Reaction products of 2-(chloromethyl)oxirane and glycerol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-02-01 - 2006-02-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Remarks:

The Swiss GLP Monitoring Authorities, November 2005

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 2-(chloromethyl)oxirane and glycerol

EC Number:

701-197-2

Molecular formula:

6 individual glycidether components (Molecular range ca. 200 - 600 g/mol)

IUPAC Name:

Reaction products of 2-(chloromethyl)oxirane and glycerol

Details on test material:

- Name of test material: 1,2,3-Propanetriol, glycidyl ethers
- Substance type: glycidyl ether
- Physical state: colourless liquid
- Analytical purity: > 90 %
- Expiration date of the lot/batch: 2006-08-31
- Stability under test conditions: stable under storage conditions
- Storage condition of test material: at room temperature (range of 20 +/- 5 °C), light protected
- Other: safety precautions: routine hygienic procedures were used to ensure the health and safety of the personnel.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: CBA/CaHsdRcc(SPF) from RCC Ltd, Laboratory Animal Service, Füllinsdorf, Switzerland
- Age at study initiation: 8-12 weeks (beginning of acclimatization)
- Weight at study initiation: 16 - 24 g (ordered)
- Housing: Individual in Makrolon type-2 cages with standard softwood bedding ("Lignocel", Schill AG, Muttenz),
- Diet (e.g. ad libitum): Pelleted standard Kliba 3433, batch no. 76/05 mouse maintenance diet (Provimi Kliba AG, Kaiseraugst) available ad libitum
- Water (e.g. ad libitum): Community tap water from Itingen, available ad libitum.
- Acclimation period: under test conditions after health examination, only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): a 12 hour fluorescent light /12 hour dark cycle with at least 8 hours music during the light period
Room temperature and humidity were monitored continuously.

Study design: in vivo (LLNA)

Vehicle:

other: Ethanol/water {7/3, v/v)

Concentration:

1 % / 2.5 % and 5 %

No. of animals per dose:

4 females

Details on study design:

RANGE FINDING TESTS:
PRE-TEST
In non-GLP solubility pre-test, the test item was tested in different vehicles: acetone/olive oil (4/1, v/v), ethanol/water (7/3, v/v) and N,N-dimethylformamide (DMF). Ethanot/water (7/3, v/v) was found to be a suitable vehicle and was selected and used in the main test.
A non-GLP local toxicity pre-test was performed for determination of concentrations for the main test. Three single animals were each treated with one of three different concentrations: 25 %, 50 % and 100 % (undiluted), in both ears on three consecutive days. One day after each topical application, slight, moderate to severe ear erythema and/or swelling was observed at all the dosing sites. One day after the third application, the animal dosed at 100 % (undiluted) died. 5 % was the highest dosing concentration in the main tests for avoiding systemic toxicity and excessive local irritation.
- Irritation: One day after each topical application, slight, moderate to severe ear erythema and/or swelling was observed at all the dosing sites. One day after the third application, the animal dosed at 100 % (undiluted) died.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT - TREATMENT PROCEDURES
TOPICAL APPLICATION
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with the test item at concentrations of 1 %, 2.5 % and 5 % in ethanol/water (7/3, v/v). The application volume, 25 µI, was spread over the entire dorsal surface (0-8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

ADMINISTRATION OF 3H-METHYL THYMIDINE
3H-methyl thymidine (3HTdR) was purchased from Amersham International (Amersham product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were administered 250 µI of 80.91 µCi/ml 3HTdR (equal to 20.2 fjC\ HTdR) by intravenous injection via a tail vein.
DETERMINATION OF INCORPORATED 3HTDR
Approximately five hours after treatment with 3HTdR all mice were euthanized by inhalation of C02(dry ice).
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing twice with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to glass scintillation vials with 10 mL of 'Irga-Safe Plus' scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a p-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The (3-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (dpm).

