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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 06.10.2016 to 16.03.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
[Specific explanation in addition to field 'Justification for data waiving']

This in vivo LLNA study was initiated on 10-10-16 (Study plan attached) and meets the requirements set out in Article 13(3), first subparagraph, and Article 13(4) shall be considered appropriate to address this standard information requirement. Therefore in vitro/in chemico skin sensitisation studies were not submitted.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Xianyang Sanjing Technology Co., ltd./160612
- Expiration date of the lot/batch: June 18, 2018
- Purity: > 99.6 % w/w


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, in a cool and dry place, away from light

In vivo test system

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeding farm VELAZ s.r.o., Lysolajské údolí 15/53, 165 00 Prague 6, Czech Republic, RČH CZ 11760500
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 10 weeks (at start of dosing)
- Weight at study initiation: 16.33 - 18.86 g (at start of dosing), in pilot experiment 17.85 – 18.34 g
- Housing: Monitored conditions, microbiologically defined background with animals in groups in macrolon cages with sterilized softwood shavings
- Diet: Pelleted standard diet for experimental animals ad libitum (Altromin, manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany). Microbiological control and content of nutrients was performed.
- Water: Drinking tap water ad libitum. Water quality corresponded to Regulation No. 252/2004 Czech Coll. Of Law, Health Ministry
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 – 70 %,
- Photoperiod (hrs dark / hrs light): Light: 12 hours light/dark cycle: 6am-6pm/6pm-6am

Study design: in vivo (LLNA)

Vehicle:
other: DAE 433 – mixture of 40 % dimethylacetamide, 30 % acetone and 30 % ethanol
Remarks:
Vehicle is used in laboratory for approximately 10 years and it elicited a consistent response over whole period.
Concentration:
Pilot and mian experiment: 50% (w/v), 5% (w/v), 0.5% (w/v)
No. of animals per dose:
Pilot experiment – 3 females
Exposed groups – 15 females (5 animals in three groups)
Positive control group – 5 females
Negative control group – 5 females
Details on study design:
PRE-SCREEN TESTS:
The test substance was administered to three animals to assess possible systemic toxicity or high irritation of skin. One animal per dose group was used in pilot experiment
The pilot experiment was conducted under the conditions identical to the main study, except the assessment of lymph node proliferation. The appropriate suspensions of the test substance (50%, 5%, 0.5% w/v) was applied to three animals in volume 25 μL to the dorsum of each ear once a day morning for 3 consecutive days. The suspensions were prepared before the start of application by mixing on magnetic stirrer and then were still mixed during application. The application was performed very slowly by micropipette. The route of administration was the same as in the main study.

Both ears of each mouse were observed for erythema and scored and subsequently ear thickness was measured using digital thickness gauge. Body weight was recorded before application and prior to termination (Day 6).

According to the results of pilot experiment, the doses used in pilot experiment were chosen also for main study.


MAIN STUDY
The test substance was administered in the form of suspension in DAE 433.
The volume of the application form was constant at all groups of animals - 25 μL of the appropriate suspension to the dorsum of each ear once a day morning for 3 consecutive days. The application was performed very slowly by micropipette. Losses caused by draining from the ear must be minimized. The application forms of test substance (suspensions) were prepared immediately before administration.

Day 1: Open application of 25μL (in the morning, by pipette) of appropriate suspension of the test substance, the vehicle alone or the positive control to the dorsum of each ear.
Days 2 and 3: The application procedure repeated as carried out on day 1.
Days 4 and 5: No treatment.
Day 6: The weight of animals was recorded. Injection 250 μL of phosphate-buffered saline (PBS) containing 7.38 x105 Bq of 3H-methyl thymidine into all test and control mice via the tail vein.
Five hours later, the animals were killed.

In Vivo Examination
Mortality : During working hours the animals were checked for general health whenever other activities were performed – twice daily during the dosing period.

Clinical Observations
Clinical observation was performed twice daily during the dosing period. All changes in behaviour and health condition of animals were recorded. E.g.: piloerection, lacrimation, appearance of skin, fur and mucous membrane, ataxia, tremors, and convulsions, aggressiveness, change in grooming activity, marked change in activity level changes in frequency and intensity of breathing such as dyspnoea, gasping, and rales etc. Efforts were made to characterize onset and duration of signs observed. During clinical observations the examination of skin irritation at application site was carried out.

Body Weight
Individual body weights were measured using an electronic balance. Weighing was performed on the first day of treatment, before dosing, and on the day of necropsy before application of the radionuclide.

Necropsy
The third day after last administration (five hours after application of radionuclide), all test animals were sacrificed by prolonged ether narcosis.

Positive control substance(s):
other: DNCB (dinitrochlorobenzene, 0.5% (w/v) solution)
Statistics:
Statistical Evaluation of Data
For statistical calculations the software Statgraphic ® Centurion (version XV, USA) was used. Statistical evaluation of measured parameters was performed by applying the parametric test for testing whether all group samples originate from the same distribution and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) for two-group comparisons.

Statistical evaluation of the body weight
As the first step the test for normality (Shapiro-Wilk test) was used. Since the smallest P-value amongst the tests performed is greater than 0,05, we cannot reject the idea that data comes from a normal distribution with 95% confidence. For normally distributed data the variance check was performed (Levene´s test) to verify if standard deviations within each group are equal. One-Way ANOVA (probability level 0.05) was used to detect whether there are any significant differences amongst the means.

