MoVNbTe mixed oxide

Endpoint summary

Description of key information

The test item was supposed to be tested with the OECD TG standardized in vitro skin sensitisation testing battery (OECD TG 442 C-E). However, OECD TG 442 C & D were not feasible with the test material due to its negligible solubility. A respective statement by the CRO testing the material was authored and attached to this dossier. Due to the fact that no decision can be made by OECD TG 442 E as stand alone assay, an in vivo skin sensitisation assay (OECD TG 406 - Buehler test) was performed. 

The test item was evaluated in the “In Vitro Skin Sensitization by Human Cell Line Activation Test (h-CLAT) Method” as per the OECD guideline for testing of chemicals, No.: 442E. The measured expression levels of CD86 and CD54 cell surface markers are used for supporting the discrimination between skin sensitisers and non-sensitisers. Study was conducted to determine the cell viability (CV-75), expression level of CD86 and CD54 cell surface markers expression using human monocytic leukaemia cell line THP-1 in the h-CLAT method. Solubility test was conducted by RPMI 1640 media and dimethyl sulphoxide. Test item formed suspension in RPMI 1640 media at 500 mg/mL. On the day of testing, test item was prepared. Eleven concentrations were prepared for dose finding assay, RPMI media was used as solvent/vehicle, the final concentrations in plate was 4.883, 9.766, 19.531, 39.063, 78.125, 156.25, 312.5, 625, 1250, 2500 and 5000µg/mL. The CV75 value of the test item was determined at 3.3105 µg/mL. Based on the CV75 of dose finding assay, eight test concentrations of 1.109, 1.331, 1.597, 1.916, 2.299, 2.759, 3.311 and 3.973µg/mL were selected for CD86/CD54 expression measurement. Upregulation of the biomarkers CD86 and/or CD54 was observed in some of the tested concentration of the test item. EC150 value for CD86 was 1.13 and 1.4 µg/mL for run 1 and 2 respectively. EC200 for CD 54 was 1.51 and 2.07 µg/mL for run 1 and 2 respectively. Based on the expression levels of CD86 and CD54, the test item can be considered as "sensitiser" within the h-Clat assay. The data generated with this method is however not sufficient to conclude in isolation on the skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA. Since further in vitro studies were not feasible, no conclusion on the sensitising properties via in vitro studies can be made. Hence, a subsequent in vivo study was performed.

The OECD TG 406 was performed as Buehler test method as it is regarded as the best option for this kind of test material. The study was conducted in two phases viz., pre study and the main study. For pre study, the dose of 25 mg and 50 mg of test item (moistened with 0.1 mL of 80% ethanol/water) were applied topically to the anterior left flank and posterior left flank respectively, whereas 75 mg and 100 mg of test item (moistened with 0.1 mL of 80% ethanol/water) were applied topically to the anterior right flank and posterior right flank respectively to each animal. No skin reactions were observed in any of the test dose approximately at 24 and 48 hours observation period after patch removal. In pre study (G1), no skin reactions were observed at the highest dose i.e. 100 mg of test item. Hence, 100 mg of test item was selected for induction and challenge phase of main study. The main study included three inductions on day 1, 8 and 15 and challenge on day 29. On induction day 1, 8 and 15, 0.1 mL of 80% ethanol/water and 100 mg of test item moistened with 0.1 mL of 80% ethanol/water were applied topically. On challenge day 29, 0.1 mL of acetone was topically applied to the anterior part of right flank, whereas 100 mg of test item moistened with 0.1 mL of acetone was topically applied to the posterior part of the right flank of all the animals. After 6 hours of contact period, the test patches were removed and the application sites were cleaned with normal saline swabs and dried. During induction phase, the test item application sites were observed for skin reactions approximately at 1 and 24 hours post removal of the test patches according to Draize method (1959) and during challenge phase the skin reactions were observed approximately at 24 and 48 hours post removal of the test patch according to Magnusson and Kligman grading scale. In induction phase, no skin reactions were observed in vehicle control and treatment group animals approximately at 1 and 24 hours of observation period of post patch removal. In challenge phase, theanimals did not reveal any skin reactions at anterior and posterior part of right flank of vehicle control and treatment group animals approximately at 24 and 48 hours of post patch removal. No clinical signs of toxicity and mortality were observed till termination. All the treated animals revealed physiologically normal increase in body weights during the observation period.

 

Under the experimental conditions employed and based on the results of the experiment, it is concluded that the test item did not show any skin sensitisation or allergic potential in Guinea pigs. Hence, the test item does not meet classification criteria according to the Globally Harmonized System (GHS) of Classification and Labelling of Chemicals.

