Dizinc pyrophosphate

Endpoint summary

Description of key information

Skin sensitisation: LLNA: not sensitising

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:

20 Jun - 27 Jul 2017

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 Jul 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Freie Hansestadt Hamburg, Behörde für Gesundheit und Verbraucherschutz

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 9 weeks
- Weight at study initiation: 28 - 34 g
- Housing: Individually housed in Makrolon cages (type II). Granulated textured wood (Granulat A2, Goldenstedt, Germany) was used as bedding material for the cages.
- Diet: Commercial diet ssniff R/M-H V1534 (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Photoperiod (hrs dark / hrs light): 12/12

- IN-LIFE DATES: From: 20 Jun 2017 To: 27 Jul 2017

Vehicle:

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

Concentration:

10, 25 and 50% (w/w)

No. of animals per dose:

6

Details on study design:

PRE-SCREEN TESTS: A preliminary experiment was carried out in 3 animals to examine the irritating potential and handling/application of the test item in order to select the appropriate concentrations. Three concentrations of 10, 25 and 50% suspended in acetone / olive oil (4:1, v/v) were examined. The mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations once daily each on three consecutive days.
- Compound solubility: A 50% concentration (w/w) of the test item in acetone/olive oil (4:1, v/v) was the maximum feasible concentration.
- Irritation: Signs of local irritation were documented for each day.
- Systemic toxicity: Mice were observed for signs of systemic toxicity.
- Ear thickness measurements: Ear thickness were measured prior to the first application of the test substance and prior to necropsy.
- Erythema scores: no erythema (0); very slight erythema (1); well defined erythema (2); moderate to severe erythema (3)

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: The alternative method was used for this study employing the lymph node weight and lymph node cell count to assess proliferation.
- Criteria used to consider a positive response: The so-called stimulation (or LLN-) indices to determine the sensitising potential (this value was fixed empirically during the interlaboratory validation of the alternative method, for details see Ehling et al. 2005a and 2005b, page 12), were calculated by dividing the average absolute lymph node weight or lymph node cell counts per group of the test item treated animals by the vehicle treated ones.
Thus, in case of no stimulating effect the index for the lymph node cell count is always below 1.4 (cut-off value). An index above 1.4 is considered positive. For lymph node weight significance at p ≤ 0.01 is considered positive (U-test according to MANN and WHITNEY).

TREATMENT PREPARATION AND ADMINISTRATION: On Day 1, the weight of each animal was individually identified. The weights and any clinical signs were recorded. In addition, ear swelling measurements were carried out at the helical edge of both ears using an Oditest micrometer. Open application of 25 μL test item concentrations of 10, 25 or 50%, the vehicle alone or the positive control (as appropriate) were administered to the dorsum of each ear for three consecutive days. 24 h after the last application, ear swelling measurements (immediately before sacrificing the mice) were carried out at the helical edge of both ears using an Oditest micrometer. The animals were euthanized by carbon dioxide (CO2) inhalation and laparotomised. Punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and immediately weighed on an analytical balance. Lateral pairs of auricular lymph nodes draining the ear tissue were excised, carefully separated from remaining fatty tissue and weighed on an analytical balance immediately following preparation. The lymph nodes were then stored on ice in PBS / 0.5% BSA and subjected to the preparation of single cell suspensions by mechanical tissue disaggregation. The cells were counted automatically in a cell counter.

JUSTIFICATION FOR THE ALTERNATIVE METHOD
The alternative method used for the study employing the lymph node weight and lymph node cell count to assess proliferation has been established by a European interlaboratory validation exercise, as described in the two publications by Ehling et al. 2005a and 2005b. This method has the advantage of (i) more simplistic experimental work, (ii) less variability, (iii) better reproducibility, (iv) faster results, (v) reduced costs. The local lymph node assay (LLNA) and modifications thereof were recently recognized by the OECD as stand-alone methods for the detection of skinsensitising potential. However, although the validity of the LLNA was acknowledged by the ICCVAM, attention was drawn to one major problem, i.e., the possibility of false positive results caused by non-specific cell activation as a result of inflammatory processes in the skin (irritation). This is based on the fact that inflammatory processes in the skin may lead to non-specific activation of dendritic cells, cell migration and non-specific proliferation of lymph node cells. Measuring cell proliferation by radioactive or non-radioactive methods, without taking the irritating properties of test items into account, leads thus to false positive reactions. By additionally measuring simple inflammatory parameters such as ear thickness or ear weight, it is possible to reliably determine the degree of response that is attributable to irritation (Vohr and Ahr, 2005). Hence, in addition, the acute inflammatory skin reaction (irritating potential) was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item employing the U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control. The stimulation indices were calculated by dividing the average ear weight and average ear thickness on test day 4 per group of the test item treated animals by the vehicle treated ones. The cut-off threshold value for ear weight was set at 1.1.

