Reaction product of n-nonanoic acid (C9), n-heptanoic acid (C7), n-pentanoic acid (C5), mixed tetraesters with pentaerythritol, and 3,5,5-trimethylhexanoic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 November to 24 November 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD, EU & US EPA test guidelines in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

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:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

not specified

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

Constituent 2

Reference substance name:

Hatcol 3331

IUPAC Name:

Hatcol 3331

Test material form:

other: Clear, colourless liquid

Details on test material:

Identification: Hatcol 3331
CAS Number: 144971-11-9
Description: Clear colourless liquid
Batch: D21287
Purity: 97.3%
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 31 December 2003
Specific Gravity: 0.969-0.976
Stability in vehicle:
-Water: Not indicated
-1% Aq. Carboxymethyl cellulose: Not indicated
-Corn oil: Not indicated
-Propylene glycol: Not indicated
-Dimethylsulfoxide: Not indicated
-Ethanol: Not indicated
-Polyethylene glycol: Not indicated
-Acetone: Not indicated

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, France
Number of animals: 20 females (four groups of five females each group) (nulliparous and non-pregnant).
Age and bodyweight: Young adult animals (approx. 9 weeks old) were selected. Bodyweight variation was +/- 20% of the sex mean.
Identification: Tail mark.
Control animals: The vehicle control animals were treated using the same vehicle and using the same procedures.
Reliability check: Similar procedures were used in the reliability test and in this study

Animal husbandry
Conditions: Animals 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.
Accommodation: Individual 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 (Macrolon II type; height 15 cm) during the acclimatisation period.
Diet: Free access to standard pelleted laboratory animal diet (from Altromin (code VRF 1), Lage, Germany).
Water: Free access to tap-water.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

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.

No. of animals per dose:

Five animals per dose concentration

Details on study design:

Preliminary irritation study
A 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 unless otherwise specified.
Four young adult animals were selected (5-14 weeks old). 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 study
Three groups of five animals were treated with three test substance concentrations respectively.
One group of five animals was treated with vehicle.
ALLOCATION
GROUP* INDUCTION
1 (1-5) Vehicle control Vehicle
2 (6-10) Experimental Lowest test substance concentration: 5%
3 (11-15) Experimental Intermediate test substance concentration: 50%
4 (16-20) Experimental Highest test substance concentration: 100%
* five females each group, animal numbers between brackets

INDUCTION - Days 1, 2 and 3
Experimental 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:
All animals: Each animal was injected via the tail vein with 0.25 ml of sterile phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (Amersham Pharmacia Biotech, NOTOX Substance 105624).
After approximately five hours, all animals were killed 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 radioactivity: A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 I'm). 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 ml 5% 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 scintillation fluid.

Radioactivity measurements: All radioactive measurements were performed using a Packard scintillation 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.

Observation
Mortality/Viability: Twice daily.
Toxicity: At least once daily.
Bodyweights: 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 following numerical scoring system. Furthermore descriptions of all other (local) effects were recorded.

GRADING IRRITATION REACTIONS
Erythema and eschar formation:
No erythema: 0
Slight erythema (barely perceptible): 1
Well-defined erythema: 2
Moderate erythema: 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth): 4
Oedema formation:
No oedema: 0
Slight oedema (barely perceptible): 1
Well-defined oedema (edges of area well-defined by definite raising): 2
Moderate oedema (raised approximately 1 millimetre): 3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure): 4


Interpretation: 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. 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).

Positive control substance(s):

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

Statistics:

A Stimulation Index (Si) is calculated for each group.

Results and discussion

Positive control results:

A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by NOTOX. In this study, performed in August 2003, females of the CBA mouse strain (from Charles River France, L'Arbresle Cedex, France) were checked for the sensitivity to ALPHA-HEXYLCINNAMICALDEHYDE, TECH. 85%.
The females were approx. 7-8 weeks old at commencement of the study. The study was based on the OECD Guideline No. 429, the EC Directive 67/548/EC, Part B.42 and on the method described by ICCVAM (NIH publication: No 99-4494, February 1999). ALPHAHEXYLCINNAMICALDEHYDE, TECH. 85% (CAS no. 101-86-0) was fabricated under lot no. 10021HF (Aldrich Chemicals Co., Germany). Concentrations used for this study were 5, 10 and 25% in Acetone/Olive oil (4:1).

CONCLUSION
The SI values calculated for the substance concentrations 5, 10 and 25% were 2.5, 7.5 and 25.4 respectively.
An EC3 value of 5.5% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 2 and 20%.
Based on these results it was concluded that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.
The raw data, protocol and report from this study are kept in the NOTOX archives. The test described above was performed in accordance with NOTOX Standard Operating Procedures and the report was audited by the QA-unit.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

5%

Results 2

Key result

Parameter:

SI

Value:

1.7

Test group / Remarks:

50%

Results 3

Key result

Parameter:

SI

Value:

2.4

Test group / Remarks:

100%

Any other information on results incl. tables

PRELIMINARY IRRITATION STUDY: Based on the results, the test highest test substance concentration selected for the main study was a 100% concentration.

 

MAIN STUDY

Induction phase: The skin effects seen after the third epidermal exposure are presented in the table.

Macroscopy: The majority of nodes were equal in size, except for the left node of animal no. 16 which was slightly enlarged when compared to the control nodes. No other macroscopic abnormalities of the nodes were noted.

Body Weights: 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 some animals, wasconsidered not toxicologically significant.

Radioactivity Measurements: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.

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

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

HATCOL 3331 should not be regarded as a skin sensitiser.

Executive summary:

Assessment for Contact Hypersensitivity to HATCOL 3331 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 (2002), Paris Cedex; EC, Council Directive 67/548/EEC, Annex IV C, B.42 (Draft) (2001); Environmental Protection Agency (EPA): Health Effects Test Guidelines OPPTS 870.2600. "Skin Sensitisation" 2003.

Test substance concentrations selected for the main study were based on the results of a preliminary study (NOTOX Project 385368).

In the main study, three groups of five experimental animals were epidermally exposed to a 5%, 50% and 100% concentration respectively on three consecutive days. Five vehicle control animals were similarly treated, but with 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.

After precipitating the DNA of the lymph node cells, radioactivity measurements were done.

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.

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.

Based on these results and according to the:

OECD Harmonized Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances (OECD, 1998), HATCOL 3331 does not have to be classified for sensitisation by skin contact.

EC criteria for classification and labelling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), HATCOL 3331 does not have to be classified and has no obligatory labelling requirement for sensitisation by skin contact.