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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
21 October to 5 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:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The test substance was cyclic trimethylolpropane formal, batch no. 3977009. 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 liquid, was stored at ambient temperature, protected from light and under nitrogen headspace. A reference sample (1 g) was retained under the same conditions as the bulk at Charles River, Edinburgh. A purity value of 99.7% was supplied by the Sponsor. Although no expiry date was provided, the Sponsor provided evidence that the shelf life of the test item was 1 year from the date of despatch (ie 03 September 2010).
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 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.

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 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, 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.
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% hexylcinnamicaldehyde, respectively.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The stimulation indices for mice treated with the test item at concentrations of 25%, 50% or 100%, when compared with the control group, were 0.9, 0.9 and 1.2, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The disintegrations per minute for mice treated with the test item at concentrations of 0% (control), 25%, 50% or 100% were 1750, 1580, 1536 and 2157, respectively.

Any other information on results incl. tables

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

# Animal 18 excluded from group mean because only one lymph node was obtained

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
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.