7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate

Endpoint summary

Description of key information

skin sensitisation (OECD 429): sensitising

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:

26 May - 12 June 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

As the test substance is a surfactant type chemical, the result of the LLNA could be indicate a false positive result.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2002

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/CaOIaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Horst, The Netherlands
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 18.6 - 21.3 g
- Housing: 4 animals per group in Makrolon cages (Type II) with wire mesh top, granulated soft wood was used as bedding
- Diet: pelleted standard diet, (Harlan, Laboratories GmbH, Borchen, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

dimethylformamide

Concentration:

Test substance: 10, 25 and 50%
The top dose was the highest technically achievable concentration.

Positive control: 5, 10 and 25%

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The highest test substance concentration, which could be technically used was a 50 % (w/w) solution in dimethylformamide. Vortexing and warming to 37 °C for about 10 minutes were necessary to dissolve the test substance.
- Irritation: To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 25 and 50% each on three consecutive days. In the pre-test clinical signs were recorded within 1 h and 24 ± 4 h after each application as well as on Day 8. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT:
- Name of test method: 3H-methyl thymidine incorporation determined by ß-scintillation
- Criteria used to consider a positive response: A stimulation index (SI) was calculated for each group using the activity of each test group divided by the activity of the vehicle control group. The first criterion for a positve response is that at least one concentration of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group. The second criterion is that the data are compatible with a conventional dose response, although allowance must be made for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance on three consecutive days. Five days after the first application an injection of 250 µL 3H-methyl thymidine (20 µCi ³HTdR per mouse) was made into the tail vein of each experimental mouse. Approximately five hours later, following injection of ³HTdR, the mice were sacrificed and draining auricular lymph nodes were excised and pooled for each group. A single cell suspension was prepared by gentle mechanical disaggregation through a 200 μm mesh stainless steel gauze. The cell suspensions were washed two times with approximately 10 mL PBS and precipitated with 5% trichloroacetic acid at 4 °C for at least 18 h. The pellets were resuspended in 1 mL of trichloroacetic acid and transferred to 10 mL of scintillation fluid.

Positive control substance(s):

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

Statistics:

Mean values and standard deviations were calculated.

The following formula was used to calculated the EC3 value:
EC3 = [(3-d)/(b-d) x (a-c)] + c

Positive control results:

The results of a reliability test with hexyl cinnamic aldehyde was performed in January 2009. The SI values calculated for the positive control concentrations 5, 10 and 25% were 1.49, 4.17 and 4.90, respectively.

Parameter:

SI

Value:

1.58

Test group / Remarks:

10% test substance

Parameter:

SI

Value:

1.7

Test group / Remarks:

25% test substance

Parameter:

SI

Value:

4.44

Test group / Remarks:

50% test substance

Parameter:

EC3

Value:

36.9

Table 1: Body weights and DPM counts.

	Body weight (g)		DPM count	DPM – BG*	DPM per lymph node**	SI value
	At start of the experiment	Before administration of ³HTdR
BG I	-	-	23	-	-	-
BG II	-	-	19	-	-	-
Vehicle Control Group	19.8 ± 1.3	21.1 ± 1.2	6433	6412	801.5	1.00
Test Group I - 10%	19.6 ± 0.8	20.7 ± 1.3	10151	10130	1266.3	1.58
Test Group II - 25%	20.5 ± 0.6	21.6 ± 0.7	10929	10908	1363.5	1.70
Test Group III - 50%	19.8 ± 0.6	22.4 ± 0.7	28518	28497	3562.1	4.44

BG: background (1 mL 5% trichloroacetic acid) in duplicate

*: The mean value was taken from the figures BG I and II

**: Since the Iymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes (8) pooled.

 

Table 2: Reliability test with hexyl cinnamic aldehyde.

	DPM count	DPM – BG*	DPM per lymph node**
BG I	33	-	-
BG II	37	-	-
Vehicle Control Group	5392	5357	669.6
5%	8026	7991	998.9
10%	22385	22350	2793.8
25%	26285	26250	3281.3

BG: background (1 mL 5% trichloroacetic acid) in duplicate

*: The mean value was taken from the figures BG I and II

**: Since the Iymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes (8) pooled.

Observation:

No deaths occurred during the study period. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

 

Body weight:

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

CLP: Skin Sens. 1B; H317

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitising potential of the test substance was investigated in a Local Lymph Node Assay (LLNA) in mice according to OECD Guideline 429 and in compliance with GLP (Vogel, 2009). The highest technically achievable test substance concentration was 50% (w/w) in dimethylformamide. To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 25 and 50% each on three consecutive days. No signs of irritation or systemic toxicity were observed at the tested concentrations. In the main study, four female CBA/CaOlaHsd mice per test group were treated with the test substance at concentrations of 10, 25 and 50% (w/w) in dimethylformamide or with vehicle alone for three consecutive days by open application on the ears (25 µL/ear). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and approximately after five hours the draining (auricular) lymph nodes were excised and pooled for each test group. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. Treatment with test substance concentrations of 10, 25 and 50% (w/w) in dimethylformamide resulted in DPM values per lymph node of 1266.3, 1363.5 and 3562.1, respectively. The SI values calculated for the substance concentrations 10, 25 and 50% were 1.58, 1.70 and 4.44, respectively. The EC3 value was calculated to be 36.9%. Based on the results, the test substance was regarded as a skin sensitizer under the conditions of the test.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on sensitisation of the test substance meet the criteria for Skin Sens. 1B (H317) according to Regulation (EC) 1272/2008.