Butanal, 4-(heptyloxy)-3-methyl-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 01 April 2010 and 19 April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD test guideline 429 using a local lymph node assay method and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

4-(heptyloxy)-3-methylbutanal

EC Number:

802-100-7

Cas Number:

1093653-57-6

Molecular formula:

C12H24O2

IUPAC Name:

4-(heptyloxy)-3-methylbutanal

Test material form:

other: Liquid

Details on test material:

Identification: TM 09-217
Chemical name: Butanal, 4-(heptyloxy)-3-methyl
Alternative chemical name: 4-(heptyloxy)-3-methylbutanal
Intended use: Fragrance ingredient in household products
Appearance: Liquid
Storage conditions: Room temperature (ca. 20°C) in the dark
Supplier: Sponsor
Sample receipt: 18 February 2010

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Seventeen healthy female CBA/Ca mice were obtained from Harlan UK Ltd. One of these
animals arrived on 01 April 2010 and was used for the preliminary investigation. The
remainder arrived on 08 April 2010 and were used for the main phase of the study.
All preliminary and main phase animals were in the weight range 15.8 to 20.9 g and
approximately eight to twelve weeks of age prior to dosing on Day 1. All the mice were
acclimatised to the experimental environment for at least five days prior to the start of the
study.
Each animal was assigned an alpha-numeric code and identified uniquely within the study by
tail marking. Each cage label was colour-coded and was identified uniquely with the study
number, dose level and animal mark.

Animal housing, diet and water supply
Animals were housed inside a barriered rodent facility (Building F21, Room 061/062). The
facility was designed and operated to minimise the entry of external biological and chemical
agents and to minimise the transference of such agents between rooms.
The mice were allocated without conscious bias to cages within the treatment groups. They
were housed individually in polycarbonate cages with woodflake bedding. The mice were
also given Nestlets and a plastic shelter for environmental enrichment. Certificates of
analysis for woodflake bedding and Nestlets were lodged in Huntingdon Life Sciences Ltd.
Archives.
The animal room was kept at positive pressure with respect to the outside by its own supply
of filtered fresh air, which was passed to atmosphere and not re-circulated. The temperature
and relative humidity controls were set to maintain the range of 19 to 23°C and 40 to 70%
respectively. Any minor deviations from these ranges would not have had an adverse effect
on the animals and would not affect the integrity or validity of the study. Artificial lighting
was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per
24 hours.
Periodic checks were made on the number of air changes in the animal rooms. Temperature
and humidity were monitored daily and records are archived with the departmental raw data.
Alarms were activated if there was any failure of the ventilation system, or temperature limits
were exceeded. A stand-by electricity supply was available to be automatically brought into
operation should the public supply fail.
The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1
Maintenance Diet). This diet contained no added antibiotic or other chemotherapeutic or
prophylactic agent.
Potable water taken from the public supply was freely available via polycarbonate bottles
fitted with sipper tubes.
Each batch of diet was analysed routinely by the supplier for various nutritional components
and chemical and microbiological contaminants. Supplier’s analytical certificates were
scrutinised and approved before any batch of diet was released for use. The quality of the
water supply is governed by regulations published by the Department for Environment, Food
and Rural Affairs. Certificates of analysis were received routinely from the water supplier.
Since the results of these various analyses did not provide evidence of contamination that
might have prejudiced the study, they are not presented.
No other specific contaminants that were likely to have been present in the diet or water were
analysed, as none that may have interfered with or prejudiced the outcome of the study was
known.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

25 and 50% v/v and as supplied

No. of animals per dose:

One female was treated with the test substance ‘as supplied’.
Groups of four mice were treated at one of three concentrations of the test substance.

Details on study design:

Test substance preparation
A vehicle trial was conducted and TM 09-217 formed a clear solution at a concentration of
50% v/v in acetone:olive oil (4:1 v/v). It was also considered suitable to dose ‘as supplied’.

Formulation
The dose level for the preliminary investigation was chosen based on the physical properties
of the test substance e.g. solubility, viscosity. The doses for the main phase of the study were
based on the results of the preliminary investigation.
Determination of the homogeneity, stability and purity of the test substance or test substance
formulations were not undertaken as part of this study.
Detailed records of test substance usage were maintained. The amount of test substance
necessary to prepare the formulations and the amount actually used were determined on each
occasion. The difference between these amounts was checked before the formulations were
dispensed.
The test substance was used as supplied.
The test substance formulations were prepared on the day of dosing at the required
concentrations.
The absorption of the test substance was not determined.

Selection of dose levels
The maximum practical concentration for pinna dosing was as supplied. Based on this
information this concentration was selected for the preliminary investigation.
The results of the preliminary investigation indicated that ‘as supplied’ was a suitable high
dose level for the main phase of the study. Based on this information the following
concentrations were selected for the main phase of the study:
25 and 50% v/v and as supplied
Prior to dose administration the Study Director authorised the choice of vehicle and dose
levels in the raw data.

Administration of test substance
Preliminary investigation
One female was treated with the test substance ‘as supplied’. The mouse was treated by daily
application of 25 μl of the test substance to the dorsal surface of each ear for three
consecutive days (Days 1-3). The test substance was applied to the dorsal surface of each ear
using an automatic micropipette. The test substance was spread over the entire dorsal surface
of the ear using the tip of the pipette.

