Dipotassium oxalate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 - 23 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 22 Jul 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayrisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Diammonium oxalate hydrate

Cas Number:

6009-70-7

Molecular formula:

C2H8N2O4.H2O

IUPAC Name:

Diammonium oxalate hydrate

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories GmbH, Venray, The Netherlands
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 17 - 21 g (main test); 17 - 23 g (pre-test)
- Housing: 5 animals per group were housed in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding.
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): At least 10
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 01 June 2016 To: 23 June 2016

Study design: in vivo (LLNA)

Vehicle:

other: aqua ad injection

Remarks:

containing 2% CMC

Concentration:

1.5, 3 and 6%

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS: 2 female mice per concentration were treated by daily application of 25 μl with a test substance concentration of 3 and 6% in aqua ad injection, respectively, to the dorsal surface of the ear, for 3 consecutive days. The body weight was recorded on Day 1 prior to dosing and on Day 6.
- Compound solubility: The test substance was insoluble in acetone/olive oil (4:1), DMSO and polyethylenglycol, respectively. The maximum, technically applicable concentration of the test substance in the vehicle was found to be 6.25% in aqua ad injectionem.
- Irritation: The animals were observed daily for local irritation at the application site.
- Systemic toxicity: The animals were observed daily for any signs of toxicity.
- Ear thickness measurements: Ear thickness measurements were performed on Day 1, 3 and 6, respectively.
- Erythema scores: Draize scoring system

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by β-scintillation
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). Before DPM/NODE values were determined, background values were subtracted. The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values (SI ≥ 3.0).
- Other: The animals were observed for signs of toxicity once daily. 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 material was applied to the entire dorsal surface of each ear of each mouse on Day 1, 2 and 3 in concentrations of 1.5, 3 and 6% in aqua ad injetionem. The irritation effects at the treatment site and any signs of systemic toxicity were assessed daily. 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. Approximately five hours later, the draining auricular lymph nodes of each ear were excised into PBS and pooled per experimental group. A single cell suspension of pooled lymph node cells was prepared 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 5% TCA and transferred to 7 mL scintillation fluid before β-scintillation counting.

Positive control substance(s):

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

Results and discussion

Positive control results:

A shared positive control hexyl cinnamic aldehyde (25%) in aqua ad injectionem was performed concomitantly using 5 animals and was considered to be a sensitizer under the conditions of the test (SI 9.8).

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Variability:

0.1

Test group / Remarks:

1.5%

Results 2

Key result

Parameter:

SI

Value:

0.9

Variability:

0.3

Test group / Remarks:

3%

Results 3

Key result

Parameter:

SI

Value:

1.4

Variability:

0.2

Test group / Remarks:

6%

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
The SI of the 1.5, 3 and 6% treatment group was 1.0, 0.9 and 1.4, respectively. None of the test substance concentrations produced an 3-fold increase in 3HTdR incorporation.

EC3 CALCULATION
None of the SI values were above 3 and it is therefore not possible to determine an EC3 concentration.

CLINICAL OBSERVATIONS
No mortality and no signs of systemic toxicity were noted in the test or negative control group. Wet fur at the application site was observed in 4/5 animals of the positive control on Day 2.

BODY WEIGHTS
Body weight changes were comparable to those observed in the corresponding control group animals over the same period.

Any other information on results incl. tables

Table 1: Summary of results

	DPM	DPM - mean background	DPM/node	Mean DPM/node	SI
Negative Control	1947	1926.6	963.3	700.5± 219.9	1.0
	697	676.6	338.3
	1534	1513.6	756.8
	1738	1717.6	858.8
	1191	1170.6	585.3
1.5%	1351	1330.6	665.3	727.9± 68.6	1.0± 0.1
	1274	1253.6	626.8
	1575	1554.6	777.3
	1562	1541.6	770.8
	1619	1598.6	799.3
3%	991	970.6	485.3	647.3± 209.9	0.9± 0.3
	1978	1957.6	978.8
	824	803.6	401.8
	1186	1165.6	582.8
	1596	1575.6	787.8
6%	2166	2145.6	1072.8	998.9± 126.3	1.4± 0.2
	1727	1706.6	853.3
	2081	2060.6	1030.3
	1738	1717.6	858.8
	2379	2358.6	1179.3

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008

Conclusions:

CLP: not classified