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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 21 to June 04, 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
animals exposed occasionally to humidity higher than 70%; no certificate of analysis, doses tested showed signs of skin irritation
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L'Arbresle Cedex, France
- Age at study initiation: 10 weeks
- Weight at study initiation: 20-23 g
- Housing: Housed individually in labeled Macrolon cages containing sterilized sawdust as bedding material
- Diet (e.g. ad libitum): Pelleted diet (SM R/M-Z SSNIFF® Spezialdiaten GmbH, Soest, Germany), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2-23.5 °C
- Humidity (%): 41-95%
- Air changes (per hour): 15/hour
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 25, 50 or 100% in acetone/olive oil (4:1 v/v)
No. of animals per dose:
Five females
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Soluble in vehicle (acetone/olive oil)
- Irritation: On Day 3, well-defined erythema (grade 2), large bald areas behind the ears, loss of flexibility of the ears and sticky test substance was noted
- Lymph node proliferation response: No data

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: Stimulation index = 3


TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of Bisphenol A epoxy diacrylate at concentrations of 0 (vehicle control), 25, 50 or 100% in acetone/olive oil (4:1 v/v) were applied to the dorsal surface of each ear on Days 1, 2 and 3. On Day 6, 250 µL phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (3H-TdR; GE Healthcare, UK) was injected into the tail vein of each experimental mouse. Five hours later, all mice were killed by intraperitoneal injection with pentobarbital Euthesate® (0.2 mL/animal) and the draining auricular lymph node of each ear was excised into PBS. A single cell suspension of lymph node cells (LNC) bilaterally from individual mouse was prepared by gentle separation through 125 µm-mesh stainless steel gauze. LNC were washed twice with an excess of PBS and precipitated with 5% trichloroacetic acid (TCA) at 4 °C. Pellets were re-suspended in 1 mL TCA and transferred to scintillation vials containing 10 mL of scintillation fluid for 3H-counting. The number of radioactive disintegrations per minute (DPM) was measured using a Packard scintillation counter (2800TR).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No data

Results and discussion

Positive control results:
EC3 = 13.8%
Mean DPM ± SEM
- Vehicle = 359 ± 87
- Test material (5 % w/v) = 628 ± 219
- Test material (10 % w/v) = 1018 ± 279
- Test material (25 % w/v) = 1302 ± 229

Stimulation Index, SI ± SEM
- Vehicle = 1.0 ± 0.3
- Test material (5 % w/v) = 1.7 ± 0.7
- Test material (10 % w/v) = 2.8 ± 1.0
- Test material (25 % w/v) = 3.6 ± 1.1

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
27
Test group / Remarks:
25%
Parameter:
SI
Value:
34
Test group / Remarks:
50%
Parameter:
SI
Value:
9.5
Test group / Remarks:
100%
Cellular proliferation data / Observations:
No irritation of the ears was seen in the control group. The irritation of the ears as shown by the animals of the test substance treated groups was considered not to have a toxicologically significant effect on the activity of the nodes.
All nodes of the control groups were considered normal in size. The majority of nodes of the test substances treated groups were considered enlarged. The largest nodes were found in the 25 and 50% dosed groups.
Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 14063, 17063 and 4958 respectively. The mean DPM.animal value for the vehicle control group was 521.
These data did not show a clear dose-response and no reliable EC3 value could be calcuulated.

Neither mortality nor any signs of systemic toxicity were observed in the test or control animals during the study. Body weights and body weight gain were normal except slight body weight loss (toxicologically not significant) in some animals.
No macroscopic abnormalities of the surrounding area were noted

Any other information on results incl. tables

PRELIMINARY IRRITATION STUDY

 

Table 1: Skin reactions after epidermal exposure and body weights

Animal number

% Test substance1

Day 1

Day 3

 

Body weight (g)

Skin reactions dorsal surface ear

Body

weight (g)

