Reaction products of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane with maleic anhydride and methacrylic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 February 2010 - 22 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with minor deviations, which had no impact on the integrity of the study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Temporary deviations from the minimum level of relative humidity occurred.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Temporary deviations from the minimum level of relative humidity occurred.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

yes

Remarks:

Temporary deviations from the minimum level of relative humidity occurred.

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane with maleic anhydride and methacrylic acid

EC Number:

701-427-1

Cas Number:

36425-16-8

Molecular formula:

Not applicable: UVCB

IUPAC Name:

Reaction products of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane with maleic anhydride and methacrylic acid

Details on test material:

- Name of test material (as cited in study report): Substance 3, NLP 500-090-6 (old name of EC 701-427-1)
- Substance type: Technical product
- Physical state: Liquid
- Composition of test material, percentage of components:
- Approximately 57.5% bisGMA,
- Approximately 14.5% monomaleic adduct of bisGMA,
- Approximately 10% polymeric methacrylates
- Lot/batch No.: 7008043
- Expiration date of the lot/batch: 20 January 2011 (allocated by NOTOX, 1 year after receipt of the test substance)
- Stability under test conditions: Stable
- Storage condition of test material: In refrigerator (2-8°C) protected from light

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source:Charles River France, L’Arbresle Cedex, France.
- Age at study initiation: Young adult animals (approx. 9 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- Housing: Macrolon cages (MIII type; height 18 cm).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: at least 5 days before the start of treatment under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3. 0ºC (actual range: 18.5 -22. 7ºC)
- Humidity (%): 40-70% (actual range: 39 - 65%)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0%, 10%, 25% and 50%

No. of animals per dose:

5 animals per group

Details on study design:

RANGE FINDING TESTS:
- Compound solubility:
- Irritation:
- Lymph node proliferation response:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group.
If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM (Reference 1). The results were evaluated according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labeling and packaging of substances and mixtures. The EC3 value (the estimated test substance concentration that will give a SI =3) was determined, using linear interpolation


TREATMENT PREPARATION AND ADMINISTRATION:
Grading Irritation Reactions:
Erythema and eschar formation:
No erythema ..............................................................................……………………………………….. ......... 0
Slight erythema (barely perceptible) ....................................................……………………………… ............ 1
Well-defined erythema ...................................................................…………………………………… ........... 2
Severe erythema (beef redness) to slight eschar formation (injuries in depth) .........……………… ............ 3
Oedema formation:
No oedema .................................................................................……………………….……………… ......... 0
Slight oedema (barely perceptible) ......................................................……………………………… ............ 1
Moderate oedema....................................…………………… ....................................................................... 2
Severe oedema… ....................................................................................................................................... 3

Positive control substance(s):

other: dimethyl formamide

Statistics:

see below

Results and discussion

Positive control results:

See Table 1 below.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.7

Test group / Remarks:

Low dose - 10% concentration

Results 2

Parameter:

SI

Value:

2.6

Test group / Remarks:

Mid dose - 25% concentration

Results 3

Parameter:

SI

Value:

4.7

Test group / Remarks:

High dose - 50% concentration

Any other information on results incl. tables

Table 1: Skin reactions, body weights and relative size auricular lymph nodes

Group	test substance1 (% w/w)	an^2	Day 1	Day 3 Skin reactions dorsal surface ear				Day 6
			bw	left		right		bw	size nodes4
			(g)^3	erythema	oedema	erythema	oedema	(g)^3	Left	right
1	0% vehicle	1	19	0	0	0	0	20	n	n
		2	20	0	0	0	0	19	n	n
		3	19	0	0	0	0	20	n	n
		4	18	0	0	0	0	18	+	n
		5	18	0	0	0	0	18	n	n
2	10%	6	20	1	0	1	0	18	n	n
		7	18	1	0	1	0	18	n	n
		8	18	1	0	1	0	18	n	n
		9	19	1	0	1	0	19	n	n
		10	18	1	0	1	0	18	n	n
3	25%	11	19	1^5	0	1^5	0	19	n	n
		12	19	1^5	0	1^5	0	19	n	n
		13	20	1^5	0	1^5	0	20	n	n
		14	20	1^5	0	1^5	0	21	n	n
		15	19	1^5	0	1^5	0	20	n	n
4	50%	16	19	1^5	0	1^5	0	18	n	+
		17	19	1^5	0	1^5	0	19	n	n
		18	21	1^5	0	1^5	0	22	+	+
		19	21	1^5	0	1^5	0	20	n	+
		20	18	1^5	0	1^5	0	18	+	+

 

   ¹Vehicle: N,N-dimethylformamide.

²Animal number.

³Body weight (grams).

⁴Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n: considered to be normal).

⁵Bald area on head and ears.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Assessment of Contact Hypersensitivity to Modified Small Vinyl Ester was tested in the murine local lymph node assay (LLN A) in accordance the guidelines (OECD 429). The results showed that Modified Small Vinyl Ester elicited a SI> or equal to 3. The data showed a dose-response and an EC3 value of 29.8% was calculated. Thus, Modified Small Vinyl Ester was sensitizing in the murine local lymph node assay.

Executive summary:

Assessment of Contact Hypersensitivity to Modified Small Vinyl Ester in the Mouse (Local Lymph Node Assay). The study was carried out in accordance with the guidelines (e.g. OECD 429). The concentrations of Modified Small Vinyl Ester selected for the main study were based on the results of a preliminary study. In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (N,N-dimethylformamide). Three days after the last exposure, all animals were injected with 3H-methylthymidine and five hours later the draining (auricular) lymph nodes were excised. After precipitation of the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

.

Slight irritation of the ears was shown by all animals treated at 10, 25% and 50%. The animals at the 25 and 50% dose groups showed also bald area on the head and ears. These skin reactions were considered not to have a toxicologically significant effect on the activity of the nodes. No oedema was observed in any of the animals examined. The auricular lymph nodes of most control animals and all animals at 10 and 25% were considered normal in size, except for one node of one control animal, which was increased in size. At the highest dose group the majority of animals showed increased size of the lymph nodes. No macroscopic abnormalities of the surrounding area were noted in any of the animals. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in some animals, was considered not toxicologically significant.

Mean DPM/animal values for the experimental groups treated with Modified Small Vinyl Ester concentrations 10, 25 and 50% were 481, 728 and 1329 DPM respectively. The mean DPM/animal value for the vehicle control group was 281 DPM. The SI values calculated for test concentrations 10, 25 and 50% were 1.7, 2.6 and 4.7, respectively. The results showed that Modified Small Vinyl Ester elicited a SI ≥ 3. The data showed dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 29.8% was calculated. Based on these results, Modified Small Vinyl Ester was sensitizing in the muirne local lymph node assay.