 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 February 2021 TO 24 June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

As per OECD Guideline No. 442E

Qualifier:

according to guideline

Guideline:

other: As per OECD Guideline No. 442E

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: EX.14402.600
- Purity test date: 87.6%
- Solubility and stability of the test substance in the solvent/vehicle: RPMI 1640 media
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: RPMI 1640 media

Details of test system:

THP-1 cell line [442E]

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design: Frozen stock of cryovial was thawed immediately at 37±1°C in the water bath.
cells were transferred into 15 mL falcon tube with 5 mL culture media, cells were centrifuged at 125g for 10 minutes. Cells pellet were transferred in to a sterile flask with RPMI-1640 supplemented with 10% Fetal Bovine Serum, antibiotics (1% Penicillin-Streptomycin) and 0.05 mM 2-Mercptoethanol
incubated at 37±1°C and 5±1% CO2 for 4 days.
On the day of testing, cells harvested from culture flask were resuspended with fresh culture medium at 2 × 106 cells/mL.
Then, cells are distributed into a 24 well flat-bottom plate with 500 µL (1 × 106 cells/well). The cells were qualified by conducting a reactivity test using the positive controls,
2,4-dinitrochlorobenzene (DNCB) and nickel sulfate (NiSO4) and the negative control, lactic acid (LA), two weeks after thawing. Both DNCB and NiSO4 produced a positive response and LA produced a negative response for both CD86 and CD54 cell surface markers.
A dose finding assay was performed to determine the CV75. The 75% cell viability (CV) of test chemical concentration are compared with RPMI media control.
The test item was prepared on the day of testing. Test item was dissolved or stably dispersed in medium as solvent/vehicle to final concentrations of 500 mg/mL (in medium) or 500 mg/mL (in DMSO).
Starting from the 500 mg/mL (in medium) stock solutions of the test chemicals, the following dilution steps was taken:
For medium as solvent/vehicle: Eleven stock solutions (eleven concentrations) were prepared, by two-fold serial dilutions using the corresponding solvent/vehicle. These stock solutions are then further diluted 50-fold into culture medium (working solutions). The top final concentration in the plate was 5000 µg/mL.

Vehicle / solvent control:

cell culture medium

Negative control:

DL-Lactic acid

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Positive control results:

DNCB was used as the positive control for CD86/CD54 expression measurement at a final single concentration in the plate was 4.0 μg/mL. To obtain a 4.0 μg/mL concentration of DNCB in the plate, a 2 mg/mL stock solution of DNCB in DMSO was prepared and further diluted 250-fold with culture medium to a 8 μg/mL working solution.
RFI values of CD86 for positive control is 412.55, 401.34 and of CD54 for positive control is 303.60, 364.43, for set 1 and set 2 respectively.

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC150, CD86 [442E]

Value:

1.13 µg/mL

Cell viability:

94.75 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC150, CD86 [442E]

Value:

1.4 µg/mL

Cell viability:

94.18

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC200, CD54 [442E]

Value:

1.51 µg/mL

Cell viability:

93.19 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC200, CD54 [442E]

Value:

2.07 µg/mL

Cell viability:

89.96 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Viability of 75 % was observed at 3.3105 µg/mL. All concentrations below showed respective higher viabilities.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: YES
- Acceptance criteria met for positive control: YES
- Acceptance criteria met for variability between replicate measurements: YES
- Range of historical values if different from the ones specified in the test guideline: NO

 

Test Item Concentrations (µg/mL)	RFI Values of test item: Set 1		RFI Values of test item: Set 2
	CD86	CD54	CD86	CD54
3.973	426.39	363.70	350.05	360.46
3.311	392.37	423.64	389.83	344.84
2.759	366.24	350.54	320.97	269.20
2.299	366.36	141.58	345.22	337.42
1.916	282.28	216.51	335.66	106.80
1.597	209.31	233.28	175.13	31.07
1.331	213.97	125.31	140.90	77.93
1.109	141.79	137.05	142.03	193.73

 

 

TABLE 1.       EC150 (CD86), EC200 (CD54) AND PREDICTION OF THE TEST ITEM

Sl. No.	Details	CD86	CD54	EC150 (CD86) µg/mL	EC200 (CD54) µg/mL
1	Set-1	RFI>150	RFI>200	1.13	1.51
2	Set-2	RFI>150	RFI>200	1.40	2.07
3	Prediction	Positive	Positive	-	-

  Refer Appendix 2 and 3

ND: Not Determined, EC: Effective Concentration.

