5-ethyl-1,3-dioxane-5-methanol

Endpoint summary

Description of key information

A LLNA is available for CTF.

The skin sensitisation potential of 5 -ethyl-1,3 -dioxane-5 -methanol (Robinson, 2010) has been
investigated using the Local Lymph Node Assay (LLNA; OECD Test Guideline 429). Under the conditions of the study, since treatment with 5 -ethyl-1,3 -dioxane-5 -methanol at concentrations of up to 100% did not achieve stimulation index values of ≥3, it was considered that the test item did not have the potential to cause sensitisation.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Sept 2009 - 28 Jan 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Specific details on test material used for the study:

CTF (Batch 3977009), a clear, colourless liquid, was received from Perstorp Holding AB on 04
September 2009 and was stored protected from light and under a nitrogen headspace, at ambient
room temperature. The purity was quoted as 99.7% and the Sponsor supplied an expiry date of 03
September 2010. A Certificate of Analysis was provided.

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 13 October 2009. They were 7 to 8 weeks old and weighed 16.4 to 20.0 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.The animals were housed in groups of 2 or 3 in polycarbonate 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, the average daily environmental temperature was within a range of approximately 21 to 22°C and average daily relative humidity was within the range of approximately 45 to 67% in the 2 rooms in which the mice were housed. 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.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 the scanning of the implants on Day 1 of the main study, the Study Director authorised the re-identification of Animals 16 and 19 by indelible tail mark.

Vehicle:

dimethylformamide

Concentration:

0, 25, 50 and 100%

No. of animals per dose:

5 female mice/dose

Details on study design:

Formulations of CTF were prepared on each day of dosing.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, bycervical 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.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 order of preference, did produce a satisfactory formulation and so was selected as the vehicle and control item for use on this study.

Positive control substance(s):

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

Statistics:

No formal statistical analysis was carried out.

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% hexylcinnamicaldehyde, respectively.

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

50%

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

100%

Parameter:

EC3

Remarks on result:

not determinable

Preliminary test: No systemic signs and no signs of local irritation were noted in either animal receiving undiluted CTF. Body weight gain was considered to be acceptable for mice of this age and strain. Based on these findings, a 100% concentration of CTF (ie undiluted CTF) 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.

Individual and Group Mean Scinitillation Counts (DPM)

Treatment	Animal No.	DPM	Group Mean DPM	Stimulation Index
Vehicle control (dimethylformamide)	1	1085	1750	1
	2	2062
	3	2507
	4	1687
	5	1410
CTF 25%	6	688	1580	0.9
	7	3134
	8	1221
	9	1616
	10	1243
CTF 50%	11	1588	1536	0.9
	12	1407
	13	2341
	14	977
	15	1365
CTF 100%	16	1749	2157	1.2
	17	1819
	18	1504#
	19	1863
	20	3198

DPM – disintegrations per minute

CTF – cyclic trimethylolpropane formal

Interpretation of results:

not sensitising

Remarks:

Migrated informationCriteria used for interpretation of results: EU

Conclusions:

Under the conditions of the study, since treatment with Cyclic Trimethylolpropane Formal at concentrations of up to 100% (ie undiluted Cyclic Trimethylolpropane Formal) 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:

This LLNA investigated the delayed contact hypersensitivity potential of the test item, Cyclic Trimethylolpropane Formal (CTF), using CBA/Ca mice. A preliminary test was conducted using 2 females. Each mouse received an open application of 25 μL of a 100% formulation of CTF (ie undiluted CTF) onto the dorsum of each ear on 3 consecutive days. There was no evidence of systemic toxicity or 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 25%, 50% and 100%, respectively, also for 3 consecutive days. The vehicle for the 25% and 50% formulations was dimethylformamide 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 5 h later the draining lymph nodes were collected in order that the 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 CTF at concentrations of 25%, 50% or 100%, when compared with the control group, were 0.9, 0.9 and 1.2, respectively. Under the conditions of the study, since treatment with Cyclic Trimethylolpropane Formal at concentrations of up to 100% (ie undiluted Cyclic Trimethylolpropane Formal) did not achieve a stimulation index of ≥3, it was considered that the test item does not have the potential to cause sensitisation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitisation potential of 5 -ethyl-1,3 -dioxane-5 -methanol (Robinson, 2010) has been investigated using the Local Lymph Node Assay (LLNA; OECD Test Guideline 429). Under the conditions of the study, since treatment with 5 -ethyl-1,3 -dioxane-5 -methanol at concentrations of up to 100% did not achieve stimulation index values of ≥3, it was considered that the test item did not have the potential to cause sensitisation.

There is no evidence from experience of use that 5 -ethyl-1,3 -dioxane-5 -methanol has the potential to cause skin sensitisation in exposed workers.

Migrated from Short description of key information:
Negative LLNA results are reported for 5-ethyl-1,3-dioxane-5-methanol in a skin sensitisation study. There is no evidence from experience of use that the substance has the potential to cause skin sensitisation in exposed workers.

Justification for selection of skin sensitisation endpoint:
Sole study providing data from a guideline compliant study.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

There is no indication from the experience of use that the substance causes respiratory sensitisation in exposed workers

Justification for classification or non-classification

Based on available information, 5 -ethyl-1,3 -dioxane-5 -methanol does not meet the criteria for classification for skin or respiratory sensitisation according to the CLP Regulation.