Bis(O,O-diisopropyl dithiophosphate)bis(cyclohexylamine) zinc

Endpoint summary

Description of key information

Skin sensitisation (OECD 429): positive

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:

24 Oct - 29 Nov 2016

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)

Version / remarks:

adopted 22 Jul 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS, B.V., Inc., The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: 2014C Teklad Global Rodent diet by Envigo RMS Limited, UK, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

- IN-LIFE DATES: From: 24 Oct 2016 To: 29 Nov 2016

Vehicle:

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

Concentration:

2.8, 5.6 and 11.1% (w/w) (equivalent to 2.5, 5 and 10% active ingredient)

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS: One female per dose was treated by daily application of 25 µL of the test item at concentrations of 11.1, 27.8 and 55.5% in acetone/olive oil 4:1 (equivalent to 10, 25 and 50% active ingredient) to the dorsal surface of each ear for three consecutive days.
- Compound solubility: The vehicle was chosen as it was suitable at the highest concentration.
- Irritation: The animals were observed once daily for local skin irritation to the application site.
- Systemic toxicity: The animals were observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6 for signs of toxicity. The body weight was recorded on Day 1 prior to dosing and on Day 6.
- Ear thickness measurements: The thickness of each ear was measured pre-dose on Day 1 and post dose on Day 3 and Day 6.
- Erythema scores: No erythema (0), very slight erythema (1), well-defined erythema (2), moderate to severe erythema (3), severe erythema to eschar formation preventing grading of erythema (4)

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by β-scintillation counting
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation is classified as a "non-sensitizer".
lf the results allowed the EC3 value could also be calculated. The EC3 value is the concentration of test item expected to cause a 3-fold increase in 3HTdR incorporation. The equation used for the calculation of EC3 is:
EC3 = c + [[(3-d)/(b-d)] x (a-c)]
a = lowest concentration giving stimulation index >3
b = actual stimulation index caused by 'a'
c = highest concentration failing to produce a stimulation index of 3
d = actual stimulation index caused by 'c'

- Other: The animals were observed for signs of toxicity twice on Day 1, 2 and 3, and once on Day 4, 5 and 6. The body weight was recorded on Day 1 prior to dosing and on Day 6 prior to termination.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of the test compound was applied to the dorsal surface of each ear of each mouse on Day 1, 2 and 3 in concentrations of 2.8, 5.6 and 11.1% (equivalent to 2.5, 5 and 10%) in acetone/olive oil. On Day 6 an injection of 250 µL phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (3H-TdR) was made into the tail vein of each experimental mouse. Five hours later, the draining auricular lymph node of each ear was excised into PBS. A single cell suspension of pooled lymph node cells was prepared per experimental group by gentle mechanical disaggregation through a 200-mesh stainless steel gauze and rinsed with PBS. The precipitates were incubated for approximately 18 h at approximately 4 °C, centrifuged, resuspended in 1 mL TCA and transferred to 10 mL scintillation fluid before β-scintillation counting.

Positive control substance(s):

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

Positive control results:

A study (study dates: 10 - 16 Nov 2016; study number: YY34MW) was performed to assess the sensitivity of the strain of mouse used at the testing facility to a known sensitizer and to show the sensitivity and reproducibility of the test. The positive control substance hexyl cinnamic aldehyde (25% (v/v) in acetone/olive oil 4:1) was considered to be a sensitizer under the conditions of the test (SI 5.66).

Key result

Parameter:

SI

Value:

4.02

Test group / Remarks:

2.8% (equivalent to 2.5% w/w active ingredient)

Key result

Parameter:

SI

Value:

3.64

Test group / Remarks:

5.6% (equivalent to 5% w/w active ingredient)

Key result

Parameter:

SI

Value:

8.94

Test group / Remarks:

11.1% (equivalent to 10% w/w active ingredient)

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
The SI of the 2.5, 5 and 10% treatment group was 4.02, 3.64 and 8.94, respectively.

EC3 CALCULATION
The EC3 value could not be determined, since no concentration failed to produce a stimulation index of 3.

CLINICAL OBSERVATIONS:
Mortality or signs of systemic toxicity were not observed in all treatment groups or in the control group.

BODY WEIGHTS
Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the control group over the same period.

Table 1: Results of the measurement of ear thickness and mean ear thickness changes - preliminary screening test

Concentration (% w/w) in acetone/olice oil 4:1	Ear thickness measurement (mm)
	Day 1 pre-dose		Day 3 post dose		Day 6
	left	right	left	right	left	right
55.5 (equivalent to 50% active ingredient)	0.22	0.21	0.24	0.23	0.30	0.29
overall mean (mm)	0.22		0.24		0.30
overall mean ear thickness change (%)	na		9.30		37.21
27.8% (equivalent to 25% active ingredient)	0.23	0.23	0.28	0.28	0.40	0.36
overall mean (mm)	0.23		0.28		0.38
overall mean ear thickness change (%)	na		19.565		65.217
11.1% (equivalent to 10% active ingredient)	0.21	0.23	0.23	0.24	0.22	0.24
overall mean (mm)	0.22		0.24		0.23
overall mean ear thickness change (%)	na		6.82		4.55

Table 2: Results of radioactivity incorporated - main test

Concentration (% w/w) in acetone/olice oil 4:1	DPM	dpm/node*	SI **	Result
Vehicle	5026.26	628.28	na	na
2.8 (equivalent to 2.5% w/w active ingredient)	20230.30	2528.79	4.02	Positive
5.6 (equivalent to 5% w/w active ingredient)	18292.23	2286.53	3.64	Positive
11.1 (equivalent to 10% w/w active ingredient)	44925.14	5615.64	8.94	Positive

DPM = disintegrations per minute

na = not applicable

* Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

** Stimulation Index of 3.0 or greater indicates a positive result

Interpretation of results:

other: Cat. 1, H317 according to Regulation (EC) 1272/2008

Conclusions:

CLP: Cat. 1, H317

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitisation potential of the registered substance was determined by a local lymph node assay according to OECD Guideline 429 and GLP (Envigo Research, 2017). Groups of four mice were exposed daily, for three consecutive days, to 2.8, 5.6 and 11.1% (w/w) (equivalent to 2.5, 5 and 10% active ingredient) of the test substance in acetone/olive oil 4:1 or to the vehicle alone, on the dorsum of both ears. Subsequently, mice were injected intravenously with [3H]-thymidine and activity measured as a function of isotope incorporation in draining auricular lymph nodes. [3H]-thymidine stimulation indices after exposure to 2.8, 5.6 and 11.1% with the test substance (equivalent to 2.5, 5 and 10% active ingredient), were 4.02, 3.64 and 8.94, respectively. Thus, under the conditions of the test, the test substance revealed skin sensitising properties.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on skin sensitisation with Zinc, bis[O,O-bis(1-methylethyl) phosphorodithioato-.kappa.S]bis(cyclohexanamine)-, (T-4)- (CAS 52585-16-7) meet the criteria for classification according to Regulation (EC) 1272/2008, therefore the test substance will be classified for skin sensitisation in Cat. 1, H317.