating nor sonication was used to improve test item solubilization since their impacts on test item integrity are not known.

TEST ITEM AND CONTROLS PREPARATION
1) Positive controls preparation
- The positive control DNCB was prepared at 8 μg/mL in DMSO as follows:
. on the treatment day, the required quantity of DNCB was mixed with DMSO at the concentration of 2 mg/mL,
. this solution was then 250-fold diluted in cRPMI in order to obtain a 8 μg/mL DNCB stock solution.
- The positive control NiSO4 was prepared at the concentration of 200 μg/mL in 0.9% NaCl as follows:
. on the treatment day, the required quantity of NiSO4 was mixed with 0.9% NaCl at the concentration of 10 mg/mL,
. this solution was then 50-fold diluted in cRPMI in order to obtain a 200 μg/mL NiSO4 stock solution.
Both positive control stock solutions were prepared within 4 hours before use, and kept at room temperature and protected from light until use.

2) Vehicle control preparation: As 0.9% NaCl was the vehicle selected at completion of the solubility assay, no vehicle control was included in the testing phases and test item formulations data was compared to data obtained from cells treated with culture media (cRPMI).

3) Test item preparation:
- All test item preparations were prepared in glass vials only. Fresh stock formulations of the test item were prepared for each run, using the vehicle and concentration identified in the "Solubility assessment section". These concentrations were the same for all runs. Test item stock formulations prepared in 0.9% NaCl were 100 x concentrated, and then 2 x concentrated formulations were prepared by 1:50 dilution in cRPMI.
- The aspect of the stock formulations was evaluated and recorded in the study files.
- The precipitation in the treatment conditions (i.e. when diluted in cRPMI) was checked and any observation was reported in the study files.
- The test item formulations were kept at room temperature and protected from light until use, i.e. within 4 hours after preparation of the stock formulations. No control of concentration was performed during the study.

TEST SYSTEM
1) Cells: The THP-1 is an immortalized human monocytic leukemia cell line derived from an acute monocytic leukemia patient. The THP-1 cell line is obtained from ATCC (Ref: TIB-202, American Type Culture Collection, Manassas, USA) by the intermediate of LGC Standards (Molsheim, France). The THP-1 cells are stored in a cryoprotective medium in a liquid nitrogen container. Cells were grown using general culture procedures. They were cultured in cRPMI medium and maintained in a humidified incubator set at 37°C, 5% CO2 and were not allowed to exceed a cell density of 1 x 106 cells/mL or more than 30 passages. The culture medium (cRPMI) was composed of RPMI 1640 with 10% FBS, 0.05 mM 2-mercaptoethanol and with penicillin and streptomycin. During cell culturing, cell viability was checked using trypan blue.

2) Cell culture for testing: For testing, THP-1 cells were seeded at a density between 0.1 x 106 cells/mL and 0.2 x 106 cells/mL, and pre-cultured in culture flasks for 48 hours to 72 hours, respectively. Cell did not exceed density of 1 x 106 cells/mL. On the day of testing, cells harvested from culture flasks were resuspended with fresh culture medium at 2 x 106 cells/mL. Then, 500 μL of cells suspension were distributed into a 24-well flat-bottom plate (i.e. 1 x 106 cells/well).

3) Reactivity check: Two weeks after thawing, a reactivity check was performed to qualify the cells of each working cell bank before testing. A reactivity check assay was performed by testing cell response after contact with Lactic Acid, DNCB and NiSO4 under an internal Citoxlab France reference number.
Results from reactivity check tests are compiled in Citoxlab France internal data, with negative and positive control data obtained during each test.
Results from the last reactivity check assay were presented in the study report (not reported in the current Endpoint Study Record).

