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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:
19 May 2016 to 28 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study was conducted according to OECD, EU and US EPA test guidelines, in an GLP accredited laboratory

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
yes
Remarks:
1. Deviations from the maximum level of daily mean relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations. The study integrity was not adversely affected by the deviation.
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
May 2008, including most recent amendments.
Deviations:
yes
Remarks:
1. Deviations from the maximum level of daily mean relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations. The study integrity was not adversely affected by the deviation.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
yes
Remarks:
1. Deviations from the maximum level of daily mean relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations. The study integrity was not adversely affected by the deviation.
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

1
Chemical structure
Reference substance name:
Heptanal, oligomeric reaction products with aniline
EC Number:
500-007-3
EC Name:
Heptanal, oligomeric reaction products with aniline
Cas Number:
9003-50-3
Molecular formula:
(C7H14O)n.(C6H7N)n
IUPAC Name:
Heptanal, oligomeric reaction products with aniline
Test material form:
liquid
Details on test material:
Test item: 207368/A
Identification: Hepteen Base®
Appearance: Clear amber liquid (determined by Charles River Den Bosch)
Batch: LT5C30Y170
Purity/Composition: 99.7%
Test item storage: At room temperature
Stable under storage conditions until: 30 November 2016 (retest date)
Specific details on test material used for the study:
Test item: 207368/A
Identification: Hepteen Base®
Appearance: Clear amber liquid (determined by Charles River Den Bosch)
Batch: LT5C30Y170
Purity/Composition: 99.7%
Test item storage: At room temperature
Stable under storage conditions until: 24 September 2016 (retest date)
pH (1% in water, indicative range) 8.0 – 7.8 (determined by Charles River Den Bosch)

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
Species: Mouse, CBA/J strain, inbred, SPF-Quality.
Recognized by the international guidelines as the recommended test system (e.g. OECD, EC, EPA).
Source: Janvier, Le Genest-Saint-Isle, France
Number of animals: 20 females (nulliparous and non-pregnant), five females per group (main study only).
Age and body weight: Young adult animals (approx. 10 weeks old) were selected.
Body weight: variation was within +/- 20% of the sex mean.
Identification: Tail mark with a marker pen.
Health inspection: At least prior to dosing. It was ensured that the animals were healthy and that the ears were intact and free from any abnormality.

Animal Husbandry
Conditions
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle: the photoperiod is between 07:00 and 19:00 hrs daily. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Accommodation
Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment. The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and
cage enrichment.

Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Water
Free access to tap water.

Diet, water, bedding and cage enrichment evaluations for contaminants and/or nutrients were performed according to facility standard procedures. There were no findings that could interfere with the study.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0%, 10%, 25%, 50%
No. of animals per dose:
5 animals per dose (Total 20)
Details on study design:
Test Item Preparation
Vehicle: Acetone/Olive oil (4:1 v/v) (Acetone p.a.: Merck, Darmstadt, Germany; Olive oil: Fagron, Nieuwerkerk a/d IJssel, The Netherlands).
Rationale: The vehicle was selected on the basis of maximizing the solubility using the test item data provided by the Sponsor and trial preparation results performed at Charles River Den Bosch.
The vehicle was chosen from the vehicles specified in the test guideline: Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol dimethylsulfoxide and 1% Pluronic© L92 in Elix water (in case an aqueous vehicle is suitable). There was no information available regarding the solubility or stability in vehicle.

Preparation: The test item preparations (w/w) were prepared within 4 hours prior to each dosing. No adjustment was made for specific gravity of the vehicle. Homogeneity was assessed by visual inspection of the solutions. Correction of the purity/composition of the test item is not applicable, since the test method requires a logical concentration range rather than specific dose levels to be dosed.

Pre-screen Test
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2) and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.

Two test item concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines. The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 11 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.

Animals were sacrificed after the final observation.

Main Study
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test.
One group of five animals was treated with the vehicle.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.

Excision of the Nodes - Day 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Tissue Processing for Radioactivity - Day 6
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter: 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

Radioactivity Measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) according to the following numerical scoring system.
In addition, a description of all other (local) effects was recorded.

Grading Irritation Reactions:
Erythema and eschar formation:
No erythema = 0
Very slight erythema (barely perceptible) = 1
Well-defined erythema = 2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth) = 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema = 4

Necropsy: No necropsy for gross macroscopic examination was performed according to study plan.


Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Linear interpolation

Results and discussion

Positive control results:
The SI values calculated for the item concentrations 5, 10 and 25% were 1.0, 1.5 and 4.4 respectively. An EC3 value of 17.8% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 14.5, 13.4, 14.1, 17.3, 9.8% and 10%.

Based on the results, it was concluded that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results
Key result
Parameter:
EC3
Value:
> 0 - <= 10
Remarks on result:
other: SI ≥ 3
Cellular proliferation data / Observations:
See "Any other information on results incl. tables"

Any other information on results incl. tables

Pre-screen Test

At a 100% test item concentration, ptosis was noted for both animals on Days 4 and 5. Very slight to well-defined erythema was observed between Days 1 and 6. Variations in ear thickness on Day 6 were more than 25% from Day 1 pre-dose values.

 

At 50%, no clinical signs of systemic toxicity was noted. Very slight to well-defined erythema and scaliness were observed between Days 2 and 6. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values.

