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Diss Factsheets

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:
Experimental start date: 11 April 2016 - Experimental completion date: 28 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (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

1
Chemical structure
Reference substance name:
3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-2,2-dimethylpropanenitrile
EC Number:
814-345-7
Cas Number:
2003244-43-5
Molecular formula:
C14H21N
IUPAC Name:
3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-2,2-dimethylpropanenitrile
Details on test material:
- Name of test material (as cited in study report): ES421 Pinyl Nitrile- Molecular weight (if other than submission substance): 203 g/mol- Physical state: Crystallized white powder- Analytical purity: 98.08% (per Certificate of Analysis)- Composition of test material, percentage of components: 2-Norpinene-2-propionitrile, alpha, alpha,6,6-tetramethyl- Purity test date: 08 February 2016- Lot/batch No.: SM15077102- Storage condition of test material: 2-8°C, protected from light
Specific details on test material used for the study:
Physical state/Appearance: white solid
Storage Conditions: approximately 4 °C in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
Mice are the preferred species of choice since quantitative methods have been developed for the measurement of skin sensitization responses in the mouse and are specified in the appropriate test guidelines.
On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were 8 to 12 weeks old.
The animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food was allowed throughout the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 C and 30 to 70%, respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
This vehicle was chosen as it produced the highest concentration that was suitable for dosing.
Concentration:
50%, 25% and 10% w/w in acetone/olive oil 4:1.
No. of animals per dose:
Groups of five mice were treated with the test item.
Details on study design:
Preliminary Screening Test:
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test item at a concentration of 50% w/w in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale included as Table A. Any clinical signs of toxicity, if present, were also recorded. The body weight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using a gauge, pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

Main Test:

-Test Item Administration:
Groups of five mice were treated with the test item at concentrations of 50%, 25% or 10% w/w in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were 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). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of five mice received the vehicle alone in the same manner.
The positive control animals were similarly treated to the test animals except that 25 µL of the positive control item, α Hexylcinnamaldehyde, tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.

-3H-Methyl Thymidine Administration:
Five days following the first topical application of the test item, vehicle control or positive control item (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmoL) giving a total of 20 µCi to each mouse.

-Observations:
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

-Terminal Procedures:
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes.
Positive control substance(s):
other: α-Hexylcinnamaldehyde, tech., 85%
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non parametric Kruskal Wallis Rank Sum and Mann Whitney U test procedures were used.

Results and discussion

Positive control results:
Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of α Hexylcinnamaldehyde, tech., 85% as a solution in acetone/olive oil 4:1 at concentrations of 5%, 10% or 25% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone and served as the vehicle control group.
The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is shoxn in Table PC1.
The concentration of α Hexylcinnamaldehyde, tech., 85% expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 20%.
α Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.98
Test group / Remarks:
Concentration: 10% w/w in acetone/olive oil 4:1
Remarks on result:
other: Negative
Key result
Parameter:
SI
Value:
1.87
Test group / Remarks:
Concentration: 25% w/w in acetone/olive oil 4:1
Remarks on result:
other: Negative
Key result
Parameter:
SI
Value:
1.09
Test group / Remarks:
Concentration: 50% w/w in acetone/olive oil 4:1
Remarks on result:
other: Negative
Cellular proliferation data / Observations:
The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table M1.
Individual clinical observations and mortality data for test and control animals are given in Table M2. There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.
Individual body weights and body weight change for test and control animals are given in Table M3. 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.

Any other information on results incl. tables

 

Table PC1: Current Positive Control Study for the Local Lymph Node Assay

Treatment

Stimulation Index

Result

5% v/v in acetone/olive oil 4:1

1.42

Negative

10% v/v in acetone/olive oil 4:1

1.53

Negative

25% v/v in acetone/olive oil 4:1

3.71

Positive

Preliminary Screening Test:

Clinical observations, body weight and mortality data are given in Table P1 and local skin irritation is given in Table P2. The ear thickness measurements and mean ear thickness changes are given in Table P3.

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Based on this information the dose levels selected for the main test were 50%, 25% and 10% w/w in acetone/olive oil 4:1.

Table P1:      Clinical Observations, Body Weight and Mortality Data – Preliminary Screening Test

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

Animal Number

Body Weight (g)

Day

1

2

3

4

5

6

Day 1

Day 6

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Pre-Dose

Post Dose

50

S-1

21.5

22.0

0

0

0

0

0

0

0

0

0

0=   No signs of systemic toxicity

Table P2:      Local Skin Irritation – Preliminary Screening Test

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

Animal Number

Local Skin Irritation

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

left

right

left

right

left

right

left

right

left

right

left

right

50

S-1

0

0

0

0

0

0

0

0

0

0

0

0

Table P3:      Measurement of Ear Thickness and Mean Ear Thickness Changes – Preliminary Screening Test

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

Animal Number

Ear Thickness Measurement (mm)

Day 1

Day 3

Day 6

pre‑dose

post dose

left

right

left

right

left

right

50

S-1

0.21

0.21

0.22

0.23

0.21

0.23

overall mean (mm)

0.210

0.225

0.220

overall mean ear thickness change (%)

na

7.143

4.762

na=  Not applicable

Table M1:      Individual Disintegrations per Minute and Stimulation Index

Treatment Group

Animal Number

dpm/
Animal
a

Mean dpm/Animal
(Standard Deviation)

