2,2-bis[[(2-ethyl-1-oxohexyl)oxy]methyl]propane-1,3-diyl bis(2-ethylhexanoate)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

19 November 2003 - 24 November 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD test guidance in compliance with GLP and reported with a valid GLP certificate. The study is read across to an analogous substance; refer to image and further information below.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2003

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:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

144971-11-9

Cas Number:

144971-11-9

IUPAC Name:

144971-11-9

Test material form:

liquid: viscous

Details on test material:

CAS Number: 144971-11-9
Description: Clear colourless liquid
Batch: D21287
Purity: >97.3%
Test substance storage: At room temperature in the dark
Stabilty under storage conditions: Not indicated
Expiry date: 31 December 2003

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Species: Mouse, CBA strain, inbred, SPF-Quality. Recognised by the international guidelines as the recommended test system (e.g. OECD, EEC, EPA) Source: Charles River France, L'Arbresle Cedex, FranceNumber of animals: 20 females (four groups offive females each group). (nulliparous and non-pregnant).Age and Bodyweight: Young adult animals (approx. 9 weeks old) were selected. Body weight variation was +/- 20% of the sex mean.Identification: Tailmark.Control animals: The vehicle control animals were treated using the same vehicle and using the same procedures.Reliability check: The results of a reliabilty test performed not more than 6 months previously or 2 months afterwards are summarised in the Appendix. Similar procedures were used in the reliabilty test and in this study.ConditionsAnimals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0± 3.0°C (actual range: 18.6 - 21.0°C), a relative humidity of 30-70% (actual range: 40 - 63%) and 12 hours artificial fluorescent light and 12 hours darkness per day.AccommodationIndividual housing in labelled Macrolon cages (type i; height 12.5 cm) containing purified sawdust as bedding material (SAWI, Jelu Werk, Rosenberg, Germany). Certificates of analysis were examined and then retained in the NOTOX archives. The acclimatisation period was at least 5 days before the start of treatment under laboratory conditions. Animals were group housed in polycarbonate cages (MacrolonII type; height 15 cm) during the acclimatisation period.DietFree access to standard pelleted laboratory animal diet (from Altromin (code VRF 1), Lage, Germany). Certificates of analysis were examined and then retained in the NOTOX archives.WaterFree access to tap-water. Certificates of quarterly analysis were examined and then retained in the NOTOX archives.

Study design: in vivo (LLNA)

Vehicle:

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

Remarks:

The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. Homogeneity was obtained to visually acceptable levels.

Concentration:

Lowest test substance concentration: 5%Intermediate test substance concentration: 50%Highest test substance concentration: 100%

No. of animals per dose:

five females each group

Details on study design:

