Registration Dossier

Diss Factsheets

Administrative data

Description of key information

A LLNA on mice according to OECD 429 is available.

The test item was considered unlikely to be a sensitizer based on the finding of this test and the corresponding guideline.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-05-16 until 2018-07-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Individual Method, 22 July 2010
Deviations:
yes
Remarks:
Pre-dose clinical observation on Day 3 (Main Test) were not recorded. This deviation was considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. QA statement)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 611220160318
- Storage stability: 31 December 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: approx. 4 °C in the dark
- Stability and homogenity under test conditions: Due to the short-term nature of the study no analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the integrity or validity of the study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: preparation of a solution in acetone/olive oil 4:1 (v/v)
- Final dilution of the stock liquid: acetone/olive oil 4:1 (v/v)

FORM AS APPLIED IN THE TEST: solution in acetone/olive oil
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: 15 to 23 g
- Housing: suspended, in solid floor polypropylene cages equipped with enrichements
- Diet: ad libitum, 2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK
- Water: ad libitum, tap water
- Acclimation period: at least 5 days
- Indication of any skin lesions: no clinical observations on day 1

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
50%, 0.5 mL test item + 0.5 mL vehicle
Concentration:
preliminary screening test: 50, 10, 2, 1 or 0.5% v/v in acetone/olive oil 4:1 (v/v)
main test: 1%, 0.5% or 0.25% v/v in acetone/olive oil 4:1 (v/v)
No. of animals per dose:
preliminary screening test: 1 (4 mice in total)
main test: 5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: given criteria: suitable for dosing if formulation is a solution or fine homogenous suspension which can be administered via a micropipette
- Irritation/Sensibilisation:
- 10% v/v: face and head was markedly swollen in the animal treated
- 1% or 0.5% v/v: no signs of visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness

- Systemic toxicity:
- 50%, 10% or 2% v/v: annimals had to be humanely killed, pre-dose on Days 1, 2 or 3, due to the occurrence of clinical signs of toxicity that were considered to approach the moderate severity limit set forth in the UK Home Office Project Licence
- 50%, 10% or 2% v/v: signs were prostration, decreased respiratory rate, labored respiration, ptosis, hunched posture, lethargy, tip toe gait and vocalization
- 1% or 0.5% v/v: no signs of systemic toxicity
- all animals showed body weight loss

Further details of the pre-screen test including results for irritation and ear thickness measurement are shown in table 1, 2 and 3.

MAIN STUDY :
- Criteria used to consider a positive response:
- proliferation response of lymph node cells expressed: (number of radioactive disintegrations / minute / animal) AND Stimulation Index = (3HTdR incorporation into lymph node cells of test nodes / recorded for the control nodes)
- test item regarded as a sensitizer: at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values (unless excessive irritation may have affected the result)
- "non-sensitizer": test item failing to produce a threefold or greater increase in 3HTdR incorporation
- EC3 value also calculated: concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation
- EC3 = c + [[(3-d)/(b-d)] x (a-c)]

a = lowest concentration giving stimulation index >3
b = actual stimulation index caused by ‘a’
c = highest concentration failing to produce a stimulation index of 3
d = actual stimulation index caused by ‘c’

TREATMENT PREPARATION:
- test item was applied to the test system within 2 hours of being formulated (freshly prepared as a solution in acetone/olive oil 4:1 (v/v))
It is assumed that the formulation was stable for this duration.
- formulations of a representative 1 mL of each prepared concentration and 1 mL of vehicle were taken daily and stored frozen in pre-labelled containers for possible future dose formulation analysis

TREATMENT ADMINISTRATION:
- mice treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3)
- test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette
- further group of five mice received the vehicle alone

- 5 days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 0.25 mL (250 μL) of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
- group mean values for disintegrations per minute, ear weight increase, lymph node weight and cell count and standard deviations
- data assessed for suitability by analysis of normality and homogeneity of variance
- normally distributed data with homogeneity of variances: parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance
- other data were assessed by non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures

Probability values (p):
P<0.001: ***
P<0.01: **
P<0.05: *
P>0.05: (not significant)
Positive control results:
The result of the current positive control study is given in table 4.
Table 5 depicts the historical control data of the positive control.
Parameter:
SI
Value:
0.87
Test group / Remarks:
0.25 % v/v in acetone/olive oil 4:1
Parameter:
SI
Value:
1.55
Test group / Remarks:
0.5 % v/v in acetone/olive oil 4:1
Parameter:
SI
Value:
4.68
Test group / Remarks:
1 % v/v in acetone/olive oil 4:1
Remarks on result:
other: false positive (see further details in chapter 'overall remarks')
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Lymph node weight and weight changes are given in table 8. The mean weight change for group 2 was noticeably different to the control group.
The lymph node cell count is given in table 9. All group mean cell counts for the test groups showed a significant increase when compared to the control group.

DETAILS ON STIMULATION INDEX CALCULATION
Details on radioactive disintegrations per minute per animal and the stimulation index are given in table 6.

