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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:
15-01-2018 to 08-02-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Justification for type of information:
Information as to the availability of the in vivo study is provided in 'attached justification'.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

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
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: July 2017 ; signature: November 2017
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
dec-1-en-4-yne
EC Number:
813-331-8
Cas Number:
24948-66-1
Molecular formula:
C10H16
IUPAC Name:
dec-1-en-4-yne
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: Approximately 4 °C, in the dark under nitrogen
- Other: Slightly yellow

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
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 applicable.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: Definitive Test: 16.0 to 21.6 grams (all females treated with test item gained body weight during the study ; where losses occurred they were within typical range for the species and strain, and specifically within the range of the vehicle control group body weight changes).
- Housing: Group housed in cages (pre-test) and solid floor polypropylene cages (main study) with soft wood bedding and cage enrichment
- Diet (e.g. ad libitum): Certified rodent diet, ad libitum
- Water: mains tap water ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: None indicated.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod: 12 hours light / 12 hours dark

- IN-LIFE DATES: 15-01-2018 to 21-01-2018

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
- Preliminary test: undiluted (100%) and 50%v/v in Acetone/Olive Oil (4:1)
- Main test: undiluted (100%), 50%v/v and 25%v/v in Acetone/Olive Oil (4:1)
No. of animals per dose:
Preliminary test: 1
Main test: 5 per dose group
Details on study design:
PRE-SCREEN TESTS / RANGE FINDING TESTS:
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse per test item concentration. The mouse was treated by daily application of 25 µL to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3) of the undiluted test item (100% v/v). The mice were observed at least twice daily on days 1, 2, 3 and 4, 5 and 6. Local skin irritation was scored daily according to the scale included in the full study report. Any clinical signs of toxicity, if present, were also recorded. The body weight of each mouse was recorded on Day 1 (prior to dosing) and on Day 6. The thickness of each ear was measured using a micrometer, pre dose and 1-hour post dose on Day 1, Days 2 and 3. Additional measurements were taken on Days 4 to 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 STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA) per OECD TG 429 and related guidelines.
- Criteria used to consider a positive response: The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser". Additionally, the data must be compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of five mice were treated with the undiluted test item or the test item at 50%v/v and 25%v/v concentrations in acetone/olive oil 4:1. 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.

3H-Methyl Thymidine Administration:
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μl of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 2.0 Ci/mol, specific activity 80 µCi/mL) 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.
- Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and prior to 3HTdR treatment and Day 6 (prior to termination).
- Ear thickness measurements: The thickness of each ear was measured using a micrometer, pre dose and post dose on Day 1, post dose on Days 2 and 3 and on Days 4 to 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.

Preparation of Single Cell Suspension:
A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labelled with the study number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re suspended in 10 mL of PBS and re pelleted. To precipitate out the radioactive material, the pellet was re suspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation:
After approximately 18 hours incubation at approximately 4 degrees Celsius, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by beta-scintillation counting. The vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using a recognised scintillation system.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
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. 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. Data was also assessed for outliers using the Grubb's test.

Results and discussion

Positive control results:
In a concurrent 'positive control study' performed according to OECD TG 429, the sensitivity of the strain of mouse used in this study was assessed using the known sensitiser, α-hexylcinnamaldehyde (85%) at 25% v/v in acetone/olive oil 4:1. The highest concentration tested showed a Stimulation Index (SI) of 8.39 and met the criteria for a 'positive' result.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
3.53
Test group / Remarks:
25% v/v in acetone/olive oil 4:1
Parameter:
SI
Value:
4.78
Test group / Remarks:
50% v/v in acetone/olive oil 4:1
Parameter:
SI
Value:
7.06
Test group / Remarks:
100% (undiluted)
Parameter:
other: disintegrations per minute (DPM)
Remarks:
mean (n=5)
Value:
5 714.46
Variability:
±1950.84
Test group / Remarks:
25% v/v in acetone/olive oil 4:1
Parameter:
other: disintegrations per minute (DPM)
Remarks:
mean (n=5)
Value:
7 679.89
Variability:
±2462.32
Test group / Remarks:
50% v/v in acetone/olive oil 4:1
Parameter:
other: disintegrations per minute (DPM)
Remarks:
mean (n=5)
Value:
11 431.34
Variability:
±383.21
Test group / Remarks:
100% (undiluted)
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
See tables. The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per individual and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

DETAILS ON STIMULATION INDEX CALCULATION
The Stimulation Index is expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group.

EC3 CALCULATION
The EC3 was by applicant calculation: extrapolated using the expression :
EC3 = 2 ^{log2(c)+(3-d)/(b-d)x[log2(a)-log2(c)]}
Where:
a = mid concentration giving a stimulation index > 3 ; = 50%
b = actual stimulation index caused by ‘a’ ; = 4.74
c = lowest concentration giving a stimulation index of > 3 ; = 25%
d = actual stimulation index caused by ‘c’ ; = 3.53
Reference: Ryan, C. et al., Extrapolating Local Lymph Node Assay EC3 Values to Estimate Relative Sensitizing Potency. Cutaneous and Ocular Toxicology, 26: 135–145. (2007).

