Reaction products of Glycerin formal and 2-Propenoic acid, 2-methyl-, methyl ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26.04.2013 to 17.07.2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of Glycerin formal and 2-Propenoic acid, 2-methyl-, methyl ester

EC Number:

945-527-5

Cas Number:

1620329-57-8

Molecular formula:

C8H12O4

IUPAC Name:

Reaction products of Glycerin formal and 2-Propenoic acid, 2-methyl-, methyl ester

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

other: Mice, CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

Test system: Mice, CBA/CaOlaHsd
Rationale: Recognised as the recommended test system.
Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
Number of animals for the pre-test: 2 females
Number of animals for the main study: 20 females
Number of animals per group: 5 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1
Age: Pre-test: 9 - 10 weeks (beginning of treatment)
Main study: 11 - 12 weeks (beginning of treatment)
Body weight: 20.1-23.2 g, Mean SD: 21.7 +/- 0.9 g
Identification: The animals were distributed into the test groups at random. All animals belonging to the same experimental group were kept in one cage.experiment. In the main experiment the animals were identified by tail tags. In the pre-experiment, animals were identified by cage
number.
Acclimation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: group
Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top
Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 + 2°C
relative humidity approx. 45-65%
artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

25, 50 and 100 %

No. of animals per dose:

Number of animals for the pre-test: 2 females
Number of animals for the main study: 20 females
Number of animals per group: 5 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1

Details on study design:

Topical application
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25, 50, and 100% in acetone/olive oil (4+1, v/v). The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

Administration of 3H-methyl-thymidine
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20.1 μCi of 3H-methyl thymidine (equivalent to 80.3 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Determination of incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal).
Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing two times 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 scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as
the number of radioactive disintegrations per minute.

Observations
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: At least once daily from experimental start to necropsy.
Body weights: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.
Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6).
Ear weights: In the pre-test after sacrifice; biopsy punches were taken from each ear.
Clinical signs (local / systemic): Clinical signs (local irritation at the application site or systemic toxicity) were recorded at least once daily.
Especially the treatment sites were observed carefully.

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 and for the DPM values (group mean DPM ± standard deviation).
The Dean-Dixon-Test was used for identification of possible outliers (performed with Microsoft Excel 2007).
However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:

see: any other information on material and methods

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1

Test group / Remarks:

control

Results 2

Parameter:

SI

Value:

0.9

Test group / Remarks:

25 % Glycerolformal methacrylate

Remarks on result:

other: negative

Results 3

Parameter:

SI

Value:

0.8

Test group / Remarks:

50 % Glycerolformal methacrylate

Remarks on result:

other: negative

Results 4

Parameter:

SI

Value:

1.2

Test group / Remarks:

100 % Glycerolformal methacrylate

Remarks on result:

other: negative

Any other information on results incl. tables

Results

Calculation and Results of Individual Data

Vehicle: acetone/olive oil (4+1, v/v)

 

Test itemconcentration			DPMvaluesmeasured	DPM-BG peranimal(2 lymph nodes)a)	S.I.b)
%	Groupno.	Animalno.
---	---	BG I	22	---	---
---	---	BG II	20	---	---
0	1	1	280	259.0	---
0	1	2	333	312.0	---
0	1	3	431	410.0	---
0	1	4	240	219.0	---
0	1	5	368	347.0	---
25	2	6	362	341.0	1.1
25	2	7	152	131.0	0.4
25	2	8	271	250.0	0.8
25	2	9	239	218.0	0.7
25	2	10	434	413.0	1.3
50	3	11	218	197.0	0.6
50	3	12	150	129.0	0.4
50	3	13	343	322.0	1.0
50	3	14	268	247.0	0.8
50	3	15	394	373.0	1.2
100	4	16	385	364.0	1.2
100	4	17	406	385.0	1.2
100	4	18	728	707.0	2.3
100	4	19	169	148.0	0.5
100	4	20	325	304.0	1.0

 

1 = Control Group

2-4 = Test Group

a)= values corrected for mean background value (BGI and BGII)

b)= Stimulation Indices relative to the mean of the control group (Group 1)

 

 

 

Calculation of Stimulation Indices per Dose Group

 

Test itemconcentration	Group Calculation
	Mean DPMperanimal (2lymphnodes)a)	SD	S.I.
Vehicle ControlGroup (acetone/olive oil (4+1,v/v))	309.4	74.6	1.0
25%Glycerinformalmethacrylate	270.6	109.4	0.9
50%Glycerinformalmethacrylate	253.6	97.1	0.8
100%Glycerinformalmethacrylate	381.6	204.2	1.2

 

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Applicant's summary and conclusion

Interpretation of results:

other: not sensitizing

Conclusions:

The test item Glycerinformal methacrylate was not a skin sensitiser under the test conditions of this study.

Executive summary:

In an OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay) Glycerolformal methacrylate was testet at concentrations of 25, 50 and 100 % in acetone/olive oil (4:1 v/v).

Stimulation Indices of 0.9, 0.8 and 1.2 were determined with the test item at concentrations of 25, 50, and 100%. A dose response was not observed.

The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

In conclusion Glycerinformal methacrylate was not a skin sensitiser under the test conditions of this study.