STUDY DESIGN
The study was divided in two successive phases. First, a Dose-Range Finding assay (DRF) was performed to assess test item toxicity. Secondly, based on cytotoxicity data obtained from the DRF, a concentration series was tested in successive runs in the main test. At each phase, all information relating to test item concentrations and run identification were given by the Study Director in the study files and no study plan amendment was issued for that purpose.
1) DOSE-RANGE FINDING TEST (DRF): The DRF consisted in two separated assays.
Treatments of DRF assays were performed at the following concentrations (final concentrations): 39.06, 78.13, 156.25, 312.50, 625, 1250, 2500 and 5000 μg/mL.
Each assay was performed as described here below:
- Test item stock solutions were prepared at eight different concentrations by 2-fold dilutions using the selected vehicle. These stock formulations were then 50-fold diluted (as 0.9%NaCl is the selected vehicle) into cRPMI to obtain working solutions.
- The working solutions were finally used for exposure by adding 500 μL of working solutions to the volume of THP-1 cell suspension in the plate (500 μL) to achieve a further 2-fold dilution. In order to avoid evaporation of volatile chemicals and cross-contamination between wells, a sealer was placed on each 24-well plate just after treatment, before putting the plastic lids back on each plate.
- The treated plates were then incubated for 24 hours ± 30 minutes in a humidified incubator set at 37°C and 5% CO2.
- At the end of the treatment phase, an inspection under microscope was performed to each well. The presence or absence of precipitate/emulsion was recorded. Cells were then cells were transferred into sample tubes and collected by centrifugation. The supernatants were discarded and the remaining cells were re-suspended with 600 μL of FACS buffer. Finally, cells were re-suspended in 200 μL FACS buffer and the plate was positioned into the plate-reader of the flow cytometer. A volume of 10 μL of Propidium Iodide (PI) solution at 12.5 μg/mL was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 μg/mL per well.

2) MAIN TEST: The main test consisted in two separated runs being performed as described here below.
- Test item stock solutions were prepared at eight different concentrations by 1.2-fold dilutions using the selected vehicle. The highest concentration corresponded to the highest achievable non-cytotoxic concentration as no CV75 was obtained. The maximum concentration in the plates was 5000 μg/mL.
- All stock formulations were then 50-fold diluted into cRPMI to obtain working solutions.
- In parallel, the working solutions of positive controls DNCB and NiSO4 were prepared as noted in "Test item and controls preparation".
- All working solutions were finally used for exposure by adding 500 μL of working solutions to the volume of THP-1 cell suspension in the plate (500 μL) to achieve a further 2-fold dilution. In order to avoid evaporation of volatile chemicals and cross-contamination between wells, a sealer was placed on each 24-well plate just after treatment, before putting the plastic lids back on each plate.
- The treated plates were then incubated for 24 hours ± 30 minutes in a humidified incubator set at 37°C and 5% CO2.
- During the main test, treatments were performed at the following final concentrations in runs A and B: 1395.41, 1674.49, 2009.39, 2411.27, 2893.52, 3472.22, 4166.67 and 5000 μg/mL.
- At the end of the treatment phase, an inspection under microscope was performed to each well. The presence or absence of precipitate/emulsion was recorded. Cells were then transferred into sample tubes and collected by centrifugation, washed twice with 1 mL FACS buffer and blocked with 600 μL of blocking solution and incubated at +4°C for 15 minutes (± 1 minute). After blocking, cells were split in three aliquots of 180 μL into a 96-well round bottom plate and centrifuged before staining with antibodies. A volume of 50
μL of FITC-labeled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies prepared in FACS buffer was added to each aliquot before incubation for 30 minutes (± 2 minutes) at +4°C.
- Finally, cells were washed with 150 μL FACS buffer two times and re-suspended in 200 μL FACS buffer.
- The plate was then positioned into the plate-reader of the flow cytometer. A volume of 10 μL of PI solution at 12.5 μg/mL was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 μg/mL per well.

Positive control results:

Positive controls gave the expected results for both runs:
- DNCB gives RFI(CD86) ≥ 150 and RFI(CD54) ≥ 200 with mean viability > 50%
- NiSO4 gives RFI(CD86) ≥ 150 and RFI(CD54) ≥ 200 with mean viability > 50%

MFI: Mean Fluorescence Intensity
RFI: Relative Fluorescence Intensity

Key result

Run / experiment:

other: A

Parameter:

other: RFI CD86

Remarks:

at all concentrations tested (cell viability ≥ 95%)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no precipitate/emulsion was noted in treated wells.

