Reaction mass of 2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol]and propylidynetrimethanol and 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant and conducted in accordance with testing guidelines.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2,6-diethyl-2,6-bis(hydroxymethyl)heptane-1,3,5,7-tetrol; 2-ethyl-2-(hydroxymethyl)propane-1,3-diol; 2-ethyl-2-{[2-ethyl-3-hydroxy-2-(hydroxymethyl)propoxy]methyl}propane-1,3-diol

EC Number:

904-155-3

Molecular formula:

C12H28O6 C12H26O5 C6H14O3

IUPAC Name:

2,6-diethyl-2,6-bis(hydroxymethyl)heptane-1,3,5,7-tetrol; 2-ethyl-2-(hydroxymethyl)propane-1,3-diol; 2-ethyl-2-{[2-ethyl-3-hydroxy-2-(hydroxymethyl)propoxy]methyl}propane-1,3-diol

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): HB TMP
- Analytical purity: 69%
- Composition of test material, percentage of components: 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol] (CAS: 23235-61-2, EC No.: 245-509-0), Propylidynetrimethanol (CAS: 77-99-6, EC No.: 201- 074-9) and 2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol] (CAS: 93983-16-5, EC No.: 301-304-9).
- Lot/batch No.: 7LTQA002
- Expiration date of the lot/batch: 8th May 2014
- Storage condition of test material: Stored at ambient temperature in the dark when not in use.

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/C

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Manston Road, Margate, Kent, UK.
- Age at study initiation: 8 - 11 weeks old
- Weight at study initiation: 15.7 - 20.3g
- Housing: All mice were housed in pairs in solid-bottomed polypropylene cages with a stainless steel grid top and an integrated food hopper. Wood shavings were used as bedding and nesting material (‘Nestlets’) was provided.
- Diet (e.g. ad libitum): SDS Rat and Mouse (modified) No. 1 Diet SQC Expanded was available ad libitum.
- Water (e.g. ad libitum): Water taken from the public supply was available from water bottles ad libitum throughout the study.
- Acclimation period: At least 8 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The average daily environmental temperature was approximately 21°C
- Humidity (%): 45% to 51%.
- Air changes (per hr): Minimum of 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle was in operation

IN-LIFE DATES: From: To:

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Preliminary study: 50% concentration used.
Main study: 10, 25 and 50% concentrations were used.

No. of animals per dose:

2 females in the preliminary study.
4 females per dose group were used in the main study.

Details on study design:

RANGE FINDING TESTS:
Preliminary testing was conducted. Two female mice received an open application of 25 μL of test item formulation onto the dorsum of each ear once daily on 3 consecutive days (Days 1 to 3), with no further treatment after the 3rd application. The animals were checked for viability twice a day and were frequently observed for signs of reaction to treatment on each day of dosing (pre-dose, immediately post dose and 1 and 2 hours after dosing) and once daily thereafter. The body weight of each individual animal was recorded on Day 1 (before the first dose) and on Day 6. On Day 1 (pre-dose) and on Day 3 (approximately 48 h after the first dose) and Day 6 the thickness of both ears of each mouse was measured using digital callipers. In addition, both ears were observed for erythema and the scores recorded.

Animals were euthanised on Day 6 and were then discarded. There were no signs of systemic toxicity and no effect on body weight was noted. Between Days 1 and 3 both mice recorded unilateral increases in ear thickness of ≥25%. However, no erythema was recorded in either mouse during the observation period.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Mouse Local Lymph Node Assay.
- Criteria used to consider a positive response:

TREATMENT PREPARATION AND ADMINISTRATION:
Formulations were prepared on each day of dosing. The test item was placed in a sealed bag and immersed in water that was heated to a maximum temperatureof 80°C until it was visibly homogeneous. The required amount of HB TMP was weighed into a pre-labelled container and formulations were made up to the final volume by adding the requisite amount of AOO. Formulations were stirred using a magnetic stirrer.

Treatment was administered on 3 consecutive days (Days 1 to 3). On each day of treatment the animals received an open application of 25 μL of the appropriate formulation onto the dorsum of each ear.

