Benzoic acid, 2,3,4,5-tetrachloro-6-cyano-, methyl ester, reaction products with 4-[2-(4-aminophenyl)diazenyl]-3-methylbenzenamine and methanol sodium salt (1:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-02-19 to 2014-04-01

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

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:

Mixture of octachloro, monomethoxy-heptachloro and bismethoxy-hexachloro derivatives of 3,3'-[(2-methyl-1,3-phenylene)diimino]bis[2,3-dihydro-1H-isoindol-1-one]

EC Number:

600-735-2

Cas Number:

106276-79-3

Molecular formula:

Mixture of C23H8Cl8N4O2, C24H11Cl7N4O3, C25H14Cl6N4O4

IUPAC Name:

Mixture of octachloro, monomethoxy-heptachloro and bismethoxy-hexachloro derivatives of 3,3'-[(2-methyl-1