1H-Isoindole-1,3(2H)-dione, 2-[[(5S)-2-oxo-3-[4-(3-oxo-4-morpholinyl)phenyl]-5-oxazolidinyl]methyl]-

Endpoint summary

Description of key information

Skin Sensitisation, LLNA/IMDS (NMRI mice, GLP, OECD TG 429, 406 and EPA OPPTS 870.2600): no non-specific (irritating) or specific immunostimulating (sensitising) properties [Bayer AG, Report No. PH-34457, 2006-05-19]

 

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:

Mar 2006

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:

2002

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

purity 99.8%

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann/Nederland, Kreuzelweg 53, N1.-5960AD Horst, Netherland
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation: 26-33 g
- Housing: During the adaptation period the animals were housed in conventional Makrolon® type II cages, up to 8 mice and during the study period in type II cages, one animal being kept in each cage. Low-dust wood granulate from J. Rettenmaier & Söhne Füllstoff-Fabriken, 73494 Rosenberg, Germany, was used as bedding.
- Diet (e.g. ad libitum): ad libitum, PROVIMI KLIBA SA 3883 maintenance diet for rats and mice (from Provimi Kliba SA, CH-4303 Kaiseraugst)
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: at least 7 days


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

Vehicle:

dimethylformamide

Concentration:

0 (vehicle control), 2, 10 and 50%

No. of animals per dose:

6 females

Details on study design:

MAIN STUDY:

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The so-called stimulation (or LLN-) index is calculated by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones. Thus, in case of no stimulating effect the index is always about 1.00 (+/- standard deviation), and the indices of vehicle treated animals are set to 1.00 (+/- standard deviation).

TREATMENT PREPARATION AND ADMINISTRATION: The test item was formulated once at day 1 of the study in DMF. The formulations were visually described as solutions. The test item in the formulation and 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 25 µL/ear.
Based on our experiences with this test system and the known properties of the test item the following concentrations were used: 0%, 2%, 10% and 50%.

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 (s; vehicle) by a one-way analysis ofvariance (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 according to Sachs can be used for both equal and unequal sample sizes. In this method of statistical processing of measurements a large number of comparisons is made, and as a result of the multiple tests the overall probability of error is considerably greater than the p values suggest (increased number of false-positive results). On the other hand, the known methods of adjusting p values lead to an excessive increase in the number of false negatives. In view of these problems the biological and toxicological relevance is also taken into consideration in the evaluation ofstatistical significance. For this reason, in the case of indices only the standard deviations between groups and difference analysis of the mean values (Scheffe‘s method) were used in the evaluation of the biological relevance.

Positive control results:

The Local Lymph Node Assay Test methodology was checked for reliability in a test on female NMRI mice using Alpha Hexyl Cinnamic Aldehyde formulated in different vehicles (PEG 400, DAE 433, DMF, MEK, Aceton/Olive Oil (4:1) and Cremophor EL/ physiological saline solution 2% v/v) at the concentrations of 3%, 10% and 30%.
The sensitivity as well as the reliability ofthe experimental technique is thus confirmed by this study. A similar check is done in regular intervals using one of the above vehicles confirming the reliability of the method.
The Local Lymph Node Assay Test methodology was checked for reliability in a test on female NMRI mice using Alpha Hexyl Cimramic Aldehyde formulated in the vehicle Acetone/Olive Oil (4:1) at the concentrations of 3%, 10% and 30%.
The results show that the test item has a clear sensitizing potential.

Key result

Parameter:

SI

Value:

1

Variability:

SD +/-32.96

Test group / Remarks:

0%

Key result

Parameter:

SI

Value:

0.95

Variability:

SD +/-15.86

Test group / Remarks:

2%

Key result

Parameter:

SI

Value:

1.08

Variability:

SD +/- 20.67

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

0.85

Variability:

SD +/- 17.61

Test group / Remarks:

50%

The "positive level" of ear swelling (i.e. increase of about 10% of the control values) has not been reached or exceeded in any dose group. No substance specific effects were determined for ear weights either.

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

Tabular summary of the LLNA/IMDS results:

	Direct LLNA		Ear swelling (in 0.01 mm)			Ear weight (in mg per 8 mm diameter punch)
Dose (%)	Weight index (mean +/- SD in %)	Cell count index (mean +/- SD in %)	day 1 (mean +/- SD in %)	day 4 (mean +/- SD in %)	Index day 4	day 4 (mean +/- SD in %)	Index day 4
0 (DMF)	1.00 +/- 20.14	1.00 +/- 32.96	17.33 +/- 4.49	17.67 +/- 5.02	1.00	11.30 +/- 8.54	1.00
2	0.97 +/- 14.37	0.95 +/- 15.86	17.50 +/- 2.98	18.17 +/- 3.95	1.03	12.05 +/- 7.80	1.07
10	1.15 +/- 22.01	1.08 +/- 20.67	17.58 +/- 5.67	18.08 +/- 3.70	1.02	11.60 +/- 5.61	1.03
50	0.88 +/- 12.12	0.85 +/- 17.61	16.67 +/- 2.95	17.00 +/- 4.34	0.96	11.26 +/- 6.51	1.00

Interpretation of results:

GHS criteria not met

Conclusions:

the test item has no skin sensitizing potential

Executive summary:

To determine the skin-sensitizing properties of Oxaphthalimid the LLNA was performed on female NMRI mice according to OECD guideline 429, 406 and EPA OPPTS 870.2600. The study was conducted with the following test substance concentrations: 2, 10 and 50%

The results showed that the test item has no sensitising potential in mice after dermal application of up to and including 50% concentration. No indication for a non-specific (irritating) activation was detected, too.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

No antigen specific activation of the cells of the immune system via dermal route was determined after application of up to and including 50% Oxaphthalimid. No indication for a non-specific (irritating) activation was detected, too. Therefore, the study does neither point to a non-specific (irritating) nor to a specific immunostimulating (sensitising) potential of the test item.

 

Justification for classification or non-classification

Based on the study results a classification according to Directive 67/548/EEC and Regulation (EC) No. 1272/2008 (CLP) is not required.