Stimulation Indexb

Result

Vehicle
acetone/olive oil 4:1

1-1

1063.44

746.81
(±297.54)

na

na

1-2

616.15

1-3

314.62

1-4

969.16

1-5

770.68

Test Item
10w/win
acetone/olive oil 4:1

2-1

447.47

732.46
(±197.06)

0.98

Negative

2-2

914.32

2-3

755.69

2-4

908.98

2-5

635.86

Test Item
25w/win
acetone/olive oil 4:1

3-1

738.33

1399.63
(±1139.77)

1.87

Negative

3-2

823.39

3-3

1036.00

3-4

972.86

3-5

3427.58

Test Item
50w/win
acetone/olive oil 4:1

4-1

491.97

817.64
(±293.26)

1.09

Negative

4-2

1001.45

4-3

1180.17

4-4

862.84

4-5

551.79

Positive Control Item
25% v/v in
acetone/olive oil 4:1

5-1

2254.18

6214.93**
(±3391.91)

8.32

Positive

5-2

8902.39

5-3

5424.75

5-4

4056.30

5-5

10437.03

The results of the statistical analysis of the data indicated there was no significant difference between the control group and the test item test groups.


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 M2:      Individual Clinical Observations and Mortality Data

Treatment Group Animal Number Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Pre-Dose Post Dose Pre-Dose Post Dose Pre-Dose Post Dose
Vehicle 1 -1 0 0 0 0 0 0 0 0 0
acetone/olive oil 4:1 1 -2 0 0 0 0 0 0 0 0 0
  1 -3 0 0 0 0 0 0 0 0 0
  1 -4 0 0 0 0 0 0 0 0 0
  1-5 0 0 0 0 0 0 0 0 0
Test Item 2 -1 0 0 0 0 0 0 0 0 0
10% w/w in 2 -2 0 0 0 0 0 0 0 0 0
acetone/olive oil 4:1  2 -3 0 0 0 0 0 0 0 0 0
  2 -4 0 0 0 0 0 0 0 0 0
  2-5 0 0 0 0 0 0 0 0 0
Test Item 3 -1 0 0 0 0 0 0 0 0 0
25% w/w in 3 -2 0 0 0 0 0 0 0 0 0
acetone/olive oil 4:1 3 -3 0 0 0 0 0 0 0 0 0
  3 -4 0 0 0 0 0 0 0 0 0
  3 -5 0 0 0 0 0 0 0 0 0
Test Item 4 -1 0 0 0 0 0 0 0 0 0
50% w/w in 4 -2 0 0 0 0 0 0 0 0 0
acetone/olive oil 4:1 4 -3 0 0 0 0 0 0 0 0 0
  4 -4 0 0 0 0 0 0 0 0 0
  4 -5 0 0 0 0 0 0 0 0 0
Positive Control Item 5 -1 0 0 0 0 0 0 0 0 0
25% v/v in 5 -2 0 0 0 0 0 0 0 0 0
acetone/olive oil 4:1 5 -3 0 0 0 0 0 0 0 0 0
  5 -4 0 0 0 0 0 0 0 0 0
  5 -5 0 0 0 0 0 0 0 0 0

0=   No signs of systemic toxicity

Table M3:      Individual Body Weights and Body Weight Change

Treatment Group

Animal Number

Body Weight (g)

Body Weight Change (g)

Day 1

Day 6

Vehicle
acetone/olive oil 4:1

1-1

20.4

20.7

0.3

1-2

19.0

19.2

0.2

1-3

22.4

21.7

-0.7

1-4

19.9

18.5

-1.4

1-5

22.1

21.2

-0.9

Test Item
10w/win
acetone/olive oil 4:1

2-1

20.1

21.9

1.8

2-2

22.6

21.6

-1.0

2-3

19.4

19.8

0.4

2-4

19.0

19.3

0.3

2-5

19.9

19.4

-0.5

Test Item
25w/win
acetone/olive oil 4:1

3-1

18.9

21.1

2.2

3-2

19.8

19.4

-0.4

3-3

22.2

24.0

1.8

3-4

19.5

19.6

0.1

3-5

20.0

20.2

0.2

Test Item
50w/win
acetone/olive oil 4:1

4-1

22.0

22.3

0.3

4-2

18.5

19.6

1.1

4-3

18.9

19.8

0.9

4-4

21.2

22.3

1.1

4-5

19.9

20.0

0.1

Positive Control Item
25% v/v in
acetone/olive oil 4:1

5-1

20.1

21.1

1.0

5-2

19.7

20.2

0.5

5-3

19.8

21.2

1.4

5-4

20.4

21.1

0.7

5-5

19.4

21.6

2.2

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be a non-sensitizer under the conditions of the test.
The test item does not meet the criteria for classification according to the Globally Harmonized Classification System.
The positive control α Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (8.32) when tested at a concentration of 25% v/v in acetone/olive oil 4:1 thus demonstrating the sensitivity and reliability of the test system.
Executive summary:

ES421 Pinyl Nitrile was considered to be a non-sensitizer according to OECD Test Guideline 429 using the LLNA method.