On Day 6 each animal received an intravenous injection (250 μL) of phosphate buffered saline (PBS) containing 19.3 μCi of [methyl-3H] thymidine into the lateral tail vein. Approximately 5 h after intravenous administration all animals were euthanised by exposure to a rising concentration of carbon dioxide and the major blood vessels were severed to exsanguinate. Each pair of draining auricular lymph nodes was collected from each animal and the animal was then discarded. A single cell suspension of lymph node cells from each paired sample was prepared by gentle disaggregation through a 200 μm mesh stainless steel gauze into conical centrifuge tubes. The lymph node cells were washed in an excess of PBS (approximately 1 mL) and the mesh was discarded. The lymph node cells were then centrifuged at approximately 1300 g for 10 min at 4°C. The supernatant was drawn off and the cells were washed a second time with approximately 1 mL PBS and then centrifuged (also at approximately 1300 g for 10 min at 4°C). Following centrifugation, the supernatant was discarded and the DNA was precipitated with approximately 1 mL 5% trichloroacetic acid at 2 to 8°C for approximately 18½ h. The resulting pellet was again centrifuged at approximately 1300 g for 10 min at 4°C and the supernatant discarded. The pellet was then re-suspended in 200 μL ‘Solvable’, an aqueous-based solubiliser, and the suspension transferred to a vial containing 10 mL scintillation fluid. Incorporation of tritiated thymidine was measured by β- scintillation counting and was expressed as disintegrations per minute (DPM).

Positive control substance(s):

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

Statistics:

Group means and standard deviations were calculated for disintegrations per minute. Group means and standard deviations were calculated for body weights, along with body weight gains. The percentage change in ear thickness of preliminary test mice was calculated. Dixon’s Q-test for the detection of a single outlier was performed on disintegrations per minute values. No other formal statistical analysis was carried out.

Results and discussion

Positive control results:

No information provided.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.1

Test group / Remarks:

Group 1 (25%)

Remarks on result:

other: The stimulation indices for mice treated with the test item at concentrations of 10%, 25% and 50%, when compared with the control group, were 1.1, 1.4 and 1.5, respectively.

Results 2

Parameter:

SI

Value:

1.4

Test group / Remarks:

Group 2 (50%)

Remarks on result:

other:

Results 3

Parameter:

SI

Value:

1.5

Test group / Remarks:

Group 3 (100%)

Remarks on result:

other:

Any other information on results incl. tables

There were no systemic signs and no signs of local irritation in any animal during the observation period. Clinical signs were restricted to wetness to the head, which was observed in all animals at the 1 h and 2 h post dose observations on Days 1 and 2. Among mice treated with the 10% concentration this sign persisted until Day 3 and among mice treated with the 25% concentration and those treated with the 50% it persisted until Day 4. However, this sign was considered to be merely an accumulation of formulation residues.

All mice gained weight during the observation period and these gains were considered to be acceptable for mice of this age and strain.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, treatment with HB TMP up to concentrations of 50% did not achieve a stimulation index of ≥3 and as a

Executive summary:

A study conducted in accordance with OECD Guideline 429 was performed to determine the ability of HB TMP to cause skin sensitisation in a mouse LLNA study using female CBA/Ca mice. A preliminary study was conducted initially, using two mice and a concentration of 50% HB TMP and vehicle (acetone:olive oil, 4:1 v/v). Both mice received an open application of 25 μL of test formulation onto the dorsum of each ear on 3 consecutive days. Both mice survived and there were no signs of systemic toxicity or excessive local skin irritation.

In the main study, three formulation concentrations of 10%, 25% and 50% in vehicle were tested, with four mice per test group. A vehicle control group was also included. Each mouse received an open application of 25 μL of test formulation onto the dorsum of each ear. Animals were treated on 3 consecutive days. Three days after the final application each animal received an intravenous injection of [methyl-3H] thymidine into the lateral tail vein. Approximately 5 h later the draining lymph nodes were collected in order that incorporation of tritiated thymidine could be assessed by scintillation counting.

Clinical observations for signs of toxic reaction, mortality and moribundity and body weights were measured.

There were no signs of systemic toxicity and body weight gains were considered to be acceptable for mice of this age and strain.

The stimulation indices (SI) for Groups 2 (25% dose group), 3 (50% dose group) and 4 (100% dose group), when compared with Group 1 (control), were 1.1, 1.4 and 1.5, respectively.

Under the conditions of the study, since treatment with HB TMP at concentrations of up to 50% did not achieve a stimulation index of ≥3, it was considered that the test item does not have the potential to cause skin sensitisation.