Reaction products of 1,3-dihydroxybenzene, 4-amino-5-hydroxynaphthalene-2,7-disulphonic acid, 4-methoxy-1-aminobenzene, 2-(4-aminoanilino)-5-nitrobenzenesulphonic acid, sodium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

published in O.J. L 193 (2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

Reaction products of 1,3-dihydroxybenzene, 4-amino-5-hydroxynaphthalene-2,7-disulphonic acid, 4-methoxy-1-aminobenzene, 2-(4-aminoanilino)-5-nitrobenzenesulphonic acid, sodium salt

EC Number:

602-814-7

Cas Number:

12269-90-8

IUPAC Name:

Reaction products of 1,3-dihydroxybenzene, 4-amino-5-hydroxynaphthalene-2,7-disulphonic acid, 4-methoxy-1-aminobenzene, 2-(4-aminoanilino)-5-nitrobenzenesulphonic acid, sodium salt

Test material form:

solid: particulate/powder

Details on test material:

Batch No.: 7012/2007
Appearance: brown powder
Composition of test substance:
Main component:
2-(4-aminoanilino)-5-nitrobenzenesulphonic acid 5.5 area %

4-[[2,4-dihydroxy-3,5-bis[[4-(4-nitro-2-sulphonatophenyl)amino]phenyl]azo]phenyl]azo]-5-
hydroxynaphtalen-2,7-disulphonate, sodium salts 13.9 area %

4-[[2,4-dihydroxy-3-[(4-methoxyphenyl)azo]-5-[[4-[(4-nitro-2-sulphonatophenyl)amino]phenyl]azo]phenyl]azo]-
5-hydroxynaphthalene-2,7-disulphonate, sodium salts 42.4 area %

4-[[2,4-dihydroxy-3,5-bis[(4-methoxyphenyl)azo]phenyl]azo]-5-
hydroxynaphthalene-2,7-disulphonate, sodium salts 18.2 area %

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test substance name: Acid Brown 235
- Other name: Korostan Brown DGR
- Source and lot/batch No. of test material: 7012/2007
- Expiration date of the lot/batch: May 2018
- Appearance: dark brown powder

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
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 10 weeks (at start of dosing)
- Weight at study initiation: 14.78 - 18.67 g (at start of dosing), in pilot experiment 14.96 - 17.59 g
- Housing: macrolon cages with sterilized softwood shavings, monitored conditions, microbiologically defined background
- Diet (e.g. ad libitum): pelleted standard diet for experimental animals ad libitum (Altromin, manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany)
- Water (e.g. ad libitum): drinking tap water ad libitum
- Acclimation period: 7 days
- Indication of any skin lesions: yes

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

Study design: in vivo (LLNA)

Vehicle:

other: DAE 433

Remarks:

dimethylacetamide - acetone - ethanol 4 : 3 : 3

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:

5 animals per dose
(1 animal per dose at pilot experiment)

Details on study design:

CHEMICALS AND MEDIA
Vehicle (for test and positive control substances): DAE 433 (dimethylacetamide - acetone – ethanol 4 : 3 : 3)
PBS – Phosphate Buffered Saline (pH 7.4, Lot No.: SLBM3210V)
Trichloroacetic acid 5% (CAS: 76-03-9Lot No.: PP/2016/03000)
3H-methyl thymidine (Lot No.: 2183600, Activity: 1mCi/37MBq)

RELIABILITY CHECK
For the demonstration of test efficiency a group of animals treated with a substance with proved positive effect is included in each test (positive effect = Stimulation Index SI ≥ 3).
The substance DNCB (dinitrochlorobenzene, 0.5% (w/v) solution) was used as the positive control. A dose level for DNCB was determined during the verification of the method. The vehicle and dosage volume were the same as in treated groups. An application form was prepared on each day of administration by dissolving an appropriate amount of the positive control substance in the vehicle to obtain a concentration of 5 mg/mL. The solution was mixed for 5 minutes with a magnetic stirrer.

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 /mL)
- 0.5% (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.

MAIN STUDY
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.
Application:
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.
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 7.70 x10^5 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.

POST MORTEM INVESTIGATIONS
Ears Weights
Immediately 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 m-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°C for 18 h. 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).

DATA TREATMENT
Mean values and standard deviations of ears weight and incorporation of 3H-methyl thymidine were computed for the test substance groups and for the positive as well as the vehicle control group. 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.

Positive control substance(s):

other: 1-Chloro-2,4-dinitrobenzene (CAS: 97-00-7 )

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 can not 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:

The positive control substance, 1-chloro-2,4-dinitrobenzene, as a contact allergen (concentration 0.5% (w/v), elicited the expected reaction SI = 7.18.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.66

Test group / Remarks:

0.5 % w/v

Results 2

Key result

Parameter:

SI

Value:

0.85

Test group / Remarks:

5 % w/v

Results 3

Key result

Parameter:

SI

Value:

0.68

Test group / Remarks:

50 % w/v

Cellular proliferation data / Observations:

see Table 1

Any other information on results incl. tables

Table 1: Experimental results

Group	Negative Control	Positive Control	50%	5%	0.5%
Activity (DPM)	543.15	3432.01	251.85	459.97	420.27
	454.49	3478.48	492.58	511.31	397.86
	399.70	2935.84	279.31	422.65	220.94
	512.40	3692.77	299.19	316.27	250.45
	394.78	3008.23	248.34	249.20	220.85
mean	460.90	3309.47	314.25	391.88	302.07
SI	1.00	7.18	0.68	0.85	0.66
Ear weight (mg)	23.50	39.02	25.22	25.20	23.80

DPM - desintegration per minute

SI - stimulation index

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, the animals exposed to the test substance, Acid Brown 235, does not elicit sensitising response in LLNA assay. Negative results in cell proliferation revealed that the test substance Acid Brown 235 could not be a contact allergen in mice.
The test substance Acid Brown 235, provides negative sensitising response in LLNA assay.

Executive summary:

The test substance, Acid Brown 235, 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 Acid Brown 235 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, was determined. The evaluation of ear weight was performed for elimination of false positive findings with certain skin irritants.

The comparison of the Stimulation Indexes and values of DPM between the treated groups and the control group revealed that the test substance did not cause a significant increase in radioisotope incorporation into the DNA of dividing lymphocytes so the result of LLNA assay is negative.

Statistically significant increase of ear weight was recorded at the middle dose level. Residues of the test substance on the ears were visible during whole study so it could cause this weight increase. The test substance did not cause 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 middle 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 test conditions, the animals exposed to the test substance, Acid Brown 235, does not elicit sensitising response in LLNA assay. Negative results in cell proliferation revealed that the test substance Acid Brown 235 could not be a contact allergen in mice.

The test substance Acid Brown 235, provides negative sensitising response in LLNA assay.