Reaction mass of propylidynetrimethanol and 2-ethylpropane-1,3-diol and 5-ethyl-1,3-dioxane-5-methanol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2009-10-28 to 2010-02-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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:

Dimethylolpropane Tech

IUPAC Name:

Dimethylolpropane Tech

Details on test material:

Dimethylolpropane tech, batch no. 3877810. One container holding approximately 1000 g of test item was received under ambient conditions at Charles River, Edinburgh on 04 September 2009. Upon arrival the test item, a colourless to slightly yellow liquid, was stored at ambient temperature with a desiccant. A reference sample (1 g) was retained under the same conditions as the bulk at Charles River, Edinburgh. A purity value of 82.2% and an expiry date of 03 September 2010 (ie one year from the date of despatch) were supplied by the Sponsor.
A read across is proposed based on structural similarities between the substances.

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 20 October 2009. They were 7 to 8 weeks old and weighed 16.1 to 19.6 g on despatch. The animals were allowed to acclimatise to the toxicology accommodation at these laboratories for at least 8 days before the start of dosing.
No formal randomisation procedure was applied. On arrival, the animals were removed from their transport box in random order and were allocated to dose group by placing them in cages labelled with at least 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.
The animals were housed in groups of 2 or 3 in cages (dimensions 36.5 x 20.7 x 14 cm) with a stainless steel grid top and an integrated food hopper. Wood shavings were used as bedding and nesting material (‘Nestlets’) was provided. Both were supplied by Datesand Limited, Manchester, UK. A wooden chewstick, supplied by Estap OÜ, 75401 Harjumaa, Estonia, 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 was monitored throughout the day and recordings were 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 of the observation period and the range for average daily relative humidity was approximately 42 to 67%. A 12 h light/dark cycle was in operation (light hours 0700 to 1900 h) 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. Each batch of diet is routinely analysed by the supplier for various nutritional components and chemical and microbiological contaminants. The quality of water supply is stipulated by legislation in Water Quality, Scotland, Regulations 2001 and certificates of analysis for dissolved materials, heavy metals, pesticide residues, pH, nitrates and nitrites are periodically provided. These analyses are based on water samples taken from these laboratories. The results of diet and water analyses did not provide evidence of contamination and so the outcome of the study was not prejudiced. Certificates relevant to this study are retained in the data.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

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 (DMF), which the OECD Guideline places second in orde

Concentration:

0, 25, 50 100%

No. of animals per dose:

Five females/dose

Details on study design:

Formulations of DMP tech at concentrations of 25% and 50% were prepared on each day of dosing. The appropriate amount of DMP tech was weighed and transferred to a suitable glass container. DMF was added and the mixture was stirred with a magnetic stirrer until a clear, colourless solution was obtained. The pH of the formulation dispensed on Day 2 of the preliminary test and also of each of the dose formulations prepared on Day 1 of the main study was pH 7.

The radioisotope was [Methyl-3H] Thymidine (Batch No. B335) was received from GE Healthcare UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, England, UK on 01 December 2008. The chemical was stored refrigerated in the dark in the radiochemistry laboratory at Charles River, Edinburgh. A purity of 98.7% was provided by the supplier.

Preliminary test: two female mice were treated with the undiluted test substance (100%). For 3 consecutive days (Days 1 to 3) animals received an open application of 25 μL of undiluted test item onto the dorsum of each ear. There was no treatment of Days 4 and 5. All animals were checked for viability early in the morning and again as late as possible on each day. 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 the kill, by cervical dislocation, on Day 6. The animals were then discarded. There were no signs of systemic toxicity or local irritation, and no effect on body weight was noted. Therefore, dose concentrations of 25%, 50% and 100% were selected as suitable non-toxic doses for administration in the main study.

Main study: For 3 consecutive days (Days 1 to 3) animals received an open application of 25 μL of undiluted test item onto the dorsum of each ear. There was no treatment of Days 4 and 5. 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 8°C for approximately 19½ 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. 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).

In both the preliminary and main studies; 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 a recent positive control study were 1.7, 2.4 and 5.0 for 5%, 10% and 25% hexyl cinnamic aldehyde, respectively.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.9

Test group / Remarks:

low dose (25% test substance)

Results 2

Parameter:

SI

Value:

0.8

Test group / Remarks:

mid dose (50% test substance)

Results 3

Parameter:

SI

Value:

1.7

Test group / Remarks:

high dose (100% test substance)

Results 4

Parameter:

other: disintegrations per minute (DPM)

Value:

3 136

Test group / Remarks:

0% (vehicle control)

Results 5

Parameter:

other: disintegrations per minute (DPM)

Value:

2 763

Test group / Remarks:

low dose (25% test substance)

Results 6

Parameter:

other: disintegrations per minute (DPM)

Value:

2 642

Test group / Remarks:

mid dose (50% test susbtance)

Results 7

Parameter:

other: disintegrations per minute (DPM)

Value:

5 471

Test group / Remarks:

high dose (100% test substance)

Cellular proliferation data / Observations:

Preliminary study: No systemic signs and no signs of local irritation were noted in either animal receiving undiluted DMP tech. Body weights were considered to be acceptable for mice of this age and strain. Based on these findings, a 100% concentration of DMP tech (i.e. undiluted DMP tech) was selected as the highest concentration for the main study.

Main study: No systemic signs were noted in any animal during the observation period. Body weight gains were considered to be acceptable for mice of this age and strain.

Any other information on results incl. tables

Table 1: Individual and Group Mean Scintillation Counts

Treatment	Animal No.	DPM	Group Mean DPM	Stimulation Index
Vehicle control (dimethylformamide)	1	3720	3136	1
	2	2595
	3	2755
	4	3697
	5	2911
DMP Tech 25%	6	3086	2763	0.9
	7	3375
	8	495
	9	2607
	10	4254
DMP Tech 50%	11	3367	2642	0.8
	12	1394
	13	1891
	14	2210
	15	4349
DMP Tech 100%	16	6835	5471	1.7
	17	5531
	18	7050
	19#	902
	20	2468

DPM – disintegrations per minute

DMP Tech – Dimethylolpropane Tech

# DPM value excluded from mean owing to spillage

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the study, since treatment with Dimethylolpropane Tech at concentrations of up to 100% (i.e. undiluted Dimethylolpropane tech) 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:

In a dermal sensitisation study (according to OECD Guideline 429) with Dimethylolpropane Tech (DMP tech) in dimethylformamide at concentrations of 25%, 50% and 100%, 5 female CBA/Ca mice per dose were tested for 3 consecutive days in the Local Lymph Node Assay. Hexy cinnamic aldehyde (CAS No. 101-86-0) served as a postive control and induced the appropriate response. Dimethylformamide was included as a vehicle control in one group of 5 females.

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 mice treated with DMP tech at concentrations of 25%, 50% or 100%, when compared with the control group, were 0.9, 0.8 and 1.7, respectively. Under the conditions of the study, treatment with Dimethylolpropane Tech at concentrations of up to 100% (i.e. undiluted Dimethylolpropane Tech) did not achieve a stimulation index of ≥3, therefore it was considered that the test item is not a dermal sensitiser.