Key result

Run / experiment:

other: A

Parameter:

other: RFI CD54

Remarks:

at all concentrations tested (cell viability ≥ 95%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not valid

Positive controls validity:

valid

Remarks on result:

other: no precipitate/emulsion was noted in treated wells,

Key result

Run / experiment:

other: B

Parameter:

other: RFI CD86

Remarks:

at all concentrations tested (cell viability ≥ 95%)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no precipitate/emulsion was noted in treated wells.

Key result

Run / experiment:

other: B

Parameter:

other: RFI CD86

Remarks:

at all concentrations tested (cell viability ≥ 95%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no precipitate/emulsion was noted in treated wells.

Other effects / acceptance of results:

1) Controls acceptance criteria valid: yes
. Viability of cells treated with cRPMI and DMSO controls should was ≥ 90%,
. in cRPMI and DMSO control wells, MFI ratio of both CD86 and CD54 to isotype control were > 105%,
. in the DMSO control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI > 150% and CD54 RFI ≥ 200%),
. in the positive controls (DNCB and NiSO4), RFI values of both CD86 and CD54 met the positive criteria (CD86 RFI ≥ 150 and CD54 RFI ≥ 200) and cell viability was more than 50%.

2) Test item acceptance criteria: no cell viability decrease below 75% was found at any tested concentration (cell viability of at least four out of eight concentrations was > 50%).

1) SOLUBILITY ASSESSMENT

Results obtained from the solubility assay are summarized in the table below:

Table 1. Solubility assay results

Vehicle	Concentration (mg/mL)	Aspect	Retained vehicle and maximum stock concentration?
0.9%NaCl	100	Colorless solution	No
	300		No
	500		Yes

 

Therefore, 0.9%NaCl was selected as vehicle, and the following test item concentrations were tested in the DRF phase: 39.06, 78.13, 156.25, 312.50, 625, 1250, 2500 and 5000μg/mL.

2) DOSE RANGE FINDING (DRF) RESULTS

Results from each DRF assay are presented in Tables 2 and 3 below:

Table 2. Dose range finding results for assay 1 (DRF1)

Concentration (μg/mL)	Viability(%)
cRPMI	97.89
39.06	97.80
78.13	97.31
156.25	96.59
312.50	96.59
625.00	96.59
1250.00	96.98
2500.00	97.19
5000.00	96.57

 

Table 3. Dose range finding results for assay 2 (DRF2)

Concentration (μg/mL)	Viability(%)
cRPMI	96.27
39.06	94.05
78.13	93.32
156.25	92.86
312.50	95.26
625.00	94.77
1250.00	93.39
2500.00	94.85
5000.00	94.84

 

The following results were obtained in both DRF assays (i.e.DRF 1 and DRF 2):

- At post-treatment observation, no abnormalities such as precipitate/emulsion or cell morphology modification was observed at any tested concentrations,

- Flow cytometry measurement afterPropidiumIodide staining revealed no cell viability decrease below 75% at any tested concentration. Therefore, no CV75 value was calculated.

Based on the results from both DRF assays, no mean CV75 was calculated, and the maximum concentration tested in the main test was therefore 5000μg/mL.

3) MAIN TEST: Individual run results (Run A and B) were presented in the study report (not presented in the current Endpoint Study Record).All acceptance criteria were reached in each run. The results for both runs aresummarisedin table 4.

Runs A and B:

- Post-treatment observations: no precipitate/emulsion was noted in treated wells,

- Run outcome: RFI CD86 and RFI CD54 did not exceed the positivity thresholds at any tested concentration. The run was therefore considered negative.

Table 4. Summary results of the main test (run A and B)

Test item	Conc. (μg/mL)	RFI for CD86		RFI for CD54		Viability(%)		Runconclusion		General conclusion
		A	B	A	B	A	B	A	B	Negative
MALTITOL	1395.41	110	92	97	74	96	97	N	N
	1674.49	94	95	89	61	96	97
	2009.39	107	79	86	70	95	97
	2411.27	101	79	51	74	96	97
	2893.52	103	79	71	57	96	97
	3472.22	94	72	80	39	95	97
	4166.67	96	81	71	48	96	97
	5000	86	83	126	70	96	96

 

Interpretation of results:

other: this test is a part of a tiered strategy for the evaluation of skin sensitisation potential in the context of an integrated approach to testing and assessment.

