4-nitrophenyl acetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Acetic acid, 4-nitrophenyl ester

IUPAC Name:

Acetic acid, 4-nitrophenyl ester

Constituent 2

Reference substance name:

4-nitrophenyl acetate

EC Number:

212-593-5

EC Name:

4-nitrophenyl acetate

Cas Number:

830-03-5

Molecular formula:

C8H7NO4

IUPAC Name:

4-nitrophenyl acetate

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): 4-Nitrophenyl Acetate

- Physical state: solid
- Analytical purity: 95 % w/w
- Impurities (identity and concentrations):
Phenol <5.0 % (w/w)
4-nitrophenol <5.0 % (w/w)
Acetic acid <1.0 % (w/w)
Water <5.0 % (w/w)

- Lot/batch No.: 201409
- Expiration date of the lot/batch: unlisted

- Storage condition of test material: During the study the test substance was stored in closed
container in the dark at laboratory temperature.

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., Unětice 139, 252 62, Czech Republic, RČH CZ 21760118
- Age at study initiation: 8-10 weeks
- Weight at study initiation: at start of dosing: 18.12 - 20.40 g, in pilot exp: 16.62 - 17.15 g
- Housing: Monitored conditions, microbiologically defined background, according to internal SOP No.40
- Diet (e.g. ad libitum): Pelleted standard diet for experimental animals ad libitum (ST – 1, VELAS a.s., Hrabanov 535, 289 22 Lysá nad Labem, CZ801080-01). Microbiological control and content of nutrients is performed according internal SOP No. 72.
- Water (e.g. ad libitum): Drinking tap water ad libitum. Water quality corresponded to Regulation No. 252/2004 Czech Coll. Of Law, Health Ministry
- Acclimation period: 15 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C, permanently monitored
- Humidity (%): 30 – 70 %, permanently monitored
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0.5, 5, 50 % w/w

No. of animals per dose:

5

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.

Results and discussion

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, which was in congruence with the expected mode of action of a contact allergen. The positive control also elicited a reaction pattern with significant increase in ear weight.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

The value of SI for positive control group was increased. The SI was ≥ 3 (13.75) – the LLNA was efficient (see Table 8). The SI for the test group treated with the test substance at the lowest dose level is below the threshold, and stimulation index (SI) is < 3 (see Table 8). The SI for the test group treated by the test substance at the middle and the highest dose level was > 3 (50% - 5.75; 5% - 4.61) (see Table 8).

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The value of DPM for positive control group was increased. The value of DPM at all dose levels is statistically significantly increased compared to negative control and dose-effect relationship is manifested.

Any other information on results incl. tables

Table 8. Individual Activities and Calculated Values

Group (Anim.No.)	NC (1-5)	PC (6-10)	50% (11-15)	5% (16-20)	0.5% (21-25)
Activity (DPM)	221.68	4123.86	1577.20	1050.97	376.19
250.84	2794.10	1164.55	959.45	405.58
317.60	3113.31	1271.17	1203.07	390.97
230.10	3565.03	1438.63	1244.99	286.90
201.18	3202.12	1567.03	1176.33	332.14
mean	244.28	3359.68*	1403.72*	1126.96*	358.36*
SD	44.69	507.82	182.18	118.32	48.49
SI	1.00	13.75	5.75	4.61	1.47

Note:

* = statistically significant on probability level < 0.05 (Mann-Whitney test)

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: not specified

Conclusions:

Under the given test conditions, the animals exposed to the test substance, 4-Nitrophenyl Acetate, elicited sensitising response in LLNA assay. Positive results in cell proliferation revealed that the test substance 4-Nitrophenyl Acetate could be a contact allergen in mice.
The test substance 4-Nitrophenyl Acetate, provides positive sensitising response in LLNA assay.

Executive summary:

The test substance, 4-Nitrophenyl Acetate, 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 4-Nitrophenyl Acetate 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.

Comparison of Stimulation Indexes between treated groups and control vehicle group revealed that the test substance 4-Nitrophenyl Acetate caused a significant increase in radioisotope incorporation into the DNA of dividing lymphocytes of animals treated by the test substance at the concentrations of 50%, 5%. The Stimulation Index of the highest and the middle treated groups is > 3 (50% - 5.75; 5% - 4.61), and the value of disintegrations per minute (DPM) is significantly increased compared to negative control.

The Stimulation Index of the lowest treated group (0.5 % w/v) is < 3, however the value of DPM is significantly increased compared to negative control and overall a dose-effect relationship is observed.

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.

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, 4-Nitrophenyl Acetate, provides positive sensitising response in LLNA assay. The SI at the highest and the middle dose levels was > 3. Dose-response relationship and statistical significance was observed simultaneously.