CLINICAL OBSERVATIONS:
There were no deaths or any signs of systemic toxicity noted in the test or control animals during the test.

BODY WEIGHTS
Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.
Details are given in table 10.

EAR WEIGHT AS INDICATOR OF IRRITATION POTENTIAL
Results of the ear weight measurement are given in table 7.
The mean increase in ear weight for group 4 was significantly different to the control group and was indicative of excessive irritation as it exceeded the cut-off value mentioned in the OECD guideline.

Table 4: Results of the current positive control study for LLNA

Concentration (% v/v) in acetone/olive oil 4:1

 

Stimulation Index

 

Result

25

5.54

Positive

Table 5: Summary of historical positive control data for the LLNA

 

Study Number

 

Start Date

Finish Date

 

Test Item

 

Concentration

 

Vehicle

Stimulation Indexa

 

Classificationb

WR55NG

09/06/17

15/06/17

α-Hexylcinnamaldehyde,tech., 85%

25% v/v

dimethyl sulphoxide

8.47

Positive

CT74KC

09/06/17

15/06/17

α-Hexylcinnamaldehyde,tech., 85%

15% v/v

ethanol/distilled water 7:3

10.60

Positive

BQ66KP

30/06/17

06/07/17

α-Hexylcinnamaldehyde,tech., 85%

15% v/v

butanone

4.28

Positive

PB44SR

30/06/17

06/07/17

α-Hexylcinnamaldehyde,tech., 85%

15% v/v

acetone

3.98

Positive

CP04BC

29/11/17

05/12/17

Phenylacetaldehyde (>90%)

5% v/v

propylene glycol

8.44

Positive

GL18PB

29/11/17

05/12/17

α-Hexylcinnamaldehyde,tech., 85%

25% v/v

dimethyl formamide

7.28

Positive

TH50YP

30/11/17

06/12/17

α-Hexylcinnamaldehyde,tech., 85%

25% v/v

acetone/olive oil 4:1

5.54

Positive

XQ67PF

30/11/17

06/12/17

α-Hexylcinnamaldehyde,tech., 85%

25% v/v

1% pluronic L92 in distilled water

6.48

Positive

MV86JM

09/05/18

15/05/18

α-Hexylcinnamaldehyde,tech., 85%

15% v/v

ethanol/distilled water 7:3

5.96

Positive

JR21XK

09/05/18

15/05/18

Phenylacetaldehyde > 90%

5% v/v

cottonseed oil

4.46

Positive

TY92VS

10/05/18

16/05/18

α-Hexylcinnamaldehyde,tech., 85%

25% v/v

dimethyl sulfoxide

4.37

Positive

VV05BT

11/05/18

17/05/18

α-Hexylcinnamaldehyde,tech., 85%

15% v/v

butanone

3.78

Positive

a = Ratio of test to control lymphocyte proliferation, b = Stimulation index greater than 3.0 indicates a positive result, na = Not applicable

Table 6: Results of the individual disintegrations per minute and calculated SI

Concentration (% v/v) in acetone/olive oil 4:1

 

Animal Number

 

dpm/ Animala

 

Mean dpm/Animal (Standard Deviation)

 

Stimulation Indexb

 

Result

 

Vehicle

1-1

1376.64

 

2323.90

(±908.65)

 

na

 

na

1-2

2723.91

1-3

3696.24

1-4

1807.51

1-5

2015.20

 

0.25

2-1

1890.31

 

2017.00

(±274.48)

 

0.87

 

Negative

2-2

2111.87

2-3

1776.91

2-4

2454.37

2-5

1851.56

 

0.5

3-1

4051.49

 

3611.31

(±1429.99)

 

1.55

 

Negative

3-2

5891.71

3-3

2960.87

3-4

2244.77

3-5

2907.73

 

1

4-1

9039.12

 

10880.05** (±1506.18)

 

4.68

 

Positive +

4-2

9690.86

4-3

11245.61

4-4

11670.32

4-5

12754.34

+ False positive (see discussion and conclusion)

dpm = Disintegrations per minute

a = Total number of lymph nodes per animal is 2

b = Stimulation Index of 3.0 or greater indicates a positive result

na = Not applicable

** = Significantly different from control group p<0.01

Table 7: Results of the measurement of ear weight and mean ear weight change

 

Concentration(% v/v) in

acetone/olive oil 4:1

 

Animal Number

 

Ear Weight (g)^

 

Mean Ear Weight (g)

Overall Mean Ear Weight Change (compared to control group)

 

Vehicle

1-1

0.0378

 

0.0412

 

na

1-2

0.0495

1-3

0.0462

1-4

0.0337

1-5

0.0389

 

0.25

2-1

0.0503

 

0.0480

 

16.49%

2-2

0.0409

2-3

0.0609

2-4

0.0435

2-5

0.0442

 

0.5

3-1

0.0527

 

0.0461

 

11.98%

3-2

0.0402

3-3

0.0485

3-4

0.0474

3-5

0.0415

 

1

4-1

0.0551

 

0.0548*

 

