Tripotassium [5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-4-sulpho-1-naphthyl)azo]naphthalene-2,7-disulphonato(5-)]cuprate(3-)

Endpoint summary

Description of key information

Under the given test conditions the animals exposed to the test substance, Reactive Blue 234, does not elicit sensitising response in LLNA assay. Negative results in cell proliferation revealed that the test substance Reactive Blue 234 could not be a contact allergen in mice. The test substance provides negative sensitising response in LLNA assay.

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:

05.08. – 18. 08. 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Council Regulation (EC) No.640/2012, published in O.J. L 193, 2012

Deviations:

no

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., Unětice 139, 252 62, Czech Republic, RČH CZ 21760118
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: 18.75 – 20.93 g
- Housing: macrolon cages with sterilized softwood shavings, monitored conditions, microbiologically defined background, according to internal SOP No.40
Cleaning and disinfection of animal room was regularly performed, as it is described in internal SOP No.10.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 21 days
- Indication of any skin lesions: no clinical changes, all animals were examined during the acclimatisation period

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
- Air changes (per hr): not specified

IN-LIFE DATES:
from: 15. 07. 2015 (acclimatization)
to: 17. 08. 2015 (necropsy)

Vehicle:

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

Concentration:

The test substance was administered in the form of suspension in DAE 433.
Concentrations of test substance in application form:
75% (w/v) 750 mg/mL
7.5% (w/v) 75 mg /mL
0.75% (w/v) 7.5 mg /mL

No. of animals per dose:

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:
- Compound solubility:
The appropriate suspensions of the test substance (75%, 7.5%, 0.75% w/v) was applied to three animals in volume 25 ul 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.
- Irritation: no erythema and skin reaction
- Systemic toxicity: no clinical symptoms of systemic toxicity
- Ear thickness measurements: without any changes, during the pathological examination the auricular lymph nodes enlargement was not detected
- Erythema scores: no erythema and skin reaction

MAIN STUDY
Note: 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.

ANIMAL ASSIGNMENT AND TREATMENT
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 (acc. to internal SOP No.42) and assigned animal numbers.

- Criteria used to consider a positive response:
Cell proliferation
Positive response: the stimulation index (SI) is ≥ 3, and the response increases in dose-related manner
Negative response: the stimulation index (SI) is < 3 without the dose – response relationship
Ambiguous response: the stimulation index is < 3, but the response increases in dose-related manner (dose–response relationship), and eventually statistical significance is observed.
Ear weight – irritation effect
If a statistically significant increase of ear weight together with clear concentration dependence of the effect is recorded, the inflammatory effect is considered as irritation induced by the test substance.

Positive result in cell proliferation reveals that the test substance could be a contact allergen. When positive irritation effect in animals is demonstrated simultaneously, the possibility can not be ruled out that the evaluation based on cell proliferation could be a false positive.


TREATMENT PREPARATION AND ADMINISTRATION:
the same as in the pre-screen test (see above)

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 at first by applying the non-parametric Kruskal-Wallis 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.

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 on application site, clonospasm and increased response to stimuli.

Key result

Parameter:

SI

Value:

< 3

Test group / Remarks:

1. (5 animals), 75% (w/v), 750 mg/mL

Key result

Parameter:

SI

Value:

< 3

Test group / Remarks:

2. (5 animals), 7.5% (w/v), 75 mg /mL

Key result

Parameter:

SI

Value:

< 3

Test group / Remarks:

3. (5 animals), 0.75% (w/v), 7.5 mg /mL

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The value of DPM and SI for positive control group was increased. The SI was ≥ 3 (9.91) – the LLNA was efficient.
The SI for the test groups treated with the test substance at all dose level is below the threshold, and stimulation index (SI) is < 3.
The value of DPM at all dose levels was not statistically significantly increased compared to negative control. On the contrary statistically significant decrease of DPM value at the lowest dose level was observed.

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index (for incorporation of 3H-methyl thymidine) was calculated by dividing mean values from exposed groups and the positive control group by the corresponding mean value of the vehicle control group. The index for the vehicle control group was set at 1 by definition.

CLINICAL OBSERVATIONS:
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.

BODY WEIGHTS
Slight reduction of body weight (in hundredth of grams) was recorded in one animal at the highest 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 highest at the lowest dose level. However, the weight fluctuations are common in mice.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the given test conditions the animals exposed to the test substance, Reactive Blue 234, does not elicit sensitising response in LLNA assay. Negative results in cell proliferation revealed that the test substance Reactive Blue 234 could not be a contact allergen in mice.
The test substance Reactive Blue 234 provides negative sensitising response in LLNA assay.

Executive summary:

The test substance, Reactive Blue 234, was tested for the assessment of skin sensitisation potential with the murine local lymph node assay. This study is a part of the test substance health hazard evaluation.

The Local Lymph Node Assay (LLNA) with the incorporation of 3H-methyl thymidine radionuclide was used. The testing was conducted according to the Method B.42 – Skin Sensitisation: Local Lymph Node Assay, Council Regulation (EC) No.640/2012, published in O.J. L 193, 2012.

In this study the contact allergenic potential of Reactive Blue 234 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 concentrations of test substance in application forms were used: 75 %, 7.5 %, 0.75 % (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 ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation Index, was determined. The evaluation of ear weight was performed for elimination of false positive findings with certain skin irritants.

Comparison of Stimulation Indexes between treated groups and control vehicle group revealed that the test substance Reactive Blue 234 did not cause a significant increase in radioisotope incorporation into the DNA of dividing lymphocytes of animals treated by the test substance at all concentrations. The Stimulation Index of the treated groups is < 3 and the value of disintegrations per minute (DPM) is not significantly increased compared to negative control.

The test substance did not cause increase of ear weight or other indication of irritation to skin at all dose levels nevertheless statistical significance was observed at the highest and the lowest dose levels.

With regard to the Confidential Test Substance Data Sheet from Sponsor and no dose-response relationship was not possible that irritation of skin was caused. Residues of the test substance on the ears were visible during whole study so it could cause this weight increase.

The animals exposed to the test substance at all doses showed no pathological and no other negative clinical symptoms of intoxication throughout the experiment.

The positive control item Dinitrochlorobenzene (DNCB) as a contact allergen (concentration 0.5% (w/v) elicited the expected reaction pattern with significant increase in Stimulation Index of cell proliferation and of ear weight. Appropriate performance of the assay in the test laboratory was then demonstrated.

Under the given test conditions, the test substance, Reactive Blue 234, provides negative sensitising response in LLNA assay. The SI at all dose levels was < 3.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available