copper sulphide and zinc chloride sulphate hydroxide, recovery products from copper and zinc dusts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 June 2020 - 25 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Type:

Constituent

Type:

Constituent

Test material form:

other: wet sticky solid

Specific details on test material used for the study:

Test Item Zinc chlorohydroxy sulphate and copper sulphide, precipitation
Batch no. 20190601
EC no. 951-963-7
CAS no. na
Appearance S olid substance
Composition Elements: 9.9 % P, 5.9 % Cl, 8.8 % Cu, 42 % Zn, 2.3 % Cd
Minerals: 70-80 % Zinc Chloride Sulphate Hydroxide Hydrate (Zn12(OH)15(SO4)3Cl3 x H2O)
10-15 % Copper Sulfide (CuS)
< 10 % Sodium Sulphate (Na2SO4)
< 1 0% Sodium Chloride (NaCl)
Purity 100 %
Homogeneity Homogenous
Solubility Water < 0.1 g/L
Expiry Date December 31, 2025
Storage Room Temperature 20 ± 5 ° C

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Anlab, Praha, Czech Republic
- Age at study initiation: 9-11 weeks old
- Weight at study initiation: 15-23g
- Housing: The animals were housed in TECNIPLAST cages from the Tecniplast Company, in Conventional animal room No: B2-611. 5 females were housed per cage.
- Diet (e.g. ad libitum): A laboratory food for mice V1534-000 R/M-H (Ssniff) was served ad libitum. The certificate of analysis is included in the raw data.
- Water (e.g. ad libitum): The animals received tap water for human consumption ad libitum. The water from the local mains was monitored for quality by testing for the microbiological and chemical quality by Waterworks Bratislava quarterly. Bottles were exchanged and cleaned once a week. The quality of drinking water is periodical monitored (including microbiological control) and recorded; certificate of analysis is included in raw data.
- Acclimation period: The animals were acclimated to the conditions identical to the condition during the experiment 5 days prior to the start of treatment.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 40-60 %
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

10, 25, 50 (% w/v).
Based on the laboratory pre-test, acetone/olive oil, 4:1 (v/v) was selected as a vehicle of choice from the recommended vehicles according to OECD Guideline No. 429.
Test substance was insoluble in all solvents used: acetone/olive oil, 4:1 (v/v), N, N-dimethyl formamide, propylene glycol and dimethyl sulfoxide. Test substance formed suspension in acetone/olive oil, 4:1 (v/v).
Each day, 0.2 g, 0.1 g and 0.04 g of Zinc chlorohydroxy sulphate and copper sulphide, precipitation (50 % w/v, 25 % w/v and 10 % w/v) were suspended in 0.4 mL of acetone/olive oil (4:1 v/v) vehicle, one hour before the application to an ear of the mice. Prepared suspension was vortexed immediately before the application to the ears of mice.

No. of animals per dose:

5 animals / dose

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Test substance was insoluble in all solvents used: acetone/olive oil, 4:1 (v/v), N, N-dimethyl formamide, propylene glycol and dimethyl sulfoxide. Test substance formed suspension in acetone/olive oil, 4:1 (v/v).

The doses were selected from the concentration series 100 %, 50 %, 25 %, 10 %, 5 %, 2.5 %
Pre-screen test:
Group Group name No. animals ID number Cage number
1 Zn, Cu 50 % (w/v) 1 1 1
2 Zn, Cu 25 % (w/v) 1 1 2
3 Zn, Cu 10 % (w/v) 1 1 3

All mice (1 mice/group) were observed daily for any clinical signs of toxicity or local irritation at the application site. Body weights were recorded on Day 1 before application and prior to the termination (Day 6). Both ears of each mouse were observed for erythema and scored (Table 1). Ear thickness was measured by a digital micrometer on Day 1 (pre-dose), Day 3 and Day 6. Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥ 25 % in day of measurement.

To determine the highest tolerated and no-irritant test concentration a Pre-screen test was performed. Zinc chlorohydroxy sulphate and copper sulphide, precipitation administered at concentrations of 50 %, 25 % and 10 % (w/v) did not cause any significant changes in monitored parameters. The daily clinical observation of the animals did not show visible clinical signs of toxicity. The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. No marked changes of body weights were observed during the test.

MAIN STUDY

Main study:
Group Group name No. animals ID number Cage number
1 Negative Control 5 1-5 1
2 Positive Control 5 6-10 2
3 Zn, Cu 10 % (w/v) 5 11-15 3
4 Zn, Cu 25 % (w/v) 5 16-20 4
5 Zn, Cu 50 % (w/v) 5 21-25 5

Day 1:
Each animal was identified, and the body weight was recorded. To the dorsum of each ear 25 µL of the appropriate dilution of the test item, positive control or the vehicle alone was applied.
Days 2 and 3:
The application procedure carried out on day 1 was repeated.
Days 4 and 5:
No treatment.
Day 6:
The body weight of each animal was recorded. 250 μL of sterile phosphate-buffered saline (PBS) containing 20 μCi (7.4×105 Bq) of tritiated (3H)-methyl thymidine was injected into all test and control mice via the tail vein.

