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

Endpoint summary

Description of key information

The test substance was determined to be a weak skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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.

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)

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

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

Positive control results:

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

Key result

Parameter:

SI

Value:

2.7

Test group / Remarks:

25 % test substance concentration

Key result

Parameter:

SI

Value:

5.1

Test group / Remarks:

50 % test substance concentration

Key result

Parameter:

SI

Value:

6.4

Test group / Remarks:

100 % test substance concentration

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.

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.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

GLP compliance:

no

Type of study:

open epicutaneous test

Justification for non-LLNA method:

No LLNA study was available as the in vivo study was conducted in 1977, before the first version of the LLNA (OECD 429) was issued in 2002.

Species:

guinea pig

Strain:

not specified

Sex:

not specified

Details on test animals and environmental conditions:

No data

Route:

epicutaneous, open

Vehicle:

other: ethanol

Concentration / amount:

100, 30, 10, 3 %

Day(s)/duration:

21 days

No.:

#1

Route:

epicutaneous, open

Vehicle:

other: ethanol

Concentration / amount:

3 %

Day(s)/duration:

on day 21

No.:

#2

Route:

epicutaneous, open

Vehicle:

other: ethanol

Concentration / amount:

3 %

Day(s)/duration:

on day 35

No.:

#3

Route:

epicutaneous, open

Vehicle:

other: ethanol

Concentration / amount:

3 %

Day(s)/duration:

on day 49

No. of animals per dose:

6-8

Details on study design:

RANGE FINDING TESTS:
One day before the induction exposure the threshold-toxic concentration of the test material was estimated by applying the substance at the the test concentrations to the skin of guinea pigs. The minimal irritant and the maximal non irritant concentration were determined.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 21 (daily for 3 weeks)
- Exposure period: The test substance was applied uncovered and not washed off.
- Site: flank
- Frequency of applications: daily
- Concentrations: 100, 30, 10 and 3 %

B. CHALLENGE EXPOSURE
- No. of exposures: 3
- Day(s) of challenge: 21, 35 and 49
- Exposure period: The test substance was applied uncovered and not washed off.
- Site: contralateral flank
- Concentrations: 3 %
- Evaluation: Skin reactions were read after 24, 48 and 72 hours.

Challenge controls:

6-8 untreated or vehicle treated animals served as challenge control.

Positive control substance(s):

not required

Key result

Reading:

1st reading

Group:

test chemical

Dose level:

100 %

No. with + reactions:

3

Total no. in group:

6

Clinical observations:

no data

Remarks on result:

positive indication of skin sensitisation

Skin sensitization after daily application for three week

Concentration [%]	Sensitization rate [Number of animals positive/total]
	Challenge on Day 21	Challenge on Day 35	Challenge on Day 49
100	3/6	3/6	2/6
30	0/6	0/6	0/6
10	0/6	0/6	0/6
3	0/6	0/6	0/6

 

Interpretation of results:

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

Conclusions:

The test substance was found to be a weak sensitizer in the OET in guinea pigs.

Executive summary:

An open epicutaneous test using guinea pigs was performed to determine the skin sensitising property of the test substance. Therefore, 0.1 mL the test substance was applied at concentrations of 100, 30, 10 and 3 % to the clipped flanks of the test animals (6 -8 per group) daily for 3 weeks. The test site remained uncovered and the test material was not washed off. Skin reactions were read afterwards and the minimal irritant concentration was determined to be 3 %, the maximal non irritant concentration was 1 %. On days 21, 35 and 49 the challenge exposures (0.025 mL of the test substance at a concentration of 3 % and some lower concentrations) were applied to the skin. Skin reactions were evaluated 24, 48 and 72 hours afterwards. As a result, in half of the animals of the high dose induction group (100 % test substance concentration) positive skin reactions were observed. As a conclusion, the test substance was determined to be a weak skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin sensitization

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 (EC3 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.

An open epicutaneous test using guinea pigs was performed to determine the skin sensitising property of the test substance. Therefore, 0.1 mL of the test substance was applied at concentrations of 100, 30, 10 and 3 % to the clipped flanks of the test animals (6 -8 per group) daily for 3 weeks. The test site remained uncovered and the test material was not washed off. Skin reactions were read afterwards and the minimal irritant concentration was determined to be 3 %, the maximal non irritant concentration was 1 %. On days 21, 35 and 49 the challenge exposures (0.025 mL of the test substance at a concentration of 3 % and some lower concentrations) were applied to the skin. Skin reactions were evaluated 24, 48 and 72 hours afterwards. As a result, in half of the animals of the high dose induction group (100 % test substance concentration) positive skin reactions were observed. As a conclusion, the test substance was determined to be a weak skin sensitizer.

Conclusion:

Both the LLNA and the in vivo test in guinea pigs concluded that the test substance is a weak skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

Based on available data on skin sensitisation, the test item is classified for skin sensitisation into category 1B and labeled with H317 (May cause an allergic skin reaction) according to Regulation (EC) No 1272/2008 (CLP), as amended for the ninth time in Regulation (EU) No 2016/1179.