Main phase
Groups of four mice were treated at one of three concentrations of the test substance. The
mice were treated by daily application of 25 μl of the appropriate concentration of the test
substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test
substance was applied to the dorsal surface of each ear using an automatic micropipette. The
test substance was spread over the entire dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.

Administration of 3H-methyl Thymidine
In the main phase of the study, five days following the first topical application of test
substance (Day 6) all mice were injected via the tail vein with 250 μl of phosphate buffered
saline containing 3H-methyl Thymidine(a) (3HTdR: 80 μCi/mL) giving a nominal 20 μCi to
each mouse. The injection into the tail vein was carried out using a plastic syringe and needle
after the mouse had been heated in a warming chamber.

(a) Specific activity 2.0 Ci/mmol, concentration 1.0 mCi/mL

Observations
Clinical signs
All animals were observed daily for signs of ill health or toxicity. The ears were also
examined for signs of irritation.

Bodyweight
The weight of the mouse in the preliminary investigation was recorded on arrival, Day 1 (first
day of dosing) and prior to termination (Day 4). These data are not reported.
The weight of each mouse in the main phase of the study was recorded on arrival (these data
are not reported), Day 1 (first day of dosing) and prior to termination (Day 6).

Terminal studies
Termination
The mouse in the preliminary investigation was humanely killed by carbon dioxide
asphyxiation on Day 4 of the study. No further investigations were carried out with this
animal.
In the main phase of the study, five hours following the administration of 3HTdR on Day 6 all
mice were humanely killed by carbon dioxide asphyxiation and the draining auricular lymph
nodes excised and pooled for each experimental group. 1.0 mL of phosphate buffered saline
was added to the pooled lymph nodes for each group. The animals were then discarded and
no further investigations were carried out. The following procedures were carried out with
the lymph nodes from these animals only.

Preparation of single cell suspensions
A single cell suspension of lymph node cells (LNC) was prepared by gentle mechanical
disaggregation through a stainless steel gauze (200 mesh size). The pooled LNC were then
washed by adding 10 mL phosphate buffered saline, pelleted at 190 x g for 10 minutes and
resuspended. The cells were washed twice again and resuspended in 3 mL trichloroacetic
acid (TCA: 5%) following the final wash.

Determination of incorporated 3H-methyl Thymidine
After overnight incubation with 5% TCA at 4°C, the precipitate was recovered by
centrifugation and resuspended in 1 mL 5% TCA and transferred to 10 mL Ultima gold
scintillation fluid on Day 7. 3HTdR incorporation was measured by β-scintillation counting.
The proliferative response of LNC was expressed as radioactive disintegrations per minute
per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes
relative to that recorded for control nodes (test/control ratio).

Positive control substance(s):

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

Results and discussion

Positive control results:

In this assay the test/control ratio obtained for HCA at 25% was 6.0. This indicates that HCA
demonstrates the potential to induce skin sensitization (delayed contact hypersensitivity) and
confirms the sensitivity of the technique to detect sensitization potential.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The Stimulation Index (SI) obtained for 25%, 50% and 100% ‘as supplied’ were 4.6, 8.7 and 11.3 respectively.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The DPM for the vehicle control, 25%, 50% and 100% as supplied of test substance were 526.38, 2435.31, 4584.13, and 5974.06, respectively.

Any other information on results incl. tables

Preliminary investigation

Mortality and clinical signs There were no deaths and no signs of ill health or toxicity observed during this study. However, wet fur on the cranial region was noted post-dose from Day 1 and prior to termination on Day 4. No signs of irritation were seen over the dosed area during the study.

Bodyweight

The animal gained weight during the study. On the basis of the results from the preliminary investigation, ‘as supplied’ was considered a suitable high dose level for the main phase of the study.

Main phase

Mortality and clinical signs

There were no deaths and no signs of ill health or toxicity observed during this study. However the following observations were noted: Vehicle control - greasy fur on the cranial region was seen post-dose in all animals from Day 1, prior to dosing in two animals from Day 2, in all animals on Day 4 and in one animal on Day 5. The sign had resolved completely for all animals by Day 6. 25% v/v - greasy fur on the cranial region was seen post-dose in all animals from Day 1, prior to dosing in three animals from Day 2 and in all animals on Days 4 and 5. The sign had resolved completely in three animals by Day 6, but was still present in one female at this time. 50% v/v - greasy fur on the cranial region was seen post-dose in all animals from Day 1, prior to dosing in all animals from Day 2 and in all animals on Days 4 and 5. The sign had resolved completely for all animals by Day 6. ‘As supplied’ – greasy fur on the cranial region was seen post-dose in all animals from Day 1, prior to dosing in all animals from Day 2 and in all animals on Days 4 and 5. The sign had resolved completely in three animals by Day 6, but was still present in one female at this time. No signs of irritation were seen over the dosed area during the study.

Bodyweight

A loss in bodyweight was recorded for one female (No. G17) in Group 5 and no bodyweight change was noted for one female (No. G4) in Group 2 and one female (No. G16) in Group 5. All remaining animals gained weight during the study.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

TM 09-217 is regarded as a potential skin sensitizer and the EC3 value is less than 25% v/v.

Executive summary:

The skin sensitisation potential of the test susbtance, TM 09-217, was assessed as a potential skin sensitizer with an EC3 value less than 25% v/v according to OECD test guideline 429.