INTERPRETATION OF RAW DATA
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporated into lymph node cells of test group relative to that recorded for control group (STIMULATION INDEX) (S.I.). Before dpm/node values were determined, mean scintillation-background dpm was subtracted from test and control raw data.
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- First, exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX (S.I.).
- Second, the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

POSITIVE CONTROL:
The contact allergenic potential of ALPHA-HEXYLCINNAMALDEHYDE was assessed in three groups each of four female mice were treated daily with the test item at concentrations of 5 %, 10 % and 25 % in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle acetone:olive oil, 4:1 (v/v) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a p-scintillation counter.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables.

Results and discussion

Positive control results:

All treated animals survived the scheduled study period. Neither clinical / local signs nor other findings were observed in any animals of the control group. On the second application day, slight to severe ear swelling was observed at both dosing sites in all mice of Groups 2-4, persisting for a total of four days or for the remainder of the in-life phase of the study, respectively. In addition, on the second application day, slight to severe ear erythema was also noted at both dosing sites in all mice of Groups 3-4, persisting for a total of four days or for the remainder of the in-life phase of the study, respectively.
The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of the strain and age.
The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured on a ß-scintillation counter.
The results obtained [STIMULATION INDEX (S.I.)] are reported below. The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.
Group 2 (Test item concentration 5 % w/v): S.I. 1,8
Group 3 (Test item concentration 10 % w/v *): S.I. 2.9 *
Group 4 (Test item concentration 25 % w/v *): 6.2 *
A clear dose response relationship was observed.
* this value was used in calculation of EC3.

Conclusion: A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the STIMULATION INDEX (S.I.). In this study S.I. of 1.8, 2.9 and 6.2 were determined with the test item at concentrations of 5 %, 10 % and 25 %, respectively, in acetone:olive oil, 4:1 (v/v). ALPHA-HEXYLCINNAMALDEHYDE was therefore found to be a skin sensitizer in the LLNA tests and an EC3 value of 10.5 % was derived.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: S.I. of test ggroup (1%): 1.1 S.I. of test group (2.5 %): 1.5 S.I. of test group (5 %): 1.6

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Disintegrations per minute (each basedon 8 lymph nodes investigated): Control group: 2529 - dpm per lymph node: 316 Test item group (1 %): 2689 - dpm per lymph node 336 Test item group (2.5 %): 3805 - dpm per lymphnode: 476 Test item group (5 %): 3979 - dpm per lymphnode: 497

Any other information on results incl. tables

CALCULATION AND RESULTS OF INDIVIDUAL DATA FOR 1,2,3 -PROPANETRIOL, GLYCIDYL ETHERS

The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H~methyl thymidine measured on a ß-scintillation counter.  The results obtained [STIMULATION INDEX (S.I.)] are reported below. The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

Group 2 (Test item concentration 1 % w/v):       S. I. value 1.1

Group 3 (Test item concentration 2.5 w/v):         S. I. value 1.5

Group 4 (Test item concentration 5 % w/v):       S. I. value 1.6

A dose response relationship was observed.

An EC3 value could not be determined because this calculation requires a S.I. value of greater than 3.

VIABILITY / MORTALITY

No deaths occurred during the study period.

CLINICAL SIGNS

No clinical signs of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS: The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of the strain and age.

Table 2: Detailed results of 1,2,3 -propanetriol, glycidyl ethers

CALCULATION AND RESULTS OF INDIVIDUAL DATA
The following results were obtained:
Vehicle:   ethanol/water {7/3, v/v)
Test item concentration % (w/v)		Measurement dpm	Calculation			Result S.I.
			dpm -BG^a)	number of lymph nodes	dpm per lymph node^b)
--	BG I	27	--	--	-	-
-	BG II	33	--	-	--	-
-	CG1	2559	2529	8	316	--
1	TG2	2719	2689	8	336	1.1
2.5	TG3	3835	3805	8	476	1.5
5	TG4	4009	3979	8	497	1.6
No findings were observed on the size of the draining lymph nodes.
BG = Background (1 ml 5 % trichloroacetic acid) in duplicate
CG = Control Group
TG = Test Item Group
S.I. = Stimulation Index
^a)  = The mean BG was calculated from the figures BG I and BG II values
^b)  = Since the lymph nodes of the animals of a dose group were pooled, dpm/node was determined by dividing the measured value by the number of lymph nodes pooled