Calculation:
EC3 = 2 ^{log2(c)+(3-d)/(b-d)x[log2(a)-log2(c)]}
EC3 = 2 ^{log2(25)+(3 - 3.53)/(4.745 - 3.53)x[log2(50)-log2(25)]}
EC3 = 18.45%

CLINICAL OBSERVATIONS:
There were no signs of clinical/systemic toxicity reported during the study. Where body weights decline occurred: they were within typical range for the species and strain, and specifically within the range of the vehicle control group body weight changes There were no signs of local skin irritation (maximum erythema score = 0) in the preliminary test (100% undiluted) and definitive test at up to maximum concentration.

Any other information on results incl. tables

In the preliminary screening test: 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.

 

In the definitive test, there were no deaths or signs of systemic toxicity. Body weights were comparable to that observed in the corresponding control group over the same period. The disintegrations per minute (DPM) and stimulation index (SI) are given in the table for all test item concentrations. There were no signs of local skin irritation (maximum erythema score = 0) at up to maximum concentration.

 

 Table 1.0 – Individual Disintegrations per Minute and Stimulation Index during the test

Test item concentration

DPM values measured / individual

Mean DPM per individual

S.D.

Group Mean S.I.

%

Group no.

Individual no.

Control 0

1

1

1559.38

 

 

 

Control 0

1

2

1166.78

 

 

 

Control 0

1

3

1533.77

 

 

 

Control 0

1

4

1209.44

 

 

 

Control 0

1

5

2629.30

1619.73

592.40

n/a

25

2

1

2922.93

 

 

 

25

2

2

8424.21

 

 

 

25

2

3

5511.00

 

 

 

25

2

4

5804.05

 

 

 

25

2

5

5910.10

5714.46

**

1950.84

3.53

50

3

1

6429.43

 

 

 

50

3

2

9375.85

 

 

 

50

3

3

5411.27

 

 

 

50

3

4

6041.57

 

 

 

50

3

5

11141.31

7679.89 **

2462.32

4.74

100

4

1

11661.85

 

 

 

100

4

2

11053.94

 

 

 

100

4

3

1096.76

 

 

 

100

4

4

11852.34

 

 

 

100

4

5

11591.80

11431.34 **

383.21

7.06

 

 

 

 

 

 

 

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

a = Total number of lymph nodes per individual is 2

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

n/a = Not applicable

* = Significantly different from control group p <0.05

** = Significantly different from control group p <0.01

*** = Significantly different from control group p <0.001

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the condition of this study, the test item is considered to be sensitising to skin. The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 18.45%.
Executive summary:

The study was performed to OECD TG 429 under GLP to assess the skin sensitisation potential of the test material in the CBA/CaOlaHsd mouse following topical application to the dorsal surface of the ear. In a preliminary screening test, single mice were treated by daily application of 25 μl of the test item at specified concentrations 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. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6. 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. The preliminary test was conducted: undiluted (100%v/v). 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 the preliminary test, concentrations of 100%v/v, 50%v/v and 25%v/v in acetone/olive oil (4:1, v/v) vehicle were selected for the main test. 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 and spread over the dorsal surface of the ear. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between Days 1 to 3 and Days 1 to 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 sensitisation. 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. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination). At test termination, five hours after administration of 3HTdR, the test organisms were humanly euthanized. For each individual animal of each group the draining auricular lymph nodes were excised and processed. The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes. The substance was regarded as sensitising if at least one concentration of the substance resulted in a 3-fold or greater increase in 3HTdR incorporation compared to control values. Any substance failing to produce a 3-fold or greater increase in 3HTdR incorporation was classified as non-sensitising.In the main test, there were no deaths or signs of systemic toxicity, and body weights were comparable to controls. A stimulation index (SI) of more than 3 was noted at the 100%v/v (undiluted), 50%v/v and 25%v/v in acetone/olive oil (4:1, v/v) test concentrations. The 100%v/v, 50%v/v and 25%v/v groups generated a SI = 7.06, 4.74 and 3.53, respectively. The test item EC3 value was calculated by log-linear extrapolation of the data. The EC3 value was determined to be 18.45%. Accordingly, the test item was considered to be sensitising under the conditions of the test. Applicant assessment indicates that the log-linear extrapolation method for determining EC3 values is indicated in reference: Ryan, C. et al., Extrapolating Local Lymph Node Assay EC3 Values to Estimate Relative Sensitizing Potency. Cutaneous and Ocular Toxicology, 26: 135–145. (2007).