 

TABLE 2.       CV-75 CONCENTRATION AND OVERALL PREDICTION OF THE TEST ITEM

 

CV-75 (µg/mL)	3.3105
Prediction	Positive

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Interpretation of results:

study cannot be used for classification

Remarks:

The data generated with this method may not be sufficient to conclude in isolation on skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA. Since further in vitro studies were not feasible, no conclusion on the sensitising properties via in vitro studies can be made. Hence, a subsequent in vivo study was performed (see Chapter 7.4.1).

Conclusions:

Based on the expression levels of CD86 and CD54, the test item in h-CLAT prediction is considered as “Sensitiser”.
The data generated with this method is however not sufficient to conclude in isolation on the skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA. Since further in vitro studies were not feasible, no conclusion on the sensitising properties via in vitro studies can be made. Hence, a subsequent in vivo study was performed (see Chapter 7.4.1).

Executive summary:

The test item was evaluated in the “In Vitro Skin Sensitization by Human Cell Line Activation Test (h-CLAT) Method” as per the OECD guideline for testing of chemicals, No.: 442E. The measured expression levels of CD86 and CD54 cell surface markers are used for supporting the discrimination between skin sensitisers and non-sensitisers. Study was conducted to determine the cell viability (CV-75), expression level of CD86 and CD54 cell surface markers expression using human monocytic leukaemia cell line THP-1 in the h-CLAT method. Solubility test was conducted by RPMI 1640 media and dimethyl sulphoxide. Test item formed suspension in RPMI 1640 media at 500 mg/mL. On the day of testing, test item was prepared. Eleven concentrations were prepared for dose finding assay, RPMI media was used as solvent/vehicle, the final concentrations in plate was 4.883, 9.766, 19.531, 39.063, 78.125, 156.25, 312.5, 625, 1250, 2500 and 5000µg/mL. The CV75 value of the test item was determined at 3.3105 µg/mL. Based on the CV75 of dose finding assay, eight test concentrations of 1.109, 1.331, 1.597, 1.916, 2.299, 2.759, 3.311 and 3.973µg/mL were selected for CD86/CD54 expression measurement. Upregulation of the biomarkers CD86 and/or CD54 was observed in some of the tested concentration of the test item. EC150 value for CD86 was 1.13 and 1.4 µg/mL for run 1 and 2 respectively. EC200 for CD 54 was 1.51 and 2.07 µg/mL for run 1 and 2 respectively.

Hence, the test item can be consideres as "sensitiser" in the h-CLAT assay. However, the data generated with this method may not be sufficient to conclude in isolation on skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA. Since further in vitro studies were not feasible, no conclusion on the sensitising properties via in vitro studies can be made. Hence, a subsequent in vivo study was performed (see Chapter 7.4.1).

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because the available in vitro test methods are not applicable for the substance and therefore an in vivo skin sensitisation study was conducted

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 November 2021 to 30 December 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

OECD Guideline for Testing of Chemicals, Section 4, Number 406, “Skin Sensitisation”, adopted on 17 July 1992 (Corrected on 14 June 2021)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

Buehler test

Justification for non-LLNA method:

Due to the negligible solubility and particulate constitution of the test item the test method according to Buehler was selected as the most appropriate test method for skin sensitisation for this test item.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.: EX. 14402. 600
- Expiration date of the batch: Oct 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: moistened with vehicle

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Adita Biosys Private Limited
Plot No.: SPL-26, 2nd Phase,
KSSIDC Industrial Estate, Madhugiri Road,
Antharasanahalli, Tumakuru – 572 106,
Karnataka, INDIA
CPCSEA Registration No. 1868/PO/RcBt/S/16/CPCSEA
- Microbiological status of animals, when known: No abnormalities Detected
- Age at study initiation: 8 to 9 weeks
- Weight at study initiation: 340.74 g to 359.06 g
- Housing: polypropylene cage (size: L 430 x B 280 x H 190 mm)
- Diet (e.g. ad libitum): Altromin diet for guinea pigs (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (e.g. ad libitum): Deep bore-well water passed through Reverse osmosis unit
- Acclimation period: Pre-study: 18 November 2021 to 22 November 2021
Main-study: 18 November 2021 to 28 November 2021
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):
- IN-LIFE DATES: From: To:

Route:

epicutaneous, occlusive

Vehicle:

other: 80% ethanol/water

Concentration / amount:

100% w/v Test Item (100 mg of test item moistened with 0.1 mL of 80% ethanol/water for induction phase)

Day(s)/duration:

Day 1, Day 8 and Day 15

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: Acetone

Concentration / amount:

100% w/v test item (100 mg of test item moistened with 0.1 mL of acetone for challenge phase)

Day(s)/duration:

Day 29

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

vehicle control - 10 animals
Test item - 20 animals

Details on study design:

RANGE FINDING TESTS: The animals did not reveal any skin reactions after application of 100 mg of test item moistened with 0.1 mL of 80% ethanol/water at 24 hours and 48 hours after patch removal in the pre-study. Hence, 100 mg of test item moistened with 0.1 mL of 80% ethanol/water was selected for induction and 100 mg test item moistened with 0.1 mL of acetone for challenge phase of the main study

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: Days 1, 8 and 15 (weekly once)
- Test groups: 1 (20 animals)
- Control group: 1 (10 animals)
- Site: left flank
- Frequency of applications: weekly once
- Duration: 3 weeks
- Concentrations: 100% w/v

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 1 day (6 hours)
- Exposure period: 6 hours
- Test groups: 1
- Control group: 1
- Site: right flank
- Concentrations: 100% w/v
- Evaluation (hr after challenge): 24 and 48

Positive control substance(s):

no

Positive control results:

The positive control was not included in this study as the reliability of the skin sensitisation was tested using 2-Mercaptobenzothiazole under study no.: BIO-ATX 3599. The positive control result is presented as Annexure 1 in study report.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

10

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

10

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100 mg

No. with + reactions:

0

Total no. in group:

10

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100 mg

No. with + reactions:

0

Total no. in group:

10

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Remarks on result:

not measured/tested

Reading:

1st reading

Hours after challenge:

48

Group:

positive control

Remarks on result:

not measured/tested

TABLE 3.   SKIN REACTIONS SCORING RECORD DURING PRE STUDY

Group, Sex & Treatment	Animal No.	Site	Dose (mg)	24hours*		48hours*
				Ery	Ede	Ery	Ede
G1, Male & test item	Ge4859	ALF	25.0	0	0	0	0
		PLF	50.2	0	0	0	0
		ARF	75.1	0	0	0	0
		PRF	100.0	0	0	0	0
	Ge4860	ALF	25.2	0	0	0	0
		PLF	50.2	0	0	0	0
		ARF	75.2	0	0	0	0
		PRF	100.1	0	0	0	0

Ery: Erythema; Ede: Oedema; ALF: Anterior Left Flank; PLF: Posterior Left Flank; ARF: Anterior Right Flank;

PRF: Posterior Right Flank; 0: No erythema/No oedema

*: Observation time points after removal of the test patch

 

TABLE 4.   SKIN REACTIONS SCORING RECORD DURING MAIN STUDY

Group, Sex & Treatment	Animal No.	Induction Phase												Challenge Phase Day 29
		Induction I - Day 1				Induction II - Day 8				Induction III - Day 15				Skin Reaction Score (Right Flank)				Sensitisation Rate (%)
		Skin Reaction Score (Left Flank)				Skin Reaction Score (Left Flank)				Skin Reaction Score (Left Flank)
		1 hr*		24 hrs*		1 hr*		24 hrs*		1 hr*		24 hrs*		24 hrs*		48 hrs*
		Ery	Ede	Ery	Ede	Ery	Ede	Ery	Ede	Ery	Ede	Ery	Ede	Post	Ant	Post	Ant
G2, Male & Vehicle Control	Ge4861	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Ge4862	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Ge4863	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Ge4864	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Ge4865	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Ge4866	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Ge4867	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Ge4868	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Ge4869	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Ge4870	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

Ery: Erythema; Ede: Oedema; hr(s): Hour(s); 0: No Erythema/Oedema; Post: Posterior part (test item); Ant: Anterior part (vehicle control); 0: No visible change for challenge scoring

*: Observation time points after removal of the test patch.

                                                                             

 

 

 

TABLE 4 (Contd…). SKIN REACTIONS SCORING RECORD DURING MAIN STUDY

Group, Sex & Treatment	Animal No.	Induction Phase												Challenge Phase Day 29
		Induction I - Day 1				Induction II - Day 8				Induction III - Day 15				Skin Reaction Score (Right Flank)					Sensitisation Rate (%)
		Skin Reaction Score (Left Flank)				Skin Reaction Score (Left Flank)				Skin Reaction Score (Left Flank)
		1 hr*		24 hrs*		1 hr*		24 hrs*		1 hr*		24 hrs*		24 hrs*			48 hrs*
		Ery	Ede	Ery	Ede	Ery	Ede	Ery	Ede	Ery	Ede	Ery	Ede	Post	Ant	Post		Ant
G3, Male & test item	Ge4871	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0	0
	Ge4872	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0
	Ge4873	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0
	Ge4874	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0
	Ge4875	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0
	Ge4876	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0
	Ge4877	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0
	Ge4878	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0
	Ge4879	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0
	Ge4880	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0
	Ge4881	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0
	Ge4882	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0
	Ge4883	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0
	Ge4884	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0
	Ge4885	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0
	Ge4886	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0
	Ge4887	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0
	Ge4888	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0
	Ge4889	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0
	Ge4890	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0		0

