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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:
October 19 - November 09, 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
1,3-Propanediol dicaprylate/dicaprate (CAS# 1072005-10-7 referred as source substance) is proposed as structural analogue for the target substance 1,3-propanediol dioctanoate (CAS# 56519-71-2). Both substances are diesters with 1,3-propanediol of similar molecular size, containing the same functional groups. Due to the lack of human health data on skin sensitisation by 1,3-propanediol dioctanoate, a skin sensitisation study with the source substance is suggested to be used for closing the respective data gap in the target substance dossier. Similarities in structure and physico-chemical properties are supposed to form a basis for the analogue approach. This study with the source substance CAS# 1072005-10-7 has been performed according to OECD and/or EC guidelines and according to GLP principles. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
Temporary deviations from the minimum level of relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations. The study integrity was not adversely affected by the deviations.
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
Temporary deviations from the minimum level of relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations. The study integrity was not adversely affected by the deviations.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,3 propanediol dicaprylate/dicaprate
- Physical state: Clear colourless liquid
- Storage condition of test material: At room temperature in the dark
- Stability under storage conditions: Stable
- Stability and solubility in acetone: Not indicated
- Stability and solubility in olive oil: Not indicated
- Smiles notation (if other than submission substance): CCCCCCCCCC(=O)OCCCOC(=O)CCCCCCC
- Physical state: liquid
- Melting point: -8 °C
- Boiling point: 358 °C
- Relative density: 0.91-0.93
- Vapour pressure: ca. 0.00001
- log Kow: 7.7
- Water solubility: 0.005 mg/L

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L‟Arbresle Cedex, France
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- Housing: Animals were individually housed in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment. The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on Day 3. On Day 6, the animals were group housed in Makrolon MIII type (height 18 cm) cages.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.
- Health inspection: A health inspection was performed prior to treatment, to ensure that the animals are in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality.

Results of analysis for diet (nutrients and contaminants), sawdust, paper and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 – 24
- Humidity (%): 39 - 64
Temporary deviations from the minimum level of relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations. The study integrity was not adversely affected by the deviations.
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 October to 09 November 2009

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Preliminary irritation study: a 100% and 50% concentration.
Main study: 0, 25, 50 and 100%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (maximally grade 2) at the highest concentration.
The test system, procedures and techniques were identical to those used during Days 1 to 3 of the main study unless otherwise specified. Two young adult animals were selected (8-14 weeks old). Each animal was treated with one concentration on three consecutive days. Approximately 3-4 hours after the last exposure, the irritation of the ears was assessed. Bodyweights were determined on Day 3. The animals were sacrificed after the final observation and no necropsy was performed.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is
calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group.
If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.
Rationale for vehicle: The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.

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

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 approximately five hours, all animals were killed by intraperitoneal injection with 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:
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) 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. Radioactive 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-treatment) and Day 6.
Clinical signs: At least once daily.
Irritation: On Day 3 (3-4 hours after treatment), the skin reactions were assessed. Skin reactions were graded according to the following numerical scoring system. Furthermore descriptions of all other (local) effects were recorded.

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

Oedema formation:
0: No oedema
1: Slight oedema (barely perceptible)
2: Moderate oedema
3: Severe oedema

Necropsy: All animals of the study were subjected to necropsy for gross macroscopic examination.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not performed.

Results and discussion

Positive control results:
The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity. See attached document 'Reliability check'.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 25, 50 and 100% were 1.3, 1.5 and 2.6 respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
The DPM value of 7871 for one animal treated at 100% was identified as outlier (well outside the experimental DPM range of 493-2076 and statistically confirmed by the Grubb test) and was therefore not used for interpretation. Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 980, 1152 and 1966 respectively. The mean DPM/animal value for the vehicle control group was 751.

Any other information on results incl. tables

Tables and figures of the Pre-screen test and Main study have been included in the attached document "LLNA tables and figures".

Results Pre-screen test:

The results of the epidermal exposures for the selection of highest test substance concentration to be tested in the main study are described in the attached LLNA table. Based on the results, the highest test substance concentration selected for the main study was a 100% concentration.

Other results - main study:

No irritation of the ears was observed in any of the controls, all animals treated at 25% and in two animals treated at 50%. The slight irritation of the ears as shown by three animals treated at 50% and all treated at 100% was considered not to have a toxicologically significant effect on the activity of the nodes.

 

All auricular lymph nodes of the controls were considered normal in size. Enlarged nodes were found among the experimental animals.

One animal treated at 50% showed enlargement and reddish discoloration of the left lymph node in combination with opacity of the left eye. Since this could have affected the outcome for this animal, the result was rejected and not used for interpretation. No macroscopic abnormalities of the surrounding area were noted in the other animals.

 

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in one animal treated at 100% was considered not toxicologically significant.

