Dipentylammonium dipentyldithiocarbamate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 March 2018 - 5 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), Testing Guidelines for Toxicology Studies, 12 NouSan No. 8147, 24 November 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

Carbamodithioic acid, dipentyl-, branched and linear, compds. with branched and linear N-pentyl-1-pentanamine

EC Number:

294-061-2

EC Name:

Carbamodithioic acid, dipentyl-, branched and linear, compds. with branched and linear N-pentyl-1-pentanamine

Cas Number:

91672-53-6

Molecular formula:

C21H46N2S2

IUPAC Name:

Branched and linear N-pentylpentan-1-aminium branched and linear dipentyldithiocarbamate

Specific details on test material used for the study:

Purity: >99% (nominal); a substance of Unknown or Variable composition, Complex reaction products of biological materials (UVCB)
Description: Amber colored viscous liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Strain: CBA/Ca (CBA/CaOlaHsd)
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 15 to 23 g
- Housing: Animals assigned to the study were group housed by dose level in suspended solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding.
- Diet and water: Free access to tap water and food was allowed throughout the study
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): At least fifteen
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0, 10, 25 and 50 % w/w

No. of animals per dose:

Two mice for the preliminary test, followed by five animals per dose in the main test.

Details on study design:

Preliminary Screening Test
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using two female mice, one mouse per test item concentration. The mice were treated by daily application of 25 µL of the undiluted test item or the test item at a concentration of 50% v/v in acetone/olive oil 4:1, to the dorsal surface of each ear for up to three consecutive days (Days 1, 2, 3). The mice were observed twice daily (pre and post dose) on Days 1 and 2 and the surviving mouse twice daily on Day 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 body weight of each mouse was recorded on Day 1 (prior to dosing) and on Day 6 or at death.

The thickness of each ear was measured using a Mitutoyo 547 300S gauge (Mitutoyo Corporation), 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 female mice were treated with the test item at concentrations of 50%, 25% or 10% v/v 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 up to 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 female mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item or vehicle (Day 6) all surviving 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.

Observations
Clinical Observations: The mice were observed twice daily (pre and post dose) on Days 1 and 2 and the surviving mice twice daily on Day 3 and once daily 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) or at death.

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all surviving mice were euthanized 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.

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 labeled 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 deg.C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by beta-scintillation counting. The "Poly Q™" 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 the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

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. 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.

Probability values (p) are presented as follows:

P<0.001 ***
P<0.01 **
P<0.05 *
P>0.05 (not significant)

Results and discussion

Positive control results:

α Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.8

Test group / Remarks:

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

Results 2

Key result

Parameter:

SI

Value:

7.08

Test group / Remarks:

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

Results 3

Key result

Parameter:

SI

Test group / Remarks:

50% w/w in acetone/olive oil 4:1

Remarks on result:

other: Early sacrifices made

Any other information on results incl. tables

Preliminary Screening Test

The animal treated with the undiluted test item was found dead, pre-dose on Day 3.

 

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 animal treated with the test item at a concentration of 50% v/v in acetone/olive oil 4:1.

Based on this information, the dose levels selected for the main test were 50%, 25% and 10% v/v in acetone/olive oil 4:1.

Main Test

Estimation of the Proliferative Response of Lymph Node Cells

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%v/v) in acetone/olive oil 4:1	Stimulation Index	Result
10	2.80	Negative
25	7.08	Positive
50	*(Early sacrifices)	*(na)

 

Clinical Observations and Mortality Data

Lethargy and ataxia were noted, pre-dose on Day 3, in animals treated with the test item at a concentration of 50% v/v in acetone/olive oil 4:1 and therefore were euthanized, due to the occurrence of clinical signs of toxicity that were considered to approach/exceed the moderate severity limit set forth in the UK Home Office Project License. 

No signs of systemic toxicity were noted in animals treated with the test item at concentrations of 25% or 10% v/v in acetone/olive oil 4:1.

Body Weight

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.

Calculation of EC₃Value

aEC₃= c + [[(3-d)/(b-d)] x (a-c)]

a	=	25
b	=	7.08
c	=	10
d	=	2.80
EC3=10+ [[(3-2.80)/(7.08-2.80)] x (25-10)] =10.7

The concentration of test item expected to cause a 3 fold increase in³HTdR incorporation (EC₃value) was calculated to be10.7%.

*=No data, animals humanely killed, pre-dose on Day 3, due to the occurrence of clinical signs of toxicity that were considered to approach/exceed the moderate severity limit set forth in the UK Home Office Project License

Na   =Not applicable

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’

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

The test item, Dipentylammonium dipentyldithiocarbamate (CAS RN 71902-20-0), was considered to be a weak skin sensitizer with an EC3 of 10.7% under the conditions of the test.

The test item is classified as a contact sensitizer (Category 1B) according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures and the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

Executive summary:

A skin sensitisation study, local lymph node assay in the mouse was performed in accordance to the standardized guidelines OECD 429 and EU Method B.42, under GLP conditions in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Following preliminary screening tests in two female mice treated with the undiluted test item or the test item at a concentration of 50% v/v in acetone/olive oil 4:1, one mouse per test item concentration, in which the undiluted test item caused mortality and no clinical signs of toxicity were noted at aconcentration of 50%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five female animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 50%, 25% or 10% v/v. A further group of five female animals was treated with acetone/olive oil 4:1 alone in the same manner. 

The test item, Dipentylammonium dipentyldithiocarbamate (CAS RN 71902-20-0),was considered to be a weak skinsensitizerwith an EC₃of 10.7% under the conditions of the test.

The test item is classified as a contact sensitizer (Category 1B) according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures and the United Nations GloballyHarmonized Systemof Classification and Labelling of Chemicals (GHS)