Conclusions:

Under the experimental conditions of this study, the test item, MALTITOL, was found to be negative in the human Cell Line Activation Test (h-CLAT) method.

Executive summary:

This GLP compliant study was performed to determine the ability of the test item to induce an increase in cell surface markers expression in the human monocytic leukaemia cell line THP-1, using the human Cell Line Activation Test (h-CLAT) method. This in vitro study was performed according to the OECD guideline 442E: in vitro skin sensitization assays addressing the Key Event on activation of dendritic cells on the adverse outcome pathway for Skin sensitization", 25 June 2018. This test is a part of a tiered strategy for the evaluation of skin sensitisation potential in the context of an integrated approach to testing and assessment.

Material and methods

A solubility assessment was first performed in the vehicle 0.9% NaCl to select the highest concentration to be used for test item formulation preparations.

Following the solubility assays, the cytotoxic potential was assessed in a Dose-Range Finding assay (DRF) in order to select sub-toxic concentrations for testing in the main test. The skin sensitizing potential of the test item was then tested in the main test in two successive runs (A and B). In each run, the test item formulations were applied to THP-1 cells and cultured in a 24-well plate for 24h ± 30 minutes at 37°C, 5% CO2 in a humidified incubator. A set of control wells was also added in each plate to guarantee the validity of each run. At the end of the incubation period, cells from each well were distributed to three wells of 96-well plate: the first well was labeled with IgG1-FITC antibodies, the second one was labeled with CD86-FITC antibodies and the third one was labelled with CD54-FITC antibodies. Then, just before flow cytometry analysis of CD86 and CD54 expression, all cells were dyed with Propidium Iodide for viability discrimination.

For each run, the Mean Fluorescence Intensity (MFI) obtained for each test sample was corrected by the isotype control IgG1 MFI value to obtain the corrected MFI. Corrected MFI value from the corresponding vehicle control was set to 100% CD54 and CD86 expression by default. Then, corrected MFI values from each test sample were compared to the corresponding vehicle control to obtain the Relative Fluorescence Index for CD86 and CD54 expression for each tested concentration (RFI CD86 and RFI CD54).

Results

Solubility assessment: The test item was found soluble in 0.9% NaCl at 500 mg/mL.

Dose-Range Finding (DRF): The test item did not decrease cell viability < 75% during both DRF runs. No mean CV75 value was therefore calculated, and the highest tested concentration retained for the main tests was 5000 μg/mL.

Main test:

All acceptance criteria were reached in each run (A and B).

No precipitate/emulsion was noted in treated well during the post-treatment observations.

Runs A and B outcome: RFI CD86 and RFI CD54 did not exceed the positivity thresholds at any tested concentration. The runs were therefore considered negative.

Conclusion

Under the experimental conditions of this study, the test item, MALTITOL, was found to be negative in the human Cell Line Activation Test (h-CLAT) method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In order to assess the skin sensitisation potential of the registered substance Maltitol, a combination of in chemico / in vitro tests performed on the target substance and in vivo read-across data was used in a Weight-of-Evidence approach as recommended in the Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance (v 6.0, July 2017).

In chemico / in vitro skin sensitisation data is available on the target substance Maltitol and the tests were performed in the context of a tiered strategy for skin sensitisation assessment. All three tests adressing the three key events of the skin sensitisation adverse outcome pathway (AOP) were conducted as the test substance felt into the applicability domain of each of them.

The Direct Peptide Reactivity Assay (DPRA) which adresses the molecular initiating event (KE1) of the skin sensitisation AOP, was performed to evaluate the in chemico reactivity of Maltitol (dissolved in milli-Q water) by monitoring peptide depletion following a 24h contact between the test item and synthetic cysteine and lysine peptides (Reliability 1; OECD 442C; CitoxLAb France; 2019). The study was considered to be valid as acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The results showed that the mean of the percentage cysteine and percentage lysine depletions was equal to 0.82%. Hence, the DPRA prediction was considered as negative and Maltitol was considered to have no or minimal peptide reactivity.