Cell suspension of lymph node cells (LNC) from pooled treatment groups were prepared by gentle mechanical disaggregation by plastic homogenizer through 200 micron-mesh gauze. The LNC were centrifuged at 600 g, 4°C for 10 min and washed twice with 10 mL of PBS. Cell suspensions were precipitated with 3 mL of 5 % trichloroacetic acid (TCA) at 4C for 18h. Pellets were re-suspended in 1 ml of TCA and transferred to scintillation vials containing 10 mL of scintillation fluid Ultima Gold LLT for 3H-counting.

Incorporation of 3H-methyl thymidine was measured by β-scintillation counting as disintegrations per minute (DPM) using liquid scintillation counter TriCarb (Perkin Elmer). The incorporation was expressed as disintegrations per minute (DPM)/treatment group.

Animals were carefully observed once daily for any clinical symptoms, either of local irritation at the application site or of systemic toxicity. All observations were systematically recorded with individual records being maintained for each animal.

The individual animal body weights were determined at the start of the test and at the scheduled day of sacrificing of the animals. Statistical analysis was performed using software IBM SPSS 19.0. Differences in body weight gain between initial body weight (Day 1) and terminal body weight (Day 6) were analysed by non-parametric Kruskal-Wallis test. Moreover, differences in body weight between treated groups and negative (vehicle) control on Day 1 and Day 6 of the study were analysed by non-parametric Kruskal-Wallis test. P – values less than 0.05 were considered significant.
Five hours later, the animals were sacrificed. The draining auricular lymph nodes from each ear was excised and pooled in PBS for each experimental group (pooled treatment group approach).

.

Positive control substance(s):

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

Statistics:

Results are expressed as the Stimulation Index (SI). The SI was obtained by dividing the pooled DPM for each treatment group by the incorporation of the pooled DPM vehicle control group; this yields a mean SI. A test item is regarded as sensitizer in the LLNA test when SI ≥ 3.
Estimated concentration three (EC3): Estimated concentration of a test substance needed to produce a stimulation index of three. EC3 value is determined by linear interpolation of points on dose- response curve, immediately above and below of SI value, according to the equation:

EC3=c+[(3-d)/(b-d)]x(a-c)
a – higher concentration, b – SI of higher concentration, c – lower concentration
d – SI of lower concentration
If all points are below the stimulation index of three, no EC3 value can be stated.

Results and discussion

Positive control results:

Signs of systemic toxicity were observed in the positive control group. On days 2, 3 and 4, changes in grooming activity. Signs of local irritation were observed in the positive control group. Slight redness was observed on days 2 and 3.
Group Lymph node weight (g) DPM SI
Positive control 0.0583 12 102 6.21

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.56

Test group / Remarks:

10% test item

Results 2

Key result

Parameter:

SI

Value:

0.96

Test group / Remarks:

25% test item

Results 3

Key result

Parameter:

SI

Value:

1.46

Test group / Remarks:

50% test item

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 50 862, 54 461 and 73 980 DPM, respectively. The mean DPM/animal value for the vehicle control group was 2 388 DPM.

EC3 CALCULATION
It was established that the EC3 value (the estimated test item concentration that will give a SI =3) is lower than 10 %.

CLINICAL OBSERVATIONS:
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals.

BODY WEIGHTS
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Any other information on results incl. tables

Clinical signs:

Regardless the dose applied, in all test substance treated mice, daily clinical observation of animals did not show visible clinical signs of local irritation or systemic toxicity.

 

Body weights:

No differences were found in body weight gain between initial body weight (Day 1) and terminal body weight (Day 6). Similarly, no significant changes in body weight between treated groups and negative (vehicle) control at Day 1 and Day 6 of the study were recorded.

Lymph Node Prolifireation:

The DPM values for the three treated groups were 3 043 (10 %w/v), 1 809 (25 %w/v) and 2 840 (50 %w/v), respectively. The SI values for the three treated groups were 1.56 (10 %w/v), 0.93 (25 %w/v) and 1.46 (50 %w/v), respectively. The EC3 value could not be calculated, since none of the tested concentrations induced SI more than the threshold value of 3.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The skin sensitizing potential of Zinc chlorohydroxy sulphate and copper sulphide, precipitation was assessed using the murine local lymph node assay. Based on the results of this study, Zinc chlorohydroxy sulphate and copper sulphide, precipitation is not considered a skin sensitizer under the conditions of this LLNA study.

Executive summary:

The skin sensitization potential of Zinc chlorohydroxy sulphate and coppersulphide, precipitation was evaluated by LLNA method, which basic underlying principle is that sensitizers induce a primary proliferation of lymphocytes in the auricular lymph nodes draining the site of chemical application.

In the present study, the test item was applied to the dorsum of each ear of five female mice (CBA/CaOlaHsd) per group over three consecutive days, at three concentrations (10 %, 25 % and 50 % w/v). All animals survived throughout the test period without showing any clinical signs of toxicity. Calculated SI values in treated groups remained under the value of 3, which is the threshold to consider the substance as a sensitizer. Therefore, it was not possible to calculate an EC3 value.

These results demonstrate that the test item Zinc chlorohydroxy sulphate and coppersulphide, precipitation was not a skin sensitizer under the test conditions of this study.