2,4-dioxo-1,3-diazetidine-1,3-bis(methyl-m-phenylene) diisocyanate

Endpoint summary

Description of key information

Metalink U has at least a moderate skin sensitising potential in the mouse LLNA (Leidenfrost, 2015).

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:

Nov 2014

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)

Deviations:

yes

Remarks:

non-radioactive alternative, measuring lymph node cell counts; in addition, measurements of ear swelling and ear weight were done to discriminate the irritating potential from the sensitizing potential of the test substance.

Principles of method if other than guideline:

Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorised in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). Information on validation of IMDS and scientific justification is given in: Vohr HW et al., Arch. Toxicol., 73, 501-509 (2000); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005); Gamer AO et al., Regul. Toxicol. Pharmacol., 52, 290-298 (2008).

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 221305
- Purity: > 98.0 %
- Expiration date of the lot/batch: 2015-01-04

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Nederland, AD Horst, The Netherlands
- Strain: HsdWin: NMRI
- Age at study initiation: not specified
- Weight at study initiation: not specified
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 40 - 70
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:

methyl ethyl ketone

Concentration:

0, 2, 10, 30 %

No. of animals per dose:

6

Details on study design:

TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the formulation, the positive control or the vehicle were applied epicutaneously onto the dorsal part of both ears of the animals. This  treatment was repeated on three consecutive days (d1, d2 and d3). The volume administered was 25µl/ear. The used concentrations were based on the experiences with the test system and the toxic properties of the test substance.
The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (d4). The appropriate organs were then removed. Lymphatic organs (the auricular lymph nodes) were transferred into physiological saline (PBS).
Investigations:
- weight of draining lymph nodes (given as weight index compared to vehicle controls)
- cell counts in draining lymph nodes (given as cell count index compared to vehicle controls)
Stimulation indices were calculated by dividing the absolute weight or number of cell counts of the substance treated lymph nodes by the vehicle treated ones.
- ear swelling (given in 0.01 mm and as index)
- ear weight (given in mg/8 mm diameter punch and as index)
- body weights

Positive control substance(s):

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

Statistics:

When it was statistically reasonable, the values from treated groups were compared with those from the control group by a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to a homogeneity testing like Cochran's test. Alternatively, if the variances are considered to be heterogenous (p<=0.05), a non-parametric Kruskal-Wallis test has been used (Kruskal-Wallis ANOVA) at significance levels of 5%. Two sided multiple test procedures were done according to Dunnett or Bonferroni-Holm, respectively. Outlying values in the LN weights were eliminated at a probability level of 99% by Nalimov's method. In addition, for the LLNA/IMDS the smallest significant differences in the means were calculated by Scheffe's method, which can be used for both equal and unequal sample sizes.

Positive control results:

A reliability test using hexyl cinnamic aldehyde showed a clear sensitising potential in the local lymph node assay (IMDS).

Key result

Parameter:

SI

Remarks:

cell count index (%)

Value:

1.51

Test group / Remarks:

2 %

Key result

Parameter:

SI

Remarks:

cell count index (%)

Value:

1.69

Test group / Remarks:

10 %

Key result

Parameter:

SI

Remarks:

cell count index (%)

Value:

1.11

Test group / Remarks:

30 %

Table 1: Summary of the LLNA/IMDS results with Metalink U (means of 6 animals per group)

Parameter investigated	Vehicle control	Dose 2 %	Dose 10 %	Dose 30 %
Stimulation index: weight of draining lymph nodes	1.00	1.32	1.70 *	1.05
Stimulation index: cell count in draining lymph nodes	1.00	1.51	1.69 *	1.11
Ear swelling in 0.01 mm on day 4 (index)	18.42 (1.00)	18.42 (1.00)	18.17 (0.99)	18.42 (1.00)
Ear weight in mg / 8 mm diameter punch on day 4 (index)	13.45 (1.00)	13.16 (0.98)	13.13 (0.98)	13.02 (0.97)

* statistically significant increase (p ≤ 0.05)

The "positive level" of ear swelling, which is 2 x 10^-2 mm increase, i.e. about 10% of the control values, has not been reached or exceeded in any dose group.

The reason for the lower stimulation indices detected at the high concentration (30 %) is not known. However, skin reactions often follow a bell-shaped reaction with respect to the concentration of the test item applied. This could be due to shifts in the optimal balance between vehicle and test item. The test item formulations were suspensions, so that it is possible, that during the application of the high concentration (30 %) the test item amount penetrating the skin is less than for the lower concentrations.

Although it is not possible to calculate an exact EC value from the data obtained, it can be assumed that the EC value is in any case below 2 %. In accordance with the classification proposed in the Technical Report No. 78 of the ECETOC (2003) this value corresponds at least to a moderate skin sensitizer.

The body weights of the animals were not affected by any treatment.

Interpretation of results:

sensitising

Remarks:

Migrated information

Executive summary:

Metalink U was investigated in the modified local lymph node assay (LLNA-IMDS) on female mice according to OECD TG 429. Concentrations of 0 (vehicle control), 2, 10 and 30 % formulated in methyl ethyl ketone were tested. Compared to vehicle treated animals there were increases regarding the cell counts (indices of 1.51, 1.69 and 1.11 %, resp.) and the weights of the draining lymph nodes (indices of 1.32, 1.70 and 1.05 %, resp.) in the low and mid dose group, which are of statistical significance in the mid dose group. The "positive level" of index 1.4 for the cell counts has been exceeded in the low and mid dose group. The "positive level" of ear swelling which is 2 x 10^-2 mm increase, i.e. about 10 % of the control values, has not been reached or exceeded in any dose group. Although it is not possible to calculate an exact EC value from the data obtained, it can be assumed that the EC value is in any case below 2 %. In accordance with the classification proposed in the Technical Report No. 78 of the ECETOC (2003) this value corresponds to a moderate skin sensitiser.

In summary, these results show that Metalink U has at least a moderate sensitising potential in mice after dermal application of a 2 % concentration. No indication for a non-specific (irritant) activation was detected.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Metalink U was investigated in the modified local lymph node assay (LLNA-IMDS) on female mice according to OECD TG 429 (Leidenfrost, 2015). Concentrations of 0 (vehicle control), 2, 10 and 30 % formulated in methyl ethyl ketone were tested. Compared to vehicle treated animals there were increases regarding the cell counts (indices of 1.51, 1.69 and 1.11 %, resp.) and the weights of the draining lymph nodes (indices of 1.32, 1.70 and 1.05 %, resp.) in the low and mid dose group, which are of statistical significance in the mid dose group. The "positive level" of index 1.4 for the cell counts has been exceeded in the low and mid dose group. The "positive level" of ear swelling which is 2 x 10^-2mm increase, i.e. about 10 % of the control values, has not been reached or exceeded in any dose group. Although it is not possible to calculate an exact EC value from the data obtained, it can be assumed that the EC value is in any case below 2 %. In accordance with the classification proposed in the Technical Report No. 78 of the ECETOC (2003) this value corresponds to a moderate skin sensitiser.

In summary, these results show that Metalink U has at least a moderate sensitising potential in mice after dermal application of a 2 % concentration. No indication for a non-specific (irritant) activation was detected.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the study results (at least a moderate skin sensitising potential detected in a LLNA) a classification with Skin Sensitisation Cat. 1A (H317: May cause an allergic skin reaction) according to Regulation (EC) No. 1272/2008 (CLP) is required.