Preliminary irritation studyA preliminary irritation study was conducted as part of NOTOX project 385368 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 (grade 3) at the highest.A series of four test substance concentrations were tested, the highest concentration being the maximum concentration that could technically be applied. The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 25%,10%,5%,2.5%,1% and if needed further lower concentrations using the same steps. The test system, procedures and techniques were identical to those used during days 1 to 3 of the main study unlessotherwise specified.Four young adult animals were selected (5-14 weeks oid). Each animal was treated with one concentration on two consecutive days. Approximately 4 hours after the last exposure, the skin was cleaned of residual test substance with water and the irritation was assessed. No necropsy was performed and no bodyweights were determined after termination.Main studyThree groups of five animals were treated with three test substance concentrations respectively. One group of five animals was treated with vehicle.Lowest test substance concentration: 5%Intermediate test substance concentration: 50%Highest test substance concentration: 100%INDUCTION - Days 1, 2 and 3Experimental animals:The dorsum surface of both ears was epidermally treated (25 µl/ear) with the test substance concentration, approximately the same time each day.Vehicle control animals:The control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle was administered.TREATMENT - Day 6:Ali animals:Each animal was injected via the tail vein with 0.25 ml of sterile phosphate buffered saline (PBS) containing 20µCi of 3H-methyl thymidine (Amersham Pharmacia Biotech, NOTOX Substance 105624).After approximately five hours, all animals were kiled by intra peritoneal injection with an overdose of pentobarbital. 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 were recorded. The nodes were pooled for each animal in 3 ml PBS.Tissue processing for radioactivityA 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 4° C. The DNA was precipitated with 3 m15% trichloroacetic acid (TCA) at 4° C for approximately 18 hours. Precipitates were recovered by centrifugation at 200g for 10 minutes, resuspended in 1 ml TCA and transferred to 10 ml of Ultima Gold (Packard) as the scintilation fluid.Radioactivity measurementsAll radioactive measurements were performed using a Packard scintilation counter (1900TR). Counting time was to a statistical precision of± 0.2% or a maximum of 5 minutes whichever comes first. The Packard 1900TR was programmed to automatically subtract background and convert CPM to DPM.ObservationMortality/Viability: Twice daily.Toxicity: At least once daily.Body weights: On days 1 (pre-treatment) and 6.Irritation: On day 3 (3-4 hours after treatment), the skin reactions were assessed. If possible, skin reactions were graded according to the numerical scoring system. Furthermore descriptions of all other (local) effects were recorded.InterpretationDPM 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. For undiluted test substance, untreated animals are used to calculate the SI.If the results indicate a SI≥3, the test substance may be regarded as a skin sensitiser, based on the test guideline and recommendations done by ICCVAM (NIH publication; No 99-4494, February 1999).The results were evaluated according to the OECD Harmonized Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances (OECD, 1998) and the EC criteria for classification and labelling of dangerous substances and preparations (Council Directive 67/548/EEC and all adaptations to technical progress and amendments of this Directive published in the Official Journal of the European Communities).If possible, an EC3 value (the estimated test substance concentration that will give a SI =3) was determined, using linear interpolation (Reference 2).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

None measured.

Results and discussion

Positive control results:

The Si values calculated for the substance concentrations 5, 50 and 100% were 1 .4, 3.5 and 3.7. Since 3/5 responses at 50 and 100% were above SI=3, it was considered that there is suffcient evidence that the test substance could elicit a SI> 3. These data showed a dose-response and an EC3 value of 39% was calculated.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The SI values calculated for the substance concentrations 5, 50 and 100% were 1.0, 1.7 and 2.4 respectively.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 50 and 100% were 192, 326 and 471 respectively.The mean DPM/animal value for the vehicle control group was 195.

Any other information on results incl. tables

Radioactivity Measurements (Individual Animals)

Group	Animal	Treatment	Induction	DPM / Animal
1	1	Vehicle Control	Acetone / Olive Oil (4:1 v/v)	101
	2			104
	3			131
	4			447
	5			194
2	1	Experimental	5% test substance	326
	2			270
	3			93
	4			80
	5			189
3	1	Experimental	50% test substance	259
	2			530
	3			462
	4			120
	5			261
4	1	Experimental	100% test substance	632
	2			515
	3			329
	4			355
	5			524

 

Calculation of Stimulation Index (SI)

Group	Treatment	Induction	Mean DPM ± SD	Mean SI ± SD
2	Experimental	5% test substance	192 ± 108	1.0 0.9
3	Experimental	50% test substance	326 ± 167	1.7 ± 0.9
4	Experimental	100% test substance	471 ± 127	2.4 ± 0.8
1	Vehicle Control	Acetone / Olive Oil (4:1 v/v)	195 ± 146	1.0

 

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated informationCriteria used for interpretation of results: EU

Conclusions:

Read across to structural analogue. Structural details are listed above. The SI values calculated for the substance concentrations 5, 50 and 100% were 1.0, 1.7 and 2.4 respectively.These data showed a dose-response but there was no indication that the test substance could elicit a SI ≥ 3.Based on these results and according to the recommendations made in the test guidelines (OECD No.429, EC B.42 and EPA OPPTS 870.2600), HATCOL 3331 should not be regarded as a skin sensitiser

Executive summary:

Read across to structural analogue. Structural details are listed above.

The SI values calculated for the substance concentrations 5, 50 and 100% were 1.0, 1.7 and 2.4 respectively.

These data showed a dose-response but there was no indication that the test substance could elicit a SI ≥ 3.

Based on these results and according to the recommendations made in the test guidelines (OECD No.429, EC B.42 and EPA OPPTS 870.2600), HATCOL 3331 should not be regarded as a skin sensitiser