    

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Analogue approach justification

 

Structure and PC parameters

The structural difference between the target and source chemicals is that one hydrocarbon chain of the source substance is two CH2 – groups longer compared to the length in target substance (C10 instead of C8). These feature accounts for differences in PC-parameters (see Data Matrix, Sec. 5 below). The melting point of the Source Substance is lower than the one of the target substance. This may be correlated with the lower symmetry of the source molecule. Both substances are liquids at ambient conditions. They are non-volatile substances of similar density, boiling at temperatures exceeding 350 °C. The logarithmic partition coefficients are in the range ca. 7-8 and water solubilities are below 0.1 mg/L.

 

 

Skin sensitisation

In the case of this endpoint, chemical structures are investigated with respect to the presence or absence of structural alerts, indicating protein binding affinity. For the two substances, the results of the relevant OECD QSAR Toolbox profiling together with Toxtree (Estimation of Toxic Hazard – A Decision Tree Approach v2.1.0) outcomes are presented in Table 1. One structural alert can be present in more than one of all four sets.

Profile

Total number of structural alerts applied

Target substance

Source substance

Protein binding by OASIS

67

No binding

No binding

Protein binding by OECD

122

No binding

No binding

Protein binding potency

104

Not possible to classify

Not possible to classify

Organic functional groups

-

Carboxylic acid ester

Ether

Methyl

Methylene

Carboxylic acid ester

Ether

Methyl

Methylene

Toxtree skin sensitisation alerts

5

No alerts identified

No alerts identified

Table 1: OECD Toolbox and Toxtree profiles with respect to Skin Sensitisation

The profiles are identical and they indicate no sensitisation potency of both substances. This allows then conclusion, that the read-across for this endpoint is justified based on the structural similarity structural alert based profiles of both, target substance and source substance.

Data matrix

 

Endpoint

Target substance

Source substance

Physical state

liquid

liquid

Melting point [°C]

6

-8 (cristallization)

Boiling point [°C]

352

358 (calc. MPBPWIN v1.43)

Relative density

0.93

0.91-0.93 (calc. SPARC v4.5)

Vapour pressure [Pa]

8E-4

ca. 1E-5 (calc. SPARC v4.5)

log Kow

7.6

7.7 (calc. KOWWIN v1.68)

Water solubility [mg/L]

< 0.01

0.005 (calc. WSKOWWIN v1.42

Skin sensitisation

RA: non-sensitising

Non-sensitising

Data are experimental determinations (Klimisch 1 or 2) unless otherwise indicated.

 

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Sufficient data was available after rejection of the results of two animals (one of the intermediate dose and one of the highest dose) to conclude that there was no indication that the test substance elicits an SI ≥ 3 when tested up to 100%. This conclusion is corroborated by the fact that the SI mean values of 4 (out of 5) animals treated at 100% are below the SI=3 threshold. Based on this evaluation 1,3 propanediol dicaprylate/dicaprate was considered not to be a skin
sensitizer.

Based on these results 1,3 propanediol dicaprylate/dicaprate would not be regarded as skin sensitizer according to the recommendations made in the test guidelines and does not have to be classified and has no obligatory labeling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures.


Based on the study results, the criteria for classification and labelling according to DSD (67/548/EEC) and CLP (EC 1272/2008) are not fulfilled. Therefore, 1,3-propanediol dioctanoate is not sensitising to the skin.
Executive summary:

Assessment of Contact Hypersensitivity to 1,3 propanediol dicaprylate/dicaprate in the Mouse (Local Lymph Node Assay, 5 females/dose) was conducted according to OECD 429 and GLP guidelines.

The slight irritation of the ears as shown by three animals treated at 50% and all treated at 100% was considered not to have a toxicologically significant effect on the activity of the nodes. No oedema was observed in any of the animals examined.

All auricular lymph nodes of the controls were considered normal in size. Enlarged nodes were found among the experimental animals.

One animal treated at 50% showed enlargement and reddish discoloration of the left lymph node in combination with opacity of the left eye. Since this could have affected the outcome for this animal, the result was rejected and not used for interpretation. No macroscopic abnormalities of the surrounding area were noted in the other animals.

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in one animal treated at 100% was considered not toxicologically significant.

The DPM value of 7871 for one animal treated at 100% was identified as outlier and was therefore not used for interpretation.

The SI values calculated for the substance concentrations 25, 50 and 100% were 1.3, 1.5 and 2.6 respectively.

Sufficient data was available after rejection of the results of two animals (one of the intermediate dose and one of the highest dose) to conclude that there was no indication that the test substance elicits an SI ≥ 3 when tested up to 100%. This conclusion is corroborated by the fact that the SI mean values of 4 (out of 5) animals treated at 100% are below the SI=3 threshold.

Based on the results 1,3 propanediol dicaprylate/dicaprate would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. It does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact