Cordierite (Mg2[Al4O3(SiO3)5])

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 08 September 2015 to 29 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 429. The study was conducted on the registered substance.

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 (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were 8 to 12 weeks old.
The animals were housed in suspended solid floor polypropylene cages furnished with softwood wood flakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were set to achieve limits of 19 to 25°C and 30 to 70%, respectively. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Vehicle:

propylene glycol

Concentration:

10, 25, and 50 (% w/w) in propylene glycol

No. of animals per dose:

4

Details on study design:

Groups of four mice were treated with the test item at concentrations of 50%, 25% or 10% w/w in propylene glycol. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.

Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse.

Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.
Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA).
Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

Positive control substance(s):

other: phenylacetaldehyde > 90% (CAS No 122-78-1) as a solution in propylene glycol at a concentration of 5% v/v

Positive control results:

Concentration (% v/v) in propylene glycol: 5
Stimulation Index: 14.82
Result: Positive

Parameter:

SI

Remarks on result:

other: Vehicle = na 10% w/w = 1.20 (negative) 25% w/w = 0.88 (negative) 50% w/w = 1.14 (negative)

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Vehicle = 12471.55 dpm (1558.94 dpm/Node) 10% w/w = 14928.52 dpm (1866.07 dpm/Node) 25% w/w = 10956.45 dpm (1369.56 dpm/Node) 50% w/w = 14272.75 dpm (1784.09 dpm/Node)

Clinical Observations, Body Weight and Mortality Data – Preliminary Screening Test

Concentration (% w/w) in propylene glycol	Animal Number	Body Weight (g)		Day
				1		2		3		4	5	6
		Day 1	Day 6	Pre-Dose	Post-Dose	Pre-Dose	Post-Dose	Pre-Dose	Post-Dose
50	S-1	19.8	19.1	0	0	0	0	0	0	0	0	0

Local Skin Irritation – Preliminary Screening Test

Concentration (% w/w) in propylene glycol	Animal Number	Day
		1		2		3		4		5		6
		left	right	left	right	left	right	left	right	left	right	left	right
50	S-1	0	0	0	0	0	0	0	0	0	0	0	0

Measurement of Ear Thickness and Mean Ear Thickness Changes – Preliminary Screening Test

Concentration (% w/w) in propylene glycol	Animal Number	Ear Thickness Measurement (mm)
		Day 1		Day 3		Day 6
		pre-dose		post dose
		left	right	left	right	left	right
50	S-1	0.20	0.20	0.23	0.23	0.22	0.20
overall mean (nm)		0.20		0.23		0.21
overall mean ear thickness change (%)		na		15.00		5.00

Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% w/w) in propylene glycol	dpm	dpm/Node	Stimulation Index	Result
Vehicle	12471.55	1558.94	na	na
10	14928.52	1866.07	1.20	Negative
25	10956.45	1369.56	0.88	Negative
50	14272.75	1784.09	1.14	Negative

Individual Clinical Observations and Mortality Data

Concentration (% w/w) in propylene glycol	Animal Number	Day 1		Day 2		Day 3		Day 4	Day 5	Day 6
		Pre-Dose	Post-Dose	Pre-Dose	Post-Dose	Pre-Dose	Post-Dose
Vehicle	1-1	0	0	0	0	0	0	0	0	0
	1-2	0	0	0	0	0	0	0	0	0
	1-3	0	0	0	0	0	0	0	0	0
	1-4	0	0	0	0	0	0	0	0	0
10	2-1	0	0	0	0	0	0	0	0	0
	2-2	0	0	0	0	0	0	0	0	0
	2-3	0	0	0	0	0	0	0	0	0
	2-4	0	0	0	0	0	0	0	0	0
25	3-1	0	0	0	0	0	0	0	0	0
	3-2	0	0	0	0	0	0	0	0	0
	3-3	0	0	0	0	0	0	0	0	0
	3-4	0	0	0	0	0	0	0	0	0
50	4-1	0	0	0	0	0	0	0	0	0
	4-2	0	0	0	0	0	0	0	0	0
	4-3	0	0	0	0	0	0	0	0	0
	4-4	0	0	0	0	0	0	0	0	0

Individual Body Weights and Body Weight Change

Concentration (% w/w) in propylene glycol	Animal Number	Body Weight (g)		Body Weight Change (g)
		Day 1	Day 6
Vehicle	1-1	17.8	18.9	1.1
	1-2	19.0	19.8	0.8
	1-3	20.9	20.9	0.0
	1-4	18.2	18.2	0.0
10	2-1	19.0	20.5	1.5
	2-2	19.2	20.2	1.0
	2-3	17.5	18.5	1.0
	2-4	18.6	19.7	1.1
25	3-1	19.2	20.1	0.9
	3-2	20.2	20.6	0.4
	3-3	19.7	20.8	1.1
	3-4	19.8	20.7	0.9
50	4-1	18.2	19.2	1.0
	4-2	18.9	19.6	0.7
	4-3	19.4	21.0	1.6
	4-4	19.4	20.7	1.3

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group provided negative results for all the concentrations tested. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Executive summary:

The in vivo skin sensitisation of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 429. A Local Lymph Node Assay (LLNA) was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the test item as a suspension in propylene glycol at concentrations of 50%, 25% or 10% w/w. A further group of four animals was treated with propylene glycol alone.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group provided negative results for all the concentrations tested. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The in vivo skin sensitisation of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 429. A Local Lymph Node Assay (LLNA) was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the test item as a suspension in propylene glycol at concentrations of 50%, 25% or 10% w/w. A further group of four animals was treated with propylene glycol alone. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group provided negative results for all the concentrations tested. The test item did not meet the criteria for classification as sensitising to the skin according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Migrated from Short description of key information:

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group provided negative results for all the concentrations tested. The test item did not meet the criteria for classification as sensitising to the skin according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Justification for selection of skin sensitisation endpoint:

Study was conducted on the registered substance according to OECD Testing Guideline 429.

Justification for classification or non-classification

The skin sensitisation study was conducted on the registered substance according to OECD Testing Guideline 429. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group provided negative results for all the concentrations tested. The test item did not meet the criteria for classification as sensitising to the skin according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.