Left

Right

Erythema

Oedema

Erythema

Oedema

1

50

23

2

0

2

0

22

23

1002

24

2

0

2

0

22

1 Vehicle: Acetone/olive oil (4:1 v/v)

2 Applied using a spatula, instead of using a pipette

3 On Day 3, large bald areas behind the ears, loss of flexibility of the ears and sticky test substance was noted

MAIN STUDY

Table 2: Skin reactions, body weights and relative size auricular lymph nodes

Group

% Test substance1

Animal2

Day 1

Day 3

Day 6

Body weight (g)3

Skin reactions dorsal surface ear

Body weight (g)3

Size nodes4

Left

Right

Erythema

Oedema

Erythema

Oedema

left

right

1

0% (vehicle)

1

21

0

0

0

0

22

n

n

2

23

0

0

0

0

21

n

n

3

21

0

0

0

0

21

n

n

4

21

0

0

0

0

21

n

n

5

22

0

0

0

0

22

n

n

2

25%

6

21

1

0

1

0

22

++

++

7

22

1

0

1

0

22

++

++

8

22

1

0

1

0

22

++

++

9

21

1

0

1

0

21

++

++

10

23

1

0

1

0

22

++

++

3

50%

11

22

1

0

2

0

21

++

++

12

22

1

0

2

0

20

++

++

13

21

2

0

2

0

20

++

++

14

23

2

0

2

0

21

++

+

15

20

2

0

2

0

21

++

++

46

100%5

16

23

2

0

2

0

23

+

+

17

20

2

0

2

0

20

+

+

18

23

2

0

2

0

22

+

+

19

23

2

0

2

0

23

+

+

20

22

2

0

2

0

21

+

+

1 Vehicle: Acetone/Olive oil (4:1 v/v)

2 Animal number

3 Body weight (g)

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

5 Applied using a spatula, instead of using a pipette

6 On Day 3, bald areas behind the ears, loss of flexibility of the ears and sticky test substance was noted

Table 3: Radioactivity measurements (individual animals)

Group

% test substance1

Animal

DPM/animal

1

0% (vehicle)

1

211

2

1193

3

628

4

431

5

140

2

25%

6

9656

7

19921

8

12257

9

17829

10

10654

3

50%

11

25946

12

7682

13

9038

14

13265

15

32483

4

100%

16

5379

17

4625

18

9283

19

1625

20

3877

1Vehicle: Acetone/Olive oil (4: 1 v/v)

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Under the test conditions, Bisphenol A epoxy diacrylate is considered to be a skin sensitizer.
Executive summary:

In a local lymph node assay performed in CBA strain mice according to OECD guideline 429 and in compliance with GLP, groups of mice (5 females/dose) were applied with 25 µL of Bisphenol A epoxy diacrylate at concentrations of 0 (vehicle control), 25, 50 or 100% in acetone/olive oil (4:1 v/v) to the dorsal surface of each ear for three consecutive days. On Day 6, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and measured for radioactivity expressed as number of disintegrations per minute (DPM) and stimulation index (SI). Historic data of a-hexylcinnamaldehyde (5, 10 and 25 % w/v) in acetone/olive oil (4:1 v/v) was used as the data for positive control group. In the preliminary screening test neither mortality nor clinical signs of toxicity were observed at concentration of 50 and 100% w/v, so 100% was selected as the highest dose for the main study.

 

In the main study, mean DPM for 0, 25, 50 or 100% Bisphenol A epoxy diacrylate were observed to be 521 ± 189, 14063 ± 2035, 17683 ± 4907 or 4958 ± 1250 dpm respectively. Stimulation index for 0, 25, 50 or 100% Bisphenol A epoxy diacrylate were observed to be 1 ± 0.5, 27 ± 10.5, 34 ± 15.5 or 9.5 ± 4.2 respectively. These results indicate that the test material could elicit a SI = 3. In absence of a clear dose-response EC3 value could not be calculated. Neither mortality nor any symptoms of systemic toxicity were observed in the vehicle and treatment groups. Also no biologically significant changes in the body weights were observed in the animals during the study. Dermal irritation reactions included slight to well defined erythema in two animals but were considered not to have a toxicologically significant effect on the nodes. Enlarged nodes were observed in the treatment groups but no macroscopic abnormalities of the surrounding area were noted.

 

Under the test conditions, Bisphenol A epoxy diacrylate is classified as ‘R43 May cause sensitisation by skin contact’, according to the criteria of Annex VI to the Directive 67/548/EEC and ‘Category 1’ in CLP Regulation (EC) N° (1272-2008).