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

Study period:

11 November to 26 November 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Proprietary GLP guideline-compliant study

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)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]

EC Number:

245-509-0

EC Name:

2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]

Cas Number:

23235-61-2

Molecular formula:

C12H26O5

IUPAC Name:

2-ethyl-2-{[2-ethyl-3-hydroxy-2-(hydroxymethyl)propoxy]methyl}propane-1,3-diol

Details on test material:

Di-Trimethylolpropane (Di-TMP), was provided by the Sponsor. The Sponsor confirmed the identity, purity and composition of the test substance.
One container holding approximately 600 g of Di-TMP was received under ambient conditions at Charles River, Edinburgh on 04 September 2009. Upon arrival the test item, described as white flakes, was stored at ambient temperature, protected from light. A purity value of 99.4% and an expiry date of 03 September 2011 were supplied by the Sponsor.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Twenty-two female (nulliparous and non-pregnant) mice of the CBA/Ca strain were used. All animals were supplied by Charles River UK Limited, Manston Road, Margate, Kent, UK and arrived at Charles River, Edinburgh on 03 November 2009. They were 7 to 8 weeks old and weighed 15.7 to 18.9 g on despatch. The animals were allowed to acclimatise for at least 8 days before the start of the study.
No formal randomisation procedure was applied. On arrival, the animals were removed from their transport box in random order and were subsequently allocated to groups by placing them in cages labelled with the study number, animal number and group.
Each animal received a subcutaneous implant which identified it individually within the study and which corresponded to that animal's number. Owing to problems with scanning of its implant on Day 1 of the main study, the Study Director authorised the re-identification of Animal 10 by indelible tail mark.
The animals were housed in groups of 2 or 3 in cages, with a stainless steel grid top and an integrated food hopper. Wood shavings were used as bedding and nesting material (‘Nestlets’) was provided. A wooden chewstick, supplied by Estap OÜ was placed in each cage as environmental enrichment. Certificates of analysis for bedding, Nestlets and chewsticks are retained in the study data. Analysis did not provide evidence of
contamination that might have prejudiced the outcome of the study.
Each cage was supplied with a water bottle.
The environment is monitored throughout the day and recordings are made every 15 min.
From animal arrival to the end of the observation period, average daily environmental temperature in the animal rooms was approximately 21°C on each day and the range for average daily relative humidity was approximately 42 to 62%. A 12 h light/dark cycle was in operation with a minimum of 15 air changes per hour.
Rat and Mouse No. 1 Maintenance Diet (Special Diets Services, PO Box 705, Witham, Essex, UK) and water taken from the public supply (Scottish Water, Edinburgh, Midlothian, UK) were available ad libitum throughout the study. The results of diet and water analyses did not provide evidence of contamination and so the outcome of the study was not prejudiced.

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Remarks:

A predose formulation trial performed at these laboratories showed that the preferred vehicle, acetone:olive oil (4:1 v/v), did not produce a formulation that was suitable for dosing. Dimethylformamide and methyl ethyl ketone were also considered to be un

Concentration:

Preliminary study: 50%
Main study: 0 (vehicle alone), 10%, 25%, 50%

No. of animals per dose:

5 females/dose

Details on study design:

A preliminary study was conducted with two females treated with 50% Di-TMP preparation. After the preliminary study, concentrations were selected for the main phase.

Preliminary study: For 3 consecutive days (Days 1-3) animals received an open application of 25 μL of a 50% concentration of test item onto the dorsum of each ear. There was no treatment on Days 4 and 5. All animals were examined for reaction to treatment. Observations were conducted frequently on each day of dosing (predose, immediately post dose and approximately 1 and 2 h after dosing) and once daily thereafter until Day 5. The animals were killed by cervical dislocation on Day 6 and were then discarded. Owing to an oversight, clinical observations were not performed on Day 6.

