2,2,2,4,4,4,4-heptakis[(2-ethylhexanoyl)oxy]cyclodimolybdoxan-2-yl 2-ethylhexanoate

Endpoint summary

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:

03 March 2011 to 17 June 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was conducted in accordance with International Guidelines (OECD 429) and in accordance with OECD Principles of Good Laboratory Practice as incorporated into the United Kingdom (Statutory Instrument for GLP).

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Sex:

female

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

25, 50 and 100 % HCAT (w/w) including a vehicle control.

No. of animals per dose:

4

Details on study design:

For 3 consecutive days (Days 1 to 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. On Day 6 each animal received an intravenous injection (250 μL) of phosphate buffered
saline (PBS) containing 21.89 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 mesh was then discarded. The lymph node 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 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 21 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’, an aqueous-based solubiliser, 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).

Positive control substance(s):

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

Statistics:

Group means and standard deviations were calculated for disintegrations per minute. Group means and standard deviations were calculated for body weights, along with body weight gains. The percentage change in ear thickness of preliminary test mice was calculated. Dixon’s Q-test for the detection of a single outlier was performed on disintegrations per minute values. No other formal statistical analysis was carried out.

Positive control results:

The stimulation indices in a recent positive control study were 1.4, 2.0 and 4.3 for formulations of hexylcinnamicaldehyde prepared at concentrations of 5%, 10% and 25%, respectively.

Parameter:

SI

Remarks on result:

other: The stimulation indices for 3 groups of mice treated with the test item at concentrations of, respectively, 25%, 50% and 100%, when compared with the control group, were 10.4, 9.4, and 8.1, respectively.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: See Table 1.

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

Group	Animal	DPM	DPM Group Mean	DPM SD	Stimulation Index
1	1	1,511	2,099	507	1
1	2	2,742
1	3	2,144
1	4	1,997
2	5	27,082	21,833	10,004	10.4
2	6	8,828
2	7	19,647
2	8	31,773
3	9	14,530	19,821	3,922	9.4
3	10	22,831
3	11	22,763
3	12	19,159
4	13	17,623	17,066	3,690	8.1
4	14	20,578
4	15	11,875
4	16	18,189

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the study, since treatment with HCAT at each of the 25%, 50% and 100% concentrations achieved a stimulation index of ≥3, it was considered that the test item has the potential to cause sensitisation.

Executive summary:

The objective of this study was to determine the delayed contact hypersensitivity potential of HCAT, an organometallic complex. This information is required by the Sponsor as part of their testing programme under the REACH Regulation.

The study was performed using female CBA/Ca mice. A preliminary test was conducted using 2 females. Each mouse received an open application of 25 μL of undiluted HCAT onto the dorsum of each ear on 3 consecutive days. There was no evidence of systemic toxicity or erythema. As a result of these findings, formulation concentrations were selected for the main study. The vehicle selected to prepare the formulations was acetone:olive oil (4:1, v/v).

The study design for the main study was as follows:

Group	Treatment	Formulation Concentration (%)	Animal
1	Acetone:olive oil (4:1 v/v)	0	1 to 4
2	HCAT	25 %	5 to 8
3	HCAT	50 %	9 to 12
4	HCAT	100 % (undiluted HCAT)	13 to 16

Treatment of main study mice was also for 3 consecutive days. Three days after the final application each animal received an intravenous injection of [methyl-3H] thymidine into the lateral tail vein. 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 signs of systemic toxicity 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 indices (SI) for the mice treated with HCAT at concentrations of 25%, 50% or 100%, when compared with the control group, were 10.4, 9.4 and 8.1, respectively.

Under the conditions of the study, since treatment with HCAT at each of the 25%, 50% and 100% concentrations achieved a stimulation index of ≥3, it was considered that the test item has the potential to cause sensitisation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

All treatments in the study produced a stimulation index of ≥3, which precluded the use of dose-response data in order to derive a DNEL. As such, a qualitative approach to risk management is implemented in order to control risks of workers to skin sensitisation.

Migrated from Short description of key information:
A sensitisation study was conducted in accordance with OECD Guideline 429: Skin Sensitisation: Local Lymph Node Assay. Since treatment with HCAT at each of the 25%, 50% and 100% concentrations achieved a stimulation index of ≥3, it was considered that the test item has the potential to cause sensitisation.

Justification for selection of skin sensitisation endpoint:
Only available study meeting quality criteria.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Migrated from Short description of key information:
No data are available for respiratory sensitisation.

Justification for classification or non-classification

HCAT is classified as a Category 1 Skin Sensitiser.