Disodium tartrate

Endpoint summary

Description of key information

Skin sensitation (OECD 429): not sensitizing

RA from source substance diammonium tartrate (CAS 3164 -29 -2)

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:

22 July 2010

Deviations:

yes

Remarks:

/ different/higher acceptable body weight loss than in TG (10 instead of 5%)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

6 July 2012

Deviations:

yes

Remarks:

/ different/higher acceptable body weight loss than in TG (10 instead of 5%)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

FORM AS APPLIED IN THE TEST (if different from that of starting material)
liquid

Species:

mouse

Strain:

CBA/Ca

Remarks:

Ola Hsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo, Venray, The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 17 - 22 g
- Housing: 5 animals per cage, 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 (sulphur acidified to a pH value of approx. 2.8), ad libitum, analysis was performed
- Acclimation period: at least 5 days
- Indication of any skin lesions: no

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 To: 23 June 2016

Vehicle:

other: aqua ad injectionem (AlleMan Pharma, lot no. 505651) containing 2% carboxymethyl cellulose (CMC, Alpha Aesar, lot no.: 10193024)

Concentration:

6.25, 12.5 and 25% (w/v)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
In the pre-screening test, two concentrations (12.5 and 25% dissolved in aqua ad injection) were selected, and 25 μL of each dose formulation or the vehicle alone were applied to the entire dorsal surface of the ears of each animal, 2 mice for each concentration (one mouse for the vehicle control), once a day for 3 consecutive days. Animals were observed daily for clinical signs of systemic toxicity and local irritation at the application site. Furthermore, body weights and ear thickness measurements were performed (before initial application and on Day 6, ear thickness was additionally measured on Day 3). None of the animals showed any abnormalities or any signs of signifcant irritation (indicated by an erythema score ≥ 3 and / or an increase of more than 25% in ear thickness). All animals showed the expected weight development. No mortality was observed.
Based on these results and considering that 25% was the maximum attainable concentration, 25% (w/v) was selected as the highest test concentration for the main study. This concentration was expected not to induce systemic toxicity, nor to induce an increase in ear thickness exceeding 25% or to induce dermal erythema with a score of 3 or more, or more than 10% body weight loss.

- Compound solubility: 25% (maximum attainable concentration)
- Irritation: no irritation observed
- Systemic toxicity: no systemic toxicity observed
- Ear thickness measurements: less than 25% increase in ear thickness was observed
- Erythema scores: no specific findings (each test group, each time point)

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by ß-scintillation
- Criteria used to consider a positive response: A substance is regarded as a sensitizer in the LLNA, if the Stimulation Index (SI) is equal to or exceeding 3

TREATMENT PREPARATION AND ADMINISTRATION:
25 μL of each dose formulation were applied to the dorsal skin of each ear of each animal once a day for 3 consecutive days. On day 6 20 μCi 3H-methyl thymidine, contained in 250 μL of PBS (= 80 μCi/mL) was administered intravenously to each mouse via the tail vein. Approximately 5 h after administration local lymph nodes were collected, minced, washed and pooled. The pooled lymph node cells (LNC) were treated with approx. 1 mL of 5% Trichloroacetic acid (TCA) at approx. 4 °C overnight (approx. 18 h) before determination of the amount of 3H-methyl thymidine incorporation on day 7 (measured in a ß-counter).

Positive control substance(s):

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

Statistics:

Mean values and standard deviations were calculated.

Positive control results:

The positive control substance (25% hexyl cinnamic aldehyde (Batch number: MKBS3936V, SIGMA-ALDRICH, Germany) in aqua ad injectionem) induced a positive reaction, determined by a DPM/lymph node of 6859.7 compared to 700.5 DPM/lymph node in the vehicle control group, leading to a SI of 9.8. No abnormal clinical signs, erythema on the ears or body weight changes were observed. Only wet fur at the application site was observed in 1 animal from Day 2-5.

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

6.25% test group

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

12.5% test group

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

25% test group

Parameter:

SI

Value:

9.8

Test group / Remarks:

positive control group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA:
No significant lymph node proliferation (SI ≥ 3) was observed for the test item at treatment concentrations of 6.25, 12.5 and 25%, compared to the control.

DETAILS ON STIMULATION INDEX CALCULATION: SI = DPM/lymph node of a treated group divided by the DPM/lymph node of the vehicle control group (The measured DPM values were corrected with the background DPM value. The average of the measured DPM values of the scintillation fluid and TCA solution was used as background DPM value.)

EC3 CALCULATION: Based on the obtained results (SI < 3) no linear regression (using SI values) was performed for dose-response analysis and no EC3 value of the test item was calculated.