Main study: For 3 consecutive days (Day 1-3) animals received an open application of 25 μL of the appropriate formulation onto the dorsum of each ear. There was no treatment on Days 4 and 5. All animals were examined for reaction to treatment. On each day of dosing the observations were conducted predose, immediately post dose and approximately 1 and 2 h post dose. Thereafter animals were observed once daily until kill on Day 6 (the day of the thymidine injection).
On Day 6 each animal received an intravenous injection (250 μL) of phosphate buffered saline (PBS) containing approximately 19 μCi of [methyl-3H] thymidine into the lateral tail vein. Approximately 5 h after intravenous administration all animals were killed 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. The lymph nodes cells were washed in excess of PBS (approximately 1 mL) and then centrifuged at approximately 1300 G for 10 min at 4°C. The supernatant was drawn off and the mesh discarded and the pellet was 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 pellet was precipitated with approximately 1 mL 5% trichloroacetic acid at 2 to 8oC for approximately 19 h. The 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’ and the suspension transferred to a vial containing 10 mL scintillation fluid (Aquasafe 500 plus liquid, Zinsser Analytic, Maidenhead, UK). Incorporation of tritiated thymidine was measured by β-scintillation counting and was expressed as disintegrations per minute (DPM).

All animals were checked for viability early in the morning and again as late as possible on each day. The body weight of each individual animal was recorded on Day 1 (before the first dose) and on Day 6.

Results were corrected for background radiation and expressed as the Stimulation Index (SI). This was obtained by dividing the mean DPM obtained from each group by the mean DPM for the control group. The SI for the control group, therefore, is one. A positive response is indicated by an SI ≥3, together with consideration of dose-response and, where appropriate, statistical significance. As there was no SI value ≥3 recorded for any group, it was not possible to calculate the estimated concentration of test item that would produce a 3-fold increase in draining lymph node cell proliferation (the EC3 value).

Positive control substance(s):

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

Statistics:

No formal statistical analysis was carried out.

Results and discussion

Positive control results:

The stimulation indices in the recent positive control study were 1.2, 1.5 and 5.0 for 5%, 10% and 25% hexylcinnamicaldehyde, respectively.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.6

Test group / Remarks:

10% test item/5 animals

Results 2

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

25% test item/5 animals

Results 3

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

50% test item/5 animals

Results 4

Parameter:

other: disintegrations per minute

Value:

1 690

Test group / Remarks:

10% test item/mean of 5 animals

Results 5

Parameter:

other: disintegrations per minute

Value:

2 749

Test group / Remarks:

25% test item/mean of 5 animals

Results 6

Parameter:

other: disintegrations per minute

Value:

2 408

Test group / Remarks:

50% test item/mean of 5 animals

Any other information on results incl. tables

No systemic signs and no signs of local irritation were noted in any animal during the observation period. Body weights were considered to be acceptable for mice of this age and strain.

Table 1. Individual and Group Mean Scintillation Counts (DPM)

Treatment	Animal	DPM	Group Mean DPM	Stimulation Index
0% (propylene glycol)	1	1135	1690	1.0
	2	1582
	3	1096
	4	2516
	5	2121
10% Di-TMP	6	1326	2749	1.6
	7	2353
	8	1948
	9	4845
	10	3275
25% Di-TMP	11	932	1724	1.0
	12	1167
	13	2130
	14	2836
	15	1554
50% Di-TMP	16	3176	2408	1.4
	17	1221
	18	3288
	19	2407
	20	1950

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 the study, since treatment with Di-Trimethylolpropane 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 sensitisation.

Executive summary:

The study investigated the delayed contact hypersensitivity potential of Di-Trimethylolpropane (Di-TMP) using CBA/Ca mice. A preliminary phase was conducted using 2 females. Each mouse received an open application of 25 μL of a 50% formulation of Di-TMP onto the dorsum of each ear on 3 consecutive days. There was no evidence of systemic toxicity and no signs of local irritation and, as a result of these findings, formulation concentrations were selected for the main study. Three groups, each consisting of 5 females, were treated in the same manner as the preliminary test mice with concentrations prepared at 10%, 25% and 50%, respectively, also for 3 consecutive days. The vehicle was propylene glycol and one group of 5 females received only this and acted as controls.

Three days after the final application each animal received an intravenous injection of [methyl-³H] thymidine into the lateral tail vein and approximately 5 h later the draining lymph nodes were collected in order that incorporation of tritiated thymidine could be assessed by scintillation counting. There were no systemic signs noted in any animal during the observation period and body weight changes were considered to be acceptable for mice of this age and strain. The stimulation index (SI) values for the mice treated with the Di-TMP at concentrations of 10%, 25% or 50%, when compared with the control group, were 1.6, 1.0 and 1.4, respectively.

Under the conditions of the study, since treatment with Di-Trimethylolpropane 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 sensitisation. Di-Trimethylolpropane did not meet the criteria for classification as a sensitiser according to the CLP Regulation.