Statistical evaluation of ears weights
As the first step the test for normality (Shapiro-Wilk test) was used. Since the smallest P-value amongst the tests performed is lower than 0,05, we can reject the idea that data comes from a normal distribution with 95% confidence. The transformation of data was performed (Box-Cox transformation). Because the normal distributed distribution was not achieved after transformation of data then the non-parametric tests (Kruskal-Wallis Test, Mann-Whitney test) were used.

Statistical evaluation of DPM
Non-parametric two-group Mann-Whitney rank test (probability level 0.05) for two-group comparisons was used for statistical evaluation of the value of DPM.

Results and discussion

Positive control results:
All animals in the positive control group showed symptoms caused by the application of DNCB: hyperaemia of skin with well defined erythema (Table 8) on application site, clonospasm and increased response to stimuli. The positive control substance DNCB produced a positive LLNA response at the exposure level expected to give an increase in the Stimulation Index SI ≥ 3 over the negative control group (SI was > 13.35; Table 9). The positive control also elicited a reaction pattern with significant increase in ear weight (Table 10). These results demonstrate that the method performed in the conditions of the laboratory has sufficient reliability.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
5.5
Test group / Remarks:
0.5%
Parameter:
SI
Value:
4.1
Test group / Remarks:
5%
Parameter:
SI
Value:
6.63
Test group / Remarks:
50%
Parameter:
SI
Value:
13.35
Test group / Remarks:
DNCB
Cellular proliferation data / Observations:
See below

Any other information on results incl. tables

Results of Pilot Experiment

Body Weight of Animals

Individual body weight of animals before administration was similar. No reduction of body weight after treatment was recorded in all animals during the pilot experiment (Table 3).

 

Mortality, Clinical Observations, Individual Ear Weights and Results of Macroscopic Necropsy

During the pilot experiment, no clinical symptoms of systemic toxicity were observed. In treated animals, no erythema or skin reactions were observed. The thickness of ears in all animals during the pilot experimentwas unchanged. The weight of ears was slightly higher at the lowest dose level compared to other dose levels. Residues of the test substance on the ears were visible during the whole study so may have caused this weight increase (Tables 4 and 5). During the pathological examination, auricular lymph nodesenlargementwas detected in all animals.

Results of Main Study

Body Weight of Animals

Individual body weight of females before administration and before necropsy was relatively well balanced (result of random selection of animals into groups; Table 6). Very slightreduction of body weight (in tenths of grams) was recorded only in two animals at the middle dose level and in one animal at the lowest dose level. Body weight increment was calculated from values of day 6 just before necropsy and day 1 before first application (Table 7). Body weight increment was lower in treated group at the lowest dose level. 

 

Mortality, Clinical Observations

No animal died during the main study.No symptoms of toxicity and no erythema on application site were observed in all animals from the negative control group and all animals administered with the test substance.

Cell Proliferation

The SI for the test groups at all dose levels was above the threshold, with an SI of > 3 (Table 9). The SI for the test group at the highest dose level was 6.63, at the middle dose level was 4.10 and at the lowest dose level was 5.50. The DPM value at all dose levels wasstatistically significantly increased compared to the negative control.

 

Irritating Effect of the Test Substance

No erythema of the skin was observed during the clinical observation at any test substance dose level. Statistically significant increases in ear weight were recorded at the middle and highest dose levels (Table 10). Residues of the test substance on the ears were visible during the whole study so may have caused this weight increase.

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
Under the given test conditions, the animals exposed to the tested concentrations of N,N′-m-phenylenedimaleimide in the LLNA assay, elicited positive results in cell proliferation. The SI value at all dose levels was > 3 and the DPM values were statistically significantly increased. The test substance N,N′-m-phenylenedimaleimide, provided a positive sensitising response in the LLNA assay and is classified as Skin sensitiser Category 1A.
Executive summary:

In a dermal sensitization study (17-3) with N′-N-phenylenedimaleimide  (99.6%) in the vehicle DAE 433, young  female BALB/c mice (5/sex) were tested in the Local Lymph Node Assay. Mice were exposed to three concentrations of N′-N-phenylenedimaleimide (50 %, 5 %, 0.5 % (w/v)) for 3 consecutive days. The positive control was 0.5% (w/v) Dinitrochlorobenzene (DNCB). Primary proliferation of lymphocytes in the lymph node was evaluated using radioactive labelling of proliferating cells. The ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation Index (SI), was determined. The evaluation of ear weight was also performed.

There were no mortalities, adverse clinical observations or changes in body weight noted. The SI for the test group at the highest dose level was 6.63, at the middle dose level was 4.10 and at the lowest dose level was 5.50. The DPM value at all dose levels was statistically significantly increased compared to the negative control. The positive control DNCB gave the appropriate response (SI > 13.35). Statistically significant increase of ear weight was recorded at the middle and highest dose levels. Residues of the test substance on the ears were visible during whole study so may have caused this weight increase. The test substance did not cause skin irritation at any dose level. The test substance, N,N′-m-phenylenedimaleimide, provided a positive sensitising response in the LLNA assay and is classified as Skin sensitiser Category 1A.

This Local Lymph Node Assay in mice is acceptable and satisfies the guideline requirement for an OECD 429 study.