Benzoic acid, 5-[(4-aminophenyl)azo]-2-hydroxy-, reaction products with 3-[(4-amino-3-methoxyphenyl)azo]benzenesulfonic acid and carbonic dichloride, sodium salts

Endpoint summary

Description of key information

Under the given test conditions, the animals exposed to the tested concentrations of the test substance Direct Yellow 44 in LLNA assay, elicited ambiguous results in cell proliferation revealed that the test substance Direct Yellow 44 could be a contact allergen in mice, but it should be proved by further testing. The test substance Direct Yellow 44, provides ambiguous 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:

09.11. – 22. 11. 2016

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., 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- Weight at study initiation: 16.11 - 18.88 g (at start of dosing), in pilot experiment 15.80 - 17.10 g- Housing: macrolon cages with sterilized softwood shavings, monitored conditions, microbiologically defined background, according to internal SOP No.40Cleaning 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: 7 days- Indication of any skin lesions: no clinical changes, all animals were examined during the acclimatisation periodENVIRONMENTAL 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 specifiedIN-LIFE DATES: from: 2.11.2016 (start of acclimatization)to: 22.11.2016 (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:50 % (w/v)500 mg/mL 5 % (w/v)50 mg /mL0.5% (w/v)5 mg /mL

No. of animals per dose:

Exposed groups – 15 females (5 animals in three groups)Positive control group – 5 femalesNegative control group – 5 females

Details on study design:

PRE-SCREEN TESTS:- Compound solubility:The appropriate suspensions of the test substance (50%, 5%, 0.5% 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. Losses caused by draining from the ear must be minimized.Both ears of each mouse were observed for erythema and scored and subsequently ear thickness was measured using digital thickness gauge- Irritation: no erythema and skin reaction- Systemic toxicity: no clinical symptoms of systemic toxicityNo reduction of body weight after treatment was recorded in all animals during the pilot experiment.- Ear thickness: The thickness of ears in all animals during pilot experiment was 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.- Erythema scores: no erythema and skin reactionThe route of administration was the same in the main study.MAIN STUDYNote: 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.ANIMAL ASSIGNMENT AND TREATMENTAnimals 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 proliferationPositive response: the stimulation index (SI) is ≥ 3 and the response increases in dose-related manner (dose-response relationship).Negative response: the stimulation index (SI) is < 3 without the dose-response relationshipAmbiguous 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 effectIf 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 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 weightAs 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 weightsAs 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 DPMNon-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:

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.Test validity criteria The test is considered valid, if the positive control substance DNCB produce a clear positive LLNA response and if the stimulation index SI is ≥ 3 over the negative control group.

Key result

Parameter:

SI

Value:

< 3

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATACell Proliferation resultsThe value of DPM and SI for positive control group was increased. The SI was ≥ 3 (7.18) – the LLNA was efficient.The SI for the test groups treated by the test substance at the all dose levels is below the threshold, stimulation index (SI) is < 3.The value of DPM at the highest dose level was statistically significantly increased and the value of DPM at the lowest dose level was statistically significantly decreased compared to negative control. The dose-effect relationship is manifested.DETAILS ON STIMULATION INDEX CALCULATIONStimulation 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 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.BODY WEIGHTSIndividual 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 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 lower in treated group at the highest dose level.

The values of the SI for the test groups treated by the test substance Direct Yellow 44 were < 3 and the value of DPM for the highest dose level was statistically significantly increased and the value of DPM for the lowest dose level was statistically significantly decreased. The dose-effect relationship was manifested.

The comparison of the Stimulation Indexes and values of DPM between the treated groups and the control group revealed that the test substance caused a statistically significant increase in radioisotope incorporation into the DNA of dividing lymphocytes so the result of LLNA assay is ambiguous.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the given test conditions, the animals exposed to the tested concentrations of the test substance Direct Yellow 44 in LLNA assay, elicited ambiguous results in cell proliferation revealed that the test substance Direct Yellow 44 could be a contact allergen in mice, but it should be proved by further testing. The test substance Direct Yellow 44, provides ambiguous sensitising response in LLNA assay.

Executive summary:

The test substance, Direct Yellow 44, 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 Method B.42 – Skin Sensitisation: Local Lymph Node Assay, Council Regulation (EC) No.640/2012, published in O.J. L 193, 2012 with respect to: OECD Test Guideline No. 429, Skin sensitisation: Local Lymph Node Assay, Adopted 22th July 2010

In this study the contact allergenic potential of Direct Yellow 44 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: 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 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 performed for elimination of false positive findings with certain skin irritants.

The SI for the test groups treated by the test substance at the all dose levels was below the threshold (SI < 3 ), but the measure of lymphocyte proliferation (DPM) at the highest dose level was statistically significantly increased and the value of DPM at the lowest dose level was statistically significantly decreased compared to negative control. The dose-effect relationship is manifested so the result of LLNA assay is ambiguous.

The test substance did not cause increase of ear weight or other indication of irritation to skin at all dose levels.

The animals exposed to the test substance at all doses showed no pathological and no other negative clinical symptoms of intoxication throughout the experiment, besides very slight weight loss in 1 animal at the highest dose level.

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 Direct Yellow 44, provides ambiguous sensitising response in LLNA assay. The SI at the all dose levels was < 3, but dose-effect relationship was manifested and the value of DPM at the highest dose level was statistically significantly increased compared to negative control.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Under the given test conditions, the animals exposed to the tested concentrations of the test substance Direct Yellow 44 in LLNA assay, elicited ambiguous results in cell proliferation revealed that the test substance Direct Yellow 44 could be a contact allergen in mice, but it should be proved by further testing. The test substance Direct Yellow 44, provides ambiguous sensitising response in LLNA assay.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available