 

Based on the systemic toxicity noted for the animals at a concentration of 100%, the highest test item concentration selected for the main study was a 50% concentration.

 

Main Study

Skin Reactions / Irritation

Very slight to well-defined erythema, scaliness and/or erythema between the ears were noted for the animals throughout the dose groups, 10%, 25% and 50% between Days 2 and 6. This was considered not to have a toxicologically significant effect on the activity of the nodes.

 

Systemic Toxicity

No mortality occurred in the animals of the main study. Hunched posture and/or piloerection were noted for most animals treated at concentrations of 25% (Days 4 and 5) and 50% (Days 3-6). No signs of systemic toxicity were noted for the 10% test item group or control

group.

 

Body weight loss was noted for the animals treated at concentrations of 25% and 50%. Body weights and body weight gain of the animals at 10% remained in the same range as controls over the study period

 

Macroscopic Examination of the Auricular Lymph Nodes and Surrounding Area

The auricular lymph nodes of the control group were considered normal in size. The auricular lymph nodes of the experimental groups were considered enlarged.

 

No macroscopic abnormalities of the surrounding area were noted for any of the animals.

 

Radioactivity Measurements and SI Values

Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 9822, 11789 and 13284 DPM, respectively. The mean DPM/animal value for the vehicle control group was 837 DPM. The SI values calculated for the test item concentrations 10, 25 and 50% were 11.7, 14.1 and 15.9, respectively.    

 

 

Main Study: Relative Size Lymph Nodes, Radioactivity Counts (DPM) and Stimulation Index (SI).

Group

TS (%)1

Animal

Size of nodes2

DPM / animal3

MeanDPM ± SEM4

mean

SI ± SEM

Left

Right

1

0

1

n

n

630

837 ± 88

1.0 ± 0.1

 

 

2

n

n

668

 

 

3

n

n

1117

 

 

4

n

n

900

 

 

5

n

n

869

2

10

6

+

+

11653

9822 ± 1178

11.7 ± 1.9

 

 

7

+

+

7482

 

 

8

+

+

12845

 

 

9

+

+

6745

 

 

10

+

+

10383

3

25

11

+

+

12534

11789 ± 2408

14.1 ± 3.2

 

 

12

+

+

20320

 

 

13

+

+

9227

 

 

14

+

+

11036

 

 

15

+

+

5829

4

50

16

+

+

8158

13284 ± 1983

15.9 ± 2.9

 

 

17

+

+

12263

 

 

18

+

+

14548

 

 

19

+

+

20073

 

 

20

+

+

11377

1TS = test item (% w/w).

2Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n:

considered to be normal).

3DPM = Disintegrations per minute.

4SEM = Standard Error of the Mean.

Positive control results:

Group1

% Alpha- Hexylcinnamaldehyde,

technical grade

mean

DPM ± SEM

SI ± SEM

1

0% (Acetone:Olive oil

(4:1 v/v))

1044 ± 160

1.0 ± 0.2

2

5%

1062 ± 154

1.0 ± 0.2

3

10%

1536 ± 275

1.5 ± 0.3

4

25%

4551 ± 643

4.4 ± 0.9

1Five females per group.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Signs of systemic toxicity were found in the two highest dose groups (25% and 50%). It was considered that this could have had a toxicologically significant effect on the activity of the nodes therefore these groups were excluded form interpretation. No signs of systemic toxicity were noted for the animals treated at 10% therefore sufficient data was available for interpretation of the study.
The results show that the test item elicits a SI ≥ 3. The EC3 value (the estimated test item concentration that will give a SI =3) was established to be between >0 and 10%.
The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed is an appropriate model for testing for contact hypersensitivity.
Based on these results:
- according to the recommendations made in the test guidelines (including all amendments), Hepteen Base would be regarded as skin sensitizer.
- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), Hepteen Base should be classified as skin sensitizer (Category 1).
- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), Hepteen Base should be classified as skin sensitizer (Category 1) and labeled as H317: May cause an allergic skin reaction
Executive summary:

Assessment of skin sensitization to Hepteen Base in the Mouse (Local Lymph Node Assay).

The study was carried out based on the guidelines described in:

OECD, Section 4, Health Effects, No.429 (2010), EC, No 440/2008;

B42: "Skin Sensitization: Local Lymph Node Assay" ;

EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

Test item concentrations selected for the main study were based on the results of a pre-screen test.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

Very slight to well-defined erythema, scaliness and/or erythema between the ears were noted for the animals throughout the dose groups, 10%, 25% and 50% between Days 2 and 6. This was considered not to have a toxicologically significant effect on the activity of the nodes.

Hunched posture and/or piloerection were noted for most animals treated at concentrations of 25% (Days 4-5) and 50% (Days 3-6). No signs of systemic toxicity were noted for the 10% test item group or control group. Body weight loss was noted for the animals treated at concentrations of 25% and 50%. Body weights and body weight gain of the animals at 10% remained in the same range as controls over the study period.

The auricular lymph nodes of the control group were considered normal in size. The auricular lymph nodes of the experimental groups were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 9822, 11789 and 13284 DPM, respectively. The mean DPM/animal value for the vehicle control group was 837 DPM. The SI values calculated for the test item concentrations 10, 25 and 50% were 11.7, 14.1 and 15.9, respectively.