Dipotassium oxalate

Endpoint summary

Description of key information

Skin sensitisation (OECD 429, GLP): not sensitising

(RA from CAS 6009-70-7)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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)

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

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)

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).

Key result

Parameter:

SI

Value:

1

Variability:

0.1

Test group / Remarks:

1.5%

Key result

Parameter:

SI

Value:

0.9

Variability:

0.3

Test group / Remarks:

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.

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

Interpretation of results:

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

Conclusions:

CLP: not classified

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Key result

Parameter:

SI

Value:

1

Variability:

0.1

Test group / Remarks:

1.5%

Remarks on result:

other: Source: 7.4.1-1

Key result

Parameter:

SI

Value:

0.9

Variability:

0.3

Test group / Remarks:

3%

Remarks on result:

other: Source: 7.4.1-1

Key result

Parameter:

SI

Value:

1.4

Variability:

0.2

Test group / Remarks:

6%

Remarks on result:

other: Source: 7.4.1-1

Interpretation of results:

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

Conclusions:

CLP: not classified

Applying the RA-A approach, similar results are expected for the target substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Justification for read-across

There are no reliable data available regarding skin sensitisation for either dipotassium oxalate monohydrate (CAS 6487-48-5) or dipotassium oxalate anhydrate (CAS 583-52-8). Read-across from an appropriate substance (diammonium oxalate monohydrate (CAS 6009-70-7) is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. in order to fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VII, 8.3. Common functional groups, structural similarities and comparable toxicological properties (according to the joint consideration in Annex VI to CLP) of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

CAS 6009-70-7

The skin sensitising potential of the source substance (CAS 6009-70-7) was evaluated in a LLNA test according to OECD 429 and in compliance with GLP (reference 7.4.1-1). The study was conducted on five female mice per dose group (1.5, 3 and 6%). 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 injection. 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, animals were sacrificed, and the draining auricular lymph nodes removed and prepared for cell suspension and scintillation counting. A vehicle control group and a shared positive control group (25% hexylcinnamaldehyde in aqua ad injection) of five animals each were run concurrently, confirming the reliability of the test system. The test substance produced a stimulation index smaller than 3 in all groups and is therefore not considered to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to dipotassium oxalate, data will be generated from information on reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

The available data on skin sensitisation from the source substance diammonium oxalate do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.

Therefore, applying the RA-A approach, the target substance dipotassium oxalate is also considered not to meet the classification criteria for skin sensitisation.