Reaction products of diazotised 5,5’-methylenedianthranilic acid, coupled with resorcinol, subsequently coupled with diazotised 4-aminobenzene-1,3-disulfonic acid, chelated with copper sulphate, sodium salt

Endpoint summary

Description of key information

Skin sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Remarks:

an in vitro or in chemico skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From September 21 to October 10, 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Reliability of the source study is 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2010

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Breeding farm VELAZ s.r.o.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 10 weeks (at start of dosing)
- Weight at study initiation: 16.43 - 17.94 g (at start of dosing), in pilot experiment 16.16 – 17.99 g
- Housing: 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 is performed according internal SOP
- Water: ad libitum
- Acclimation period: 7 days

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

OTHER INFORMATION
- Prophylactic arrangement: Cleaning and disinfection of animal room was regularly performed.
- Cage identification: By cage number, study number and group specific colour.
- Animal identification: By felt tip marking (from 1 to 5 per each group and group specific colour)

Vehicle:

other: DAE 433 – mixture of 40 % dimethylacetamide, 30 % acetone and 30 % ethanol

Concentration:

50% (w/v) -500 mg/mL
5% (w/v) -50 mg /mL
0.5% (w/v) -5 mg /mL

No. of animals per dose:

Number of animals per group:
Pilot experiment – 3 female
Exposed groups – 15 females (5 animals in three groups)
Positive control group – 5 females
Negative control group – 5 females
Total: 28 animals

Details on study design:

PILOT EXPERIMENT
The test substance was administered to three animals to assess a possible systemic toxicity or high irritation of skin. One animal per dose group was used in pilot experiment.The test substance was administered in the form of suspension in DAE 433.
Concentrations of test substance in application form: 50% (w/v)500 mg/mL 5% (w/v)50 mg /mL0.5% (w/v)5 mg /mL
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.Concentrations of test substance in application forms: 50% (w/v), 500 mg/mL;5% (w/v), 50 mg /mL; 0.5% (w/v), 5 mg /mL

MAIN STUDY
Animal Check-in and Allocation to Groups
Animals were subjected to a clinical examination (health check) shortly after arrival. No clinical changes were recorded.
After acclimatization the animals have been randomly allocated to the dose groups and assigned animal numbers.

a. Experimental Schedule
Day 1:Open application of 25 μL (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 7.43 x105 Bq of 3H-methyl thymidine into all test and control mice via the tail vein. Five hours later, the animals were killed.

b. 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.

c. Post Mortem Investigations
Ears WeightsImmediately after scheduled humane kill of animals, the both ears were cut off and circular pieces from the apical area of each ear with a diameter of 8 mm (=0.5 cm2) were excised using a disposable punch and weighed together on analytical scales. Incorporation of 3H-methyl Thymidine

The tissue of both of the lymph nodes was prepared by gentle mechanical disaggregation through 100 μm-mesh nylon gauze with concomitant pooling in 1 mL PSB (Phosphate Buffered Saline). The pairs of lymph nodes were analysed separately for each animal per group. Cells were washed twice with an excess of PBS and precipitated with 5% trichloroacetic acid (TCA) at 4 oC for 18h. The pellets were re-suspended in 1 mL TCA and transferred to scintillation vials containing 10 mL of scintillation fluid. Incorporation of 3H-methyl thymidine was measured by β-scintillation counting (Beckmann LS 6500) as disintegrations per minute (DPM).

TREATMENT PREPARATION AND ADMINISTRATION: 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.

Positive control substance(s):

other: Dinitrochlorobenzene (DNCB)

Statistics:

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.The parametric tests were used for statistical evaluation of the body weight and the ears weights. As the first step the test for normality (Shapiro-Wilk test) was used. If the data were not normally distributed than the transformation of data was performed (Box-Cox transformation). If still the normal distributed distribution was not achieved then non-parametric tests (Kruskal-Wallis Test, Mann-Whitney test) were used. 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. In case of significant differences the post hoc statistical testing was performed (Fisher's least significant difference - LSD test).
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.

Positive control results:

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.

Parameter:

EC3

Value:

ca. 44.42

Parameter:

SI

Value:

ca. 3.12

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.

Mortality, Clinical Observations, Individual Ear Weightsand Results of Macroscopic Necropsy

During the pilot experiment no clinical symptoms of systemic toxicity were observed.

In treated animals no erythema and skin reaction were observed. The thickness of ears in all animals during pilot experimentwas without any changes.

Weight of ears was slightly higher at the highest and middle dose levels compared to lower dose level. Residues of the test substance on the ears were visible during whole study so it could cause this weight increase. During the pathological examination the auricular lymph nodes enlargement was not 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). Very slight reduction of body weight (in tenths of grams) was recorded only in one animal at the middle dose level.

Body weight increment was calculated from values of day 6 just before necropsy and day 1 before first application. Body weight increment was lower in treated group at the middle 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 by the test substance.

All animals in the positive control group showed symptoms caused by the application of DNCB: hyperaemia of skin with well defined erythema on application site, clonospasm and increased response to stimuli.

Irritating Effect of the Test Substance

In the positive control group, the weight of ear target was significantly increased against negative control group.

No erythema of skin was observed during the clinical observation at all dose levels. 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 it could cause this weight increase.

Interpretation of results:

other: Category 1B (indication of skin sensitising potential) based on CLP criteria

Conclusions:

Skin sensitising

Executive summary:

Method

The test substance was tested for the assessment of skin sensitisation potential with the murine local lymph node assay, according to the OECD Guideline 429.

In this study the contact allergenic potential of the test substance was evaluated after topical application to female BALB/c mice. Mice were exposed to three concentrations of test substance suspended in vehicle DAE 433 (mixture of 40 % dimethylacetamide, 30 % acetone and 30 % ethanol) for 3 consecutive days. 

In pilot experiment the following concentrationsof test substance in application forms were used: 50 %, 5 %, 0.5 % (w/v). According to the results of pilot experiment the same doses were confirmed for main study.  

Primary proliferation of lymphocytes in the lymph node draining the site of application was evaluated using radioactive labelling of proliferating cells.

The evaluation of ear weight was performed for elimination of false positive findings with certain skin irritants.

Results

The EC3 value calculated for the test item is 44.42 % with a SI = 3.12.

Conclusion

Skin sensitising

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

SKIN SENSITIZATION

Sub-category 1A

Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.

Specific criteria:

Local lymph node assay-EC3 value ≤ 2 %

Guinea pig maximisation test-≥ 30 % responding at ≤ 0,1 % intradermal induction dose or ≥ 60 % responding at > 0,1 % to ≤ 1 % intradermal induction dose

Buehler assay - ≥ 15 % responding at ≤ 0,2 % topical induction dose or ≥ 60 % responding at > 0,2 % to ≤ 20 % topical induction dose

Sub-category 1B

Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.

Local lymph node assay - EC3 value > 2 %

Guinea pig maximisation test- ≥ 30 % to < 60 % responding at > 0,1 % to ≤ 1 % intradermal induction dose or ≥ 30 % responding at > 1 % intradermal induction dose.

Buehler assay - ≥ 15 % to < 60 % responding at > 0,2 % to ≤ 20 % topical induction dose or ≥ 15 %responding at > 20 % topical induction dose.

Based on animal test (Local lymph node assay) results performed, according to the paragraph 3.4. of the CLP Regulation n. 1272/2008, the test substance is classified in Sub Category 1B, as the EC3 value is≤ 2% (EC3 = 44.42 %; SI = 3.12).