  Ery: Erythema; Ede: Oedema; hr(s): Hour(s); 0: No Erythema/Edema; Post: Posterior part (test item); Ant: Anterior part (vehicle control); 0: No visible change for challenge scoring

 *: Observation time points after removal of the test patch.    

 

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions employed and based on the results of the experiment, it is concluded that the test item did not show any skin sensitisation or allergic potential in Guinea pigs. Hence, the test item does not meet classification criteria according to the Globally Harmonized System (GHS) of Classification and Labelling of Chemicals.

Executive summary:

The study was conducted to evaluate the skin Sensitisation potential of the test item in Guinea Pigs by Buehler Test Method.

The study was conducted in two phases viz., pre study and the main study.For pre study, approximately 24 hours before treatment, fur from the right and left flank region was clipped closely using an electric hair clipper exposing an area of approximately 80 sq. cm.The dose of 25 mg and 50 mg of test item (moistened with 0.1 mL of 80% ethanol/water) were applied topically to the anterior left flank and posterior left flank respectively, whereas 75 mg and 100 mg of test item (moistened with 0.1 mL of 80% ethanol/water) were applied topically to the anterior right flank and posterior right flank respectively to each animal. No skin reactions were observed in any of the test dose approximately at 24 and 48 hours observation period after patch removal.

In pre study (G1), no skin reactions were observed at the highest dose i.e. 100 mg of test item. Hence, 100 mg of test item was selected for induction and challenge phase of main study.

The main study comprised of two groups, G2 (Vehicle control) and G3 (treatment). The main study included three inductions on day 1, 8 and 15 and challenge on day 29. Approximately 24 hours prior to initiation of the treatment, fur from the left and right flank was clipped closely using an electric hair clipper exposing an area of approximately 80 sq. cm. for induction and challenge phase of the experiment respectively.

On induction day 1, 8 and 15, 0.1 mL of 80% ethanol/water and 100 mg of test item moistened with 0.1 mL of 80% ethanol/water were applied topically on left flank region to G2 and G3 group animals respectively. On challenge day 29, 0.1 mL of acetone was topically applied to the anterior part of right flank, whereas 100 mg of test item moistened with 0.1 mL of acetone was topically applied to the posterior part of the right flank of all the animals. The test patches were covered by approximately 4×6 cm2cotton gauze and held in place with non-irritating adhesive tape and crepe bandage. After 6 hours of contact period, the test patches were removed and the application sites were cleaned with normal saline swabs and dried. During induction phase, the test item application sites were observed for skin reactions approximately at 1 and 24 hours post removal of the test patches according to Draize method (1959) and during challenge phase the skin reactions were observed approximately at 24 and 48 hours post removal of the test patch according to Magnusson and Kligman grading scale.

All the animals were observed once daily for clinical signs of toxicity and twice daily for mortality/morbidity during the experimental period. Individual animal body weight was recorded at receipt and on day 1 (before start of the treatment) and at termination for the pre study and main study animals.

In induction phase, no skin reactions were observed in vehicle control and treatment group animals approximately at 1 and 24 hours of observation period of post patch removal.

In challenge phase, theanimals did not reveal any skin reactions at anterior and posterior part of right flank of vehicle control and treatment group animals approximately at 24 and 48 hours of post patch removal.

No clinical signs of toxicity and mortality were observed till termination. All the treated animals revealed physiologically normal increase in body weights during the observation period.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the expression levels of CD86 and CD54, the test item in h-CLAT prediction is considered as “Sensitiser”. The data generated with this method is however not sufficient to conclude in isolation on the skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA. Since further in vitro studies were not feasible, no conclusion on the sensitising properties via in vitro studies can be made. Hence, a subsequent in vivo study was performed.

The subsequent in vivo skin sensitising study did not show any skin sensitisation or allergic potential in Guinea pigs. Hence, the test item does not meet classification criteria according to the Globally Harmonized System (GHS) of Classification and Labelling of Chemicals.