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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:
14 November - 20 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
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
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

1
Reference substance name:
Reaction products of 3-methyl-5-[(1S,4aS,8aS)-5,5,8a-trimethyl-2-methylenedecahydro-1-naphthalenyl]-1-penten-3-ol, cyclized
Molecular formula:
Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)
IUPAC Name:
Reaction products of 3-methyl-5-[(1S,4aS,8aS)-5,5,8a-trimethyl-2-methylenedecahydro-1-naphthalenyl]-1-penten-3-ol, cyclized
Test material form:
liquid: viscous
Details on test material:
Appearance: Off white paste

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: Not reported
- Age at study initiation: ca. 10 weeks
- Weight at study initiation: all within ± 20 % of the mean sex value
- Housing: Makrolon cage (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment. The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days
- Indication of any skin lesions: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24 ºC
- Humidity (%): 40 - 70 %
- Air changes (per hr): min. 10
- Photoperiod (hrs dark / hrs light): 12 / 12
- IN-LIFE DATES: Not reported

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
2, 5, 10 % w/w
No. of animals per dose:
5 per study group
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility:solubilised in acetone:olive oil (4:1 v/v)
- Irritation: Yes
- Systemic toxicity: Yes
- Ear thickness measurements: Yes
- Erythema scores: Yes

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Mouse LLNA
- Criteria used to consider a positive response: Stimulation Index (SI) - an SI result greater than or equal to 3 indicates that the test item is a skin sensitiser.

TREATMENT PREPARATION AND ADMINISTRATION:

Test item and positive control preparation

The test item and positive control item preparations (w/w) were prepared within 4 hours prior to each dosing. Test item preparations were stirred for 30 minutes during preparation. No adjustment was made for specific gravity of the vehicle. Homogeneity was assessed by visual inspection of the solutions. Correction of the purity/composition of the test item is not applicable, since the test method requires a logical concentration range rather than specific dose levels to be dosed.

Induction

The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The vehicle and positive control animals were treated in the same way as the experimental animals, except that the vehicle and/or positive control item was administered instead of the test item.



Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.

Results and discussion

Positive control results:
The positive control group added to the study showed that the vehicle is suitable for eliciting a SI>3 in this batch of animals and with the procedures used for this study.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.8
Test group / Remarks:
2 % w/w
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
5 % w/w
Key result
Parameter:
SI
Value:
2.2
Test group / Remarks:
10 % w/w
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA

N/A

DETAILS ON STIMULATION INDEX CALCULATION:

Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 5 and 10% were 368, 471 and 966 DPM, respectively. The mean DPM/animal value for the vehicle control group was 443 DPM and a mean DPM/animal value of 1840 DPM was obtained from the positive control group. The SI values calculated for the test item concentrations 2, 5 and 10% were 0.8, 1.1 and 2.2, respectively.

EC3 CALCULATION

> 10 % w/w

CLINICAL OBSERVATIONS:

None (none obesrved in pre-screen test at 10 % w/w test group).

BODY WEIGHTS:

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Any other information on results incl. tables

Table 2: Body weights and skin reaction

group

TS1

(%)

animal

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

bw

(g)2

erythema3

erythema

erythema

erythema

erythema

erythema

bw

(g)

left

right

left

right

left

right

left

right

left

right

left

right

1

0

1

20.0

0

0

0

0

0

0

0

0

0

0

0

0

19.3

 

 

2

19.8

0

0

0

0

0

0

0

0

0

0

0

0

20.1

 

 

3

19.3

0

0

0

0

0

0

0

0

0

0

0

0

20.5

 

 

4

19.6

0

0

0

0

0

0

0

0

0

0

0

0

20.7

 

 

5

20.0

0

0

0

0

0

0

0

0

0

0

0

0

21.6

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2

2

6

20.6

0

0

0

0

0

0

0

0

0

0

0

0

20.7

 

 

7

21.1

0

0

0

0

0

0

0

0

0

0

0

0

22.0

 

 

8

20.5

0

0

0

0

0

0

0

0

0

0

0

0

21.6

 

 

9

23.2

0

0

0

0

0

0

0

0

0

0

0

0

23.2

 

 

10

22.5

0

0

0

0

0

0

0

0

0

0

0

0

21.9

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3

5

11

20.8

0

0

0

0

0

0

0

0

0

0

0

0

20.0

 

 

12

21.4

0

0

0

0

0

0

0

0

0

0

0

0

21.9

 

 