The KeratinoSens test adressing the second key event of the skin sensitisation AOP (keratinocyte activation, KE2) was performed to evaluate the potential of the registered substance to activate the Nrf2 transcription factor in the KeratinoSens cell line (Reliability 1; OECD 442D; CitoxLAb France; 2019). Two independant valid runs were performed with all acceptance criteria fulfilled for both positive and negative controls. Maltitol was dissolved in DMSO and both runs were performed using the following concentrations: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM. For both runs no precipitate or emulsion was observed in any well at the end of the treatment period and no noteworthy decrease in cell viability was noted (i.e. cell viability > 70%). The results of this test showed no statistically significant gene-fold induction above the threshold of 1.5 in comparison to the negative control at any tested concentration. The Imax values were < 1.5 and the test item Maltitol was thus negative in the KeratinoSens assay.

The h-CLAT study that adresses the third key event (dendritic cells activation; KE3) was performed to determine the ability of Maltitol to induce an increase in cell surface markers expression in the human monocytic leukaemia cell line THP-1 (Reliability 1; OECD 442E; CitoxLAb France; 2019). The skin sensitizing potential of the test item was tested in two successive runs (A and B). In each run, the test item formulations (in 0.9% NaCl) were applied to THP-1 cells and cultured in a 24-well plate for 24h ± 30 minutes at 37°C, 5% CO2 in a humidified incubator. As Maltitol did not decrease cell viability in the dose-range finding test, the highest tested concentration was 5000 µg/mL. A set of control wells was also added in each plate to guarantee the validity of each run. Corrected Mean Fluorescence Intensity values (MFI) from each test sample were compared to the corresponding vehicle control to obtain the Relative Fluorescence Index for CD86 and CD54 expression for each tested concentration (RFI CD86 and RFI CD54). All acceptance criteria were reached in each run (A and B) and no precipitate or emulsion was noted in treated well during the post-treatment observations. The results of this test showed that RFI CD86 and RFI CD54 did not exceed the positivity thresholds at any tested concentration. Thus, Maltitol was found to be negative in the h-CLAT test.

According to the results of the in chemico / in vitro tests adressing the three key events of the skin sensitisation AOP, Maltitol was found to be negative.

An in vivo skin sensitization study (Reliability 1; OECD 429; Seibersdorf Labor GmbH, 2010) is available on the relevant analogue substance Syrups, wheat, hydrolyzed starch (No CAS / EC 931-687-3) which consists primarily of D-glucose, maltose and maltodextrins. The preliminary solubility testing showed that the highest concentration suitable for application of the test substance can be achieved with dimethylformamide (DMF) as vehicle was 14.9%, w/w. Three concentrations of Syrups, wheat, hydrolyzed starch (5, 10 and 14.9% in DMF) were administered to the dorsal surfaces of the ears of mice (5 mice/ group). Negative and positive control groups were treated the same way as the test substance but with DMF or Hexyl cinnamic aldehyde (HCA) respectively. Each animal was treated for 3 consecutive days. 3 days after the last administration, the proliferation of the lymphocytes of the draining lymph nodes was measured by the determination of amounts of incorporated radioactive ³H-methyl thymidine (3HTdR). Negative and positive control results were as expected. The Stimulation Indexes (SI) of the test substance were all clearly below 3 (0.7, 0.6 and 0.7 for the low, mid and high dose groups respectively). Hence, under the conditions of this test, the analogue substance Syrups, wheat, hydrolyzed starch was not found to be a skin sensitizer.

According to the weight of evidence approach and based on the negative results of the three in chemico / in vitro tests performed on the target substance in combination with the negative result of the relevant in vivo read across study, the registered substance Maltitol is not considered to be a skin sensitizer

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin sensitisation: According to the negative results obtained with the registered substance in the three key in chemico / in vitro skin sensitisation studies OECD 442 C, OECD 442 D, OECD 442 E guidelines, CitoxLab France, 2019), together with the negative result in the in vivo read-across key study (OECD 429 test guideline, Seibersdorf Labor GmbH, 2010), the substance Maltitol is not classified for skin sensitisation according to the CLP criteria.

Respiratory sensitisation: no data available.