CLINICAL OBSERVATIONS:
No mortality or symptoms of systemic toxicity were observed in any treatment group. No signs of irritation (indicated by an erythema score ≥ 3) or any other local effect were observed in any treatment group. Topical application of the test substance led only to a slight increase in the mean DPM values of the the 25% treatment group. The following values (DPM/lymph node) were obtained: 700.5, 690.8, 652.9 and 861.5 in vehicle control (aqua ad injectionem), 6.25, 12.5 and 25% treatment group, respectively.

BODY WEIGHTS:
All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.

Table 1: Stimulation index in mice after application of the vehicle, test substance or positive control substance

Compound	Concentration [%]	Animal No.	DPM/animal - mean background	DPM/lymph node	Stimulation index
Vehicle (aqua ad injectionem)	100	1	1926.6	963.3	1.0
		2	676.6	338.3
		3	1513.6	756.8
		4	1717.6	858.8
		5	1170.6	585.3
		Mean ± SD	1401 ± 439.8	700.5 ± 219.9
Test substance (in aqua ad injectionem)	6.25	6	734.6	367.3	0.5
		7	1900.6	950.3	1.4
		8	1308.6	654.3	0.9
		9	1608.6	804.3	1.1
		10	1355.6	677.8	1.0
		Mean ± SD	1381.6 ± 386.1	690.8 ± 193.1	1.0 ± 0.3
	12.5	11	1087.6	543.8	0.8
		12	984.6	492.3	0.7
		13	1556.6	778.3	1.1
		14	1365.6	682.8	1.0
		15	1534.6	767.3	1.1
		Mean ± SD	1305.8 ± 232.2	652.9 ± 116.1	0.9 ± 0.2
	25	16	799.6	399.8	0.6
		17	983.6	491.8	0.7
		18	1717.6	858.8	1.2
		19	2370.6	1185.3	1.7
		20	2743.6	1371.8	2.0
		Mean ± SD	1723 ± 756.4	861.5 ± 378.2	1.2 ± 0.5
Positive control (HCA)	25	21	1926.6	963.3	14.4
		22	676.6	338.3	9.4
		23	1513.6	756.8	11.7
		24	1717.6	858.8	8.0
		25	1170.6	585.3	5.5
		Mean ± SD	13719.4 ± 4265.8	6859.7 ± 2132.9	9.8 ± 3.0

DPM = disintegrations per minute

HCA = hexyl cinnamic aldehyde

SD = standard deviation

 

Table 2: Body weight of mice after application of the vehicle, test substance or positive control substance

Compound	Concentration [%]	Animal No.	Body weight
			Start of Study [g]	End of Study [g]	Change [g]
Vehicle (Aqua ad injectionem)	100	1	19	20	1
		2	21	21	0
		3	22	23	1
		4	20	20	0
		5	21	21	0
Test substance	6.25	6	21	22	1
		7	18	18	0
		8	21	21	0
		9	22	22	0
		10	19	19	0
	12.5	11	18	20	2
		12	19	21	2
		13	18	18	0
		14	18	18	0
		15	20	21	1
	25	16	17	17	0
		17	18	19	1
		18	19	18	-1
		19	18	19	1
		20	21	21	0
Positive control (HCA)	25	21	18	19	1
		22	18	20	2
		23	20	21	1
		24	19	21	2
		25	19	20	1

HCA = hexyl cinnamic aldehyde

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:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

6.25% test group

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

12.5% test group

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

25% test group

Parameter:

SI

Value:

9.8

Test group / Remarks:

positive control group

Interpretation of results:

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

Conclusions:

CLP: not classified
In a study according to OECD 429 with the source substance no skin sensitsation potential was observed (SI values were below 3). As explained in the analogue justification, this result is considered to be valid also for the target substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Justification for read-across

 

There are no data on Sensitisation available for disodium tartrate (CAS 868 -18 -8). To fulfil the standard data requirements defined in Regulation (EC) No. 1907/2006, Annex VII, 8.3., readacross from an appropriate substance is conducted in accordance with Regulation (EC) No. 1907/2006, Annex XI. According to Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met”. In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across) “to avoid the need to test every substance for every endpoint”.

 

For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read across, with regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substances 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).

 

As no experimental/measured data are available on skin sensitsation of disodium tartrate (CAS 868 -18 -8), read-across to reliable data on the analogue substance diammonium tartrate (CAS 3164 -29 -2) was conducted.

 

Skin sensitisation

The skin sensitising properties of the test substance were tested in a study according to OECD 429 and EU Method B.42 and in compliance with GLP (reference 7.4.1-1). The mice were exposed to different concentrations of the test substance (6.25, 12.5 and 25%) once a day for 3 consecutive days. The resulting Stimulation indices of 1, 0.9 and 1.2 for 6.25, 12.5 and 25% of the test substance, respectively, were below 3. Therefore, diammonium tartrate is not regarded as skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the analogue read-across approach, the available data on sensitisation do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.