13

20.3

1

1

1

0

0

0

0

0

0

0

0

0

20.1

 

 

14

20.6

0

0

0

0

0

0

0

0

0

0

0

0

21.4

 

 

15

19.4

1

0

1

1

1

0

0

0

0

0

0

0

19.3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4

10

16

22.4

1

1

1

1

1

1

1

1

1

1

1

0

20.8

 

 

17

21.3

0

1

1

1

1

1

1

1

1

1

1

1

21.2

 

 

18

22.6

0

0

0

0

1

1

1

1

1

1

1

1

22.1

 

 

19

22.6

0

0

0

0

1

1

1

1

1

1

1

0

20.6

 

 

20

21.1

0

1

1

1

0

0

1

1

1

1

0

1

20.8

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5

HCA

21

19.5

1

1

1

1

1

1

1

1

1s

1

1s

1s

19.3

 

25

22

24.0

1

1

1

2

2

2

1

1

1

1

1

1

22.1

 

 

23

21.7

1

1

1

1

1

1

1

1

1

1

1

1

20.9

 

 

24

20.3

1

1

1

1

1

1

1

1

1

1s

1s

1s

20.8

 

 

25

19.4

1

1

1

1

1

2

1

1

1

1s

1

1s

19.8

s.Scaliness

1  TS = test item (% w/w).

2  Body weight (grams).

3  Grading erythema and eschar formation(Left = dorsal surface of left ear; right = dorsal surface of right ear):

   0 = No erythema

   1 = Very slight erythema (barely perceptible)

2 = Well-defined erythema

 

Table 3: Relative size lymph nodes, radioactivity counts and stimulation index

group

TS1

(%)

animal

Size nodes2

DPM3/ animal

mean

DPM ± SEM4

mean

SI ± SEM

left

right

1

0

1

n

n

414

443

±

62

1.0

±

0.2

 

 

2

n

n

462

 

 

3

n

n

236

 

 

4

n

n

618

 

 

5

n

n

483

 

 

 

 

 

 

 

 

 

 

 

 

2

2

6

n

n

291

368

±

23

0.8

±

0.1

 

 

7

n

n

431

 

 

8

n

n

364

 

 

9

+

+

394

 

 

10

n

n

359

 

 

 

 

 

 

 

 

 

 

 

 

3

5

11

n

n

309

471

±

78

1.1

±

0.2

 

 

12

n

n

564

 

 

13

n

n

264

 

 

14

n

n

560

 

 

15

n

n

660

 

 

 

 

 

 

 

 

 

 

 

 

4

10

16

+

+

523

966

±

157

2.2

±

0.5

 

 

17

+

+

919

 

 

18

+

+

758

 

 

19

+

+

1260

 

 

20

+

+

1369

 

 

 

 

 

 

 

 

 

 

 

 

5

HCA

21

+

+

426

1840

±

546

4.2

±

1.4

 

25

22

+

+

711

 

 

23

+

+

3217

 

 

24

+

+

2193

 

 

25

+

+

2653

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was not considered to be a skin sensitiser.
Executive summary:

The skin sensitising potential of the test item was established in accordance with OECD guideline 429 using the mouse local lymph node assay method.

 

Test item concentrations selected for the main study were based on the results of a pre-screen test.

 

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 2, 5 or 10% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v)) and another group of five animals received a positive control item (HCA).

 

Three days after the last exposure, all animals were injected with3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating 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.

 

The very slight erythema of the ears as shown by several animals treated at 5% between Days 1 and 3 and all animals treated at 10% between Days 1 and 6 was considered not to have a toxicologically significant effect on the activity of the nodes.

 

The very slight to well-defined erythema and/or scaliness as shown by all animals of the positive control group between Days 1 and 6 was considered not to have a toxicologically significant effect on the activity of the nodes.

 

Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Except for the ear thickness of ear of one positive control animal on Day 2 only which was enlarged by 27%. This incidental finding was considered not to have a toxicologically significant effect on the activity of the nodes.

 

The majority of auricular lymph nodes were considered normal in size, except for the nodes in one animal treated at 2%, all animals treated at 10% and all positive control animals, which were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

 

Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 5 and 10% were 368, 471 and 966 DPM, respectively. The mean DPM/animal value for the vehicle control group was 443 DPM and a mean DPM/animal value of 1840 DPM was obtained from the positive control group. The SI values calculated for the test item concentrations 2, 5 and 10% were 0.8, 1.1 and 2.2, respectively.