Reaction mass of (2E)-Tridec-2-enenitrile and (2Z)-Tridec-2-enenitrile and (3E)-Tridec-3-enenitrile and (3Z)-Tridec-3-enenitrile

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-09-29 to 2015-10-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

yes

Remarks:

relative humidity was at times outside the protocol range. Study outcome was not affected.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

yes

Remarks:

relative humidity was at times outside the protocol range. Study outcome was not affected.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

Reaction mass of (2E)-Tridec-2-enenitrile and (2Z)-Tridec-2-enenitrile and (3E)-Tridec-3-enenitrile and (3Z)-Tridec-3-enenitrile

EC Number:

919-489-5

Molecular formula:

C13H23N

IUPAC Name:

Reaction mass of (2E)-Tridec-2-enenitrile and (2Z)-Tridec-2-enenitrile and (3E)-Tridec-3-enenitrile and (3Z)-Tridec-3-enenitrile

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: young adult animals
- Weight at study initiation: 20.3 - 25.3 g
- Housing: 1-5 per cage in polycarbonate box with bedding
- Diet: PMI Feeds Inc., ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 35-92

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

25 and 50 %

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS: No pre-test was performed.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: Stimulation index and EC3 -value

TREATMENT PREPARATION AND ADMINISTRATION:
The following test groups were included in the study: positive control, vehicle control, test group I (25 % test item), test group II (50 % test item), test group III (100 %). Each group consisted of 5 female CBA mice. The test animals received an open application of 25 µL of appropriate dilution (25 or 50 %, 100 %) of test item in the vehicle to the dorsum of both ears.
The vehicle group was treated with the vehicle only. The positive control group was treated with 100 % alpha-hexylcinnamaldehyde. All test and control animals were given a 2-day rest period on days 4 and 5.
On day 6, all animals were injected in the tail vein with 250 µL of 0.01 M phosphate-buffered saline (PBS), pH 7.4 at 25 °C containing 20 µCi of (methyl-3H)Thymidine. Five hours after injection, animals were sacrificed with an overdose of CO2, the draining auricular lymph nodes excised and pairs from each indicvidual animal processed.
A single cell suspension was prepared by mechanical disintegration through 200 mesh stainless steel gauze. Cells were washed twice and precipitated with 5 % trichloracetic acid. The pellets were resuspended in 1 mL TCA and transferred to 10 mL of scinitllation fluid. Incorporation of tritiated thymidine was measured by liquid scintillation counting as disintegrations per minute (DPM) from paired lymph nodes of each animal, and mean DPM/animal was calculated for each group. Background DPM values, determined by blanks, were automatically subtracted by the scintillation counter.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

A one-way parametric analysis of variance (ANOVA) with Dunnett's Multiple Comparison Test, using GraphPad InStat version 3.06 for Windows 95, GraphPad Software, San Diego California USA, was performed on DPM counts. If test groups showed a Stimulation Index (SI) of >3, then extrapolated EC3 was calculated from SI values at low % and either mid or high % concentrations per following formula:
If at least one concentration shows SI of <3, then the formula is: EC3 = c+ [3-d)/(b-d)] x (a-c)
where a = the dose concentration with higher SI;
b= the higher SI value
c= the dose concentration with lower SI,
d= the lower SI value using SI values closest to 3, one above and one below

Results and discussion

Positive control results:

The positive control item produced a stimulation index of >= 3, and is therefore considered a sensitizer.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.7

Test group / Remarks:

25 % test substance concentration

Results 2

Key result

Parameter:

SI

Value:

5.1

Test group / Remarks:

50 % test substance concentration

Results 3

Key result

Parameter:

SI

Value:

6.4

Test group / Remarks:

100 % test substance concentration

Results 4

Key result

Parameter:

SI

Value:

7.1

Test group / Remarks:

Positive control

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
For the 25 % test substance group the mean DPM count was 6366 (±3201). For the 50 % test substance group the mean DPM count was 12075 (±1487). For the 100 % test substance group the mean DPM count was 14998 (±4996). For the vehicle control group the mean DPM count was 2351 (±1895). For the positive control group the mean DPM count was 16498 (±5131).

EC3 CALCULATION
The extrapolated EC3 was 27.98 % using the 25 % and 50 % concentrations.

CLINICAL OBSERVATIONS:
All animals appeared to be normal for the study duration.

BODY WEIGHTS
All test group animals exhibited weight gain during the study.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

The test substance produced a stimulation index >=3 in two groups of test animals, and is therefore considered a sensitizer. As the EC3 value was greater than 10 %, the substance was assessed as a weak sensitizer.

Executive summary:

To assess the sensitizing potential of the test substance, a local lymph node assay in the mouse was conducted according to OECD 429. Three test groups consisted of 5 female CBA mice each. The animals received an open application of 25 µL of test item in the vehicle (25 or 50 %, 100 %) to both ears. All test and control (vehicle and positive) animals were given a 2-day rest period on days 4 and 5. On day 6, all animals were injected in the tail vein with 250 µL of 0.01 M phosphate-buffered saline containing 20 µCi of (methyl-3H)Thymidine. Five hours after injection, animals were sacrificed, the draining auricular lymph nodes excised and processed. Incorporation of tritiated thymidine was measured by liquid scintillation counting as disintegrations per minute (DPM) from paired lymph nodes of each animal, and mean DPM/animal was calculated for each group. As a result, the positive control item produced a stimulation index of >= 3, and is therefore considered a sensitizer. The test substance produced a stimulation index >=3 (27.98 %) in two groups (II and III) of test animals, and is therefore considered a sensitizer. As the EC3 value was greater than 10 %, the substance was assessed as a weak sensitizer.