Reference
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: First round; Toxicology 212, 60-68(2005a);
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round; Toxicology 212, 69-79 (2005b).
Vohr, H.-W. and Ahr, H.-J.: The local lymph node assay too sensitive? Arch. Toxicol. 79: 721-728 (2005)

Positive control substance(s):

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

Statistics:

For lymph node weight significance at p ≤ 0.01 is considered positive (U-test according to MANN and WHITNEY). A possible concentration-response-relationship for the lymph node weight was examined by linear regression analysis employing PEARSON's correlation coefficient. A U-test was performed also for cell count.

Positive control results:

The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid.

Key result

Parameter:

SI

Remarks:

Lymph node cell count

Value:

1.145

Test group / Remarks:

10% (w/w)

Key result

Parameter:

SI

Remarks:

Lymph node cell count

Value:

1.185

Test group / Remarks:

25% (w/w)

Key result

Parameter:

SI

Remarks:

Lymph node cell count

Value:

1.321

Test group / Remarks:

50% (w/w)

Key result

Parameter:

SI

Remarks:

Ear weight (Punch biopsies)

Value:

1.102

Test group / Remarks:

10% (w/w)

Key result

Parameter:

SI

Remarks:

Ear weight (Punch biopsies)

Value:

1.07

Test group / Remarks:

25% (w/w)

Key result

Parameter:

SI

Remarks:

Ear weight (Punch biopsies)

Value:

1.057

Test group / Remarks:

50% (w/w)

Key result

Parameter:

SI

Remarks:

Lymph node weight

Value:

1.054

Test group / Remarks:

10% (w/w)

Key result

Parameter:

SI

Remarks:

Lymph node weight

Value:

1.087

Test group / Remarks:

25% (w/w)

Key result

Parameter:

SI

Remarks:

Lymph node weight

Value:

1.239

Test group / Remarks:

50% (w/w)

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
The stimulation indices of the lymph node cell count did not exceed the threshold level of 1.4. Hence, the test item is classified as not sensitising. The threshold level for the ear weight of 1.1 was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted. The marginal increase of ear weight in the 10% treatment group is considered to be coincidental as no concentration response relationship was noted.

CLINICAL OBSERVATIONS:
No signs of local or systemic intolerance were recorded.

BODY WEIGHTS
The animal body weight was not affected by the treatment.

Table 1: Summary of Results

Group	Concentration Dizinc pyrophosphate	Lymph node Cell count mean (n=6) per mL	Stimulation Index threshold: 1.4	Lymph node weight mean (n=6) [mg]	Stimulation Index	Ear weight (Punch biopsies) mean (n=12) [mg]	Stimulation Index threshold: 1.1	Ear thickness (TD 4) mean (n=12) [µm]	Stimulation Index
1	vehicle (negative control)	7833333	1.000	7.7	1.000	13.1	1.000	196.67	1.000
2	10%	8966667	1.145	8.2	1.065	14.4	1.102	204.17	1.038
3	25%	9283333	1.185	8.3	1.087	14.0	1.070	207.50	1.055
4	50%	10350000	1.321	9.5	1.239	13.8	1.057	208.33	1.059
5	positive control (HCA)	14366667**	1.834	12.8**	1.674	14.6**	1.115	220.00	1.119

HCA: α-hexyl cinnamic aldehyde

** significantly increased compared to negative control (U-test according to MANN and WHITNEY, at p ≤ 0.01)

Interpretation of results:

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Reliable data is available in order to examine the skin sensitisation potential of the test substance (LPT, 2017). The LLNA according to OECD 429 was performed. However, an alternative method was used employing the lymph node weight and lymph node cell count to assess proliferation of lymphocytes. In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test substance. Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the respective vehicle treated ones. Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive. Three concentrations of test substance (10%, 25% and 50%), suspended in acetone/olive oil (4:1, v/v) (w/w), were tested in six female NMRI mice per group and compared to a vehicle control group. Acetone/olive oil (4:1, v/v) was selected as it is recommended by the OECD guideline and provided suitable suspensions of the test substance both for administration and adherence to the mouse ear of such high concentrations.

A 50% concentration (w/w) of the test substance in acetone/olive oil (4:1, v/v) was the maximum feasible concentration.

In the main study treatment with the test substance at concentrations of 10%, 25% or 50% did not reveal any statistically increase values for the lymph node cell count. The stimulation indices of the lymph node cell count did not exceed the treshold level of 1.4. Therefore, the test substance is not classified as skin sensitising. The threshold level for the ear weight of 1.1 was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted. The positive control group caused increases in lymph node cell cound and lymph node weight (statistically significant at p <= 0.01). Therefore, the study can be regarded as valid. No signs of local or systemic intolerance were recorded. The animal body weight was not affected by the treatment.

In conclusion, under the present test conditions, the test substance at concentrations of 10%, 25% and 50% in acetone/olive oil did not reveal any skin sensitising properties in the local lymph node assay.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on skin sensitisation does not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.