CALCULATION AND RESULTS OF INDIVIDUAL DATA FOR ALPHA-HEXYLCINNAMALDEHYDE

The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H~methyl thymidine measured on a ß-scintillation counter. The results obtained [STIMULATION INDEX (S.I.)] are reported below. The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

Group 2 (Test item concentration 5 % w/v):               S. I. value 1.8

Group 3 (Test item concentration 10 %w/v *):             S. I. value 2.9*

Group 4 (Test item concentration 25 % w/v*):              S. I. value 6.2*

EC3 = 10.5 %

A dose response relationship was observed.

* this value was used in calculation of EC3.

An EC3 value could not be determined because this calculation requires a S.I. value of greater than 3.

Table 3:Detailed results of the positive control substance alpha-hexylcinnamaldehyde

CALCULATION AND RESULTS OF INDIVIDUAL DATA
The following results were obtained:
Vehicle:   acetone:olive oil, 4:1 (v/v)
Test item concentration (w/v)			Measurement dpm	Calculation					Result S.l.
				dpm - BG^a)		number of lymph nodes		dpm per lymph node^b)
-	BG I		31	-		-		--	--
-	BG II		20	-		-		-	-
-	CG 1		4713	4687		8		586	-
5	TG 2		8513	8487		8		1061	1.8
10	TG 3		13636	13610		8		1701	2.9
25	TG 4		29235	29209		8		3651	6.2
BG = Background {1 ml 5 % trichloroacetic acid) in duplicate
CG = Control Group
TG = Test Item Group
S.I. = Stimulation Index
^a)  = The mean BG was calculated from the figures BG I and BG II values
^b)  = Since the lymph nodes of the animals of a dose group were pooled, dpm/node was determined by dividing the measured value by the number of lymph nodes pooled

Applicant's summary and conclusion

Interpretation of results:

other: Not sensitising according to EU GHS Criteria

Conclusions:

The study was performed according to the OECD Guideline 429 with no deviations and according to the good laboratory practice principles, it is considered to be of the highest quality (reliability Klimisch 1). The criteria of validity of the test system are fulfilled. The test material did not induce a sensitisation. The test material was considered to be not sensitising under the conditions of the test.
Under the conditions of the test, the test material did not produced sensitisation and was classified as a non-sensitizer.
The test material did not meet the criteria for classification as a sensitizer according to the European regulation (EC) No. 1272/2008. No symbol and risk phrase are required.

Executive summary:

1,2,3 -propanetriol, glycidyl ethers was investigated for its sensitising potential in a local lymph node assay test in mice (Wang-Fan, 2006, according to OECD 429, reliable without restrictions, Klimisch 1). Three groups each of four female mice were treated daily with the test item at concentrations of 1 %, 2.5 % and 5 % in ethanol/water (7/3, v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle ethanol/water (7/3, v/v) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a scintillation counter. All treated animals survived the scheduled study period. Neither clinical / local signs nor other findings were observed. The estimated concentration of test item required to produce a Stimulation Index (S.I.) of 3 is referred to as the EC3 value. A S. I. value of 1.1 was determiend for animals treated with 1 %, while a S. I. value of 1.5 and 1.6 for animals treated with 2.5 and 5 % was reported, respectively. A dose response relationship was observed.

An EC3 value could not be determined because this calculation requires a S.I. value of greater than 3. A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the S.I. In conclusion, the test item does not show allergenic potential when tested up to the concentration of 5 % in ethanol/water (7/3, v/v).