32.95%

4-2

0.0426

4-3

0.0692

4-4

0.0548

4-5

0.0521

^ = Combined weight of two biopsy punches, one per ear

na = Not applicable

* = Significantly different from control group p<0.05

Table 8: Results of the lymph node weight measurement and the change compared to controls

 

Concentration(% v/v) in

acetone/olive oil 4:1

 

Animal Number

 

Lymph Nodes Weight

(g)

 

Mean Lymph Node Weight (g)

Overall Mean Lymph Node Weight Change (compared to control

group)

 

Vehicle

1-1

0.0245

 

0.1120

 

Na

1-2

0.1996

1-3

0.0690

1-4

0.2384

1-5

0.0668

 

0.25

2-1

0.1783

 

0.1518

 

35.54%

2-2

0.0234

2-3

0.2224

2-4

0.0880

2-5

0.2467

 

0.5

3-1

0.1184

 

0.1086

 

-3.04%

3-2

0.0821

3-3

0.1246

3-4

0.0199

3-5

0.1978

 

1

4-1

0.0156

 

0.1027

 

-8.31%

4-2

0.0350

4-3

0.2187

4-4

0.2303

4-5

0.0137

Na = Not applicable 

Table 9: Results of the lymph node cell count

Concentration (% v/v) in acetone/olive

oil 4:1

 

Animal Number

Cell Count A

(X 105

cells/mL)

Cell Count B

(X 105

cells/mL)

Mean Cell Count (X 105

cells/mL)

 

Group Mean Cell

 

% Change

 

Vehicle

1-1

5.7

6.9

6.3

 

5.4

 

Na

1-2

4.5

4.2

4.4

1-3

5.1

5.7

5.4

1-4

5.3

4.9

5.1

1-5

6.7

4.9

5.8

 

0.25

2-1

6.7

6.1

6.4

 

6.34*

 

17.4

2-2

6.4

6.7

6.6

2-3

7.0

6.0

6.5

2-4

6.0

6.8

6.4

2-5

6.4

5.2

5.8

 

0.5

3-1

6.8

6.6

6.7

 

9.04**

 

58.5

3-2

7.9

8.1

8.0

3-3

11.4

9.8

10.6

3-4

10.1

10.9

10.5

3-5

9.2

9.6

9.4

 

1

4-1

10.9

10.1

10.5

 

12.9**

 

138.9

4-2

10.8

12.4

11.6

4-3

16.3

14.2

15.3

4-4

14.3

15.7

15.0

4-5

11.4

12.8

12.1

* = Significantly different from control group p<0.05

** = Significantly different from control group p<0.01

Table 10: Results of the body weight measurements and body weight change

Concentration (% v/v) in acetone/olive oil 4:1

 

Animal Number

Body Weight (g)

 

Body Weight Change (g)

 

Day 1

 

Day 6

 

Vehicle

1-1

19.6

19.6

0.0

1-2

18.2

18.1

-0.1

1-3

19.3

18.4

-0.9

1-4

18.6

18.8

0.2

1-5

16.2

16.4

0.2

 

0.25

2-1

20.7

20.1

-0.6

2-2

19.2

19.6

0.4

2-3

20.3

20.0

-0.3

2-4

19.8

19.9

0.1

2-5

21.8

23.0

1.2

 

0.5

3-1

20.2

19.5

-0.7

3-2

18.5

18.6

0.1

3-3

17.8

17.8

0.0

3-4

18.1

18.0

-0.1

3-5

19.4

19.0

-0.4

 

1

4-1

19.0

18.7

-0.3

4-2

19.2

19.4

0.2

4-3

21.1

19.9

-1.2

4-4

20.7

21.2

0.5

4-5

18.3

18.8

0.5

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered unlikely to be a skin sensitizer under the conditions of the test.

The mean ear weight increase in the 1% dose group reflected that the test item was excessively irritant (increase of 32.95%) at this concentration, so the cut-off of 25% mentioned in the OECD guideline was exceeded. Therefore it is likely a false positive result for sensitization was obtained. The lower doses caused clearly negative stimulation indices (lower than a 3-fold increase, indices of 0.87 and 1.55 respectively).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A LLNA was performed according to OECD 429 to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 1% v/v, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50μL (25μL per ear) of the test item as a solution in acetone/olive oil 4:1 (v/v) at concentrations of 1%, 0.5% or 0.25% v/v. A further group of five animals was treated with acetone/olive oil 4:1(v/v) alone.

 

Concentration (% v/v) in acetone/olive oil 4:1

Stimulation Index

Result

0.25

0.87

Negative

0.5

1.55

Negative

1

4.68

Positive*

 

* The mean ear weight increase in the 1% dose group reflected that the test item was excessively irritant (increase of 32.95%) at this concentration, so the cut-off of 25% mentioned in the OECD guideline was exceeded. Therefore it is likely a false positive result for sensitization was obtained.

The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 0.7%.

A count of the lymph node cells were made and attached to the study entry in IUCLID.

 

The test item was considered unlikely to be a sensitizer under the conditions of the test.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008, as amended for the thirteenth time in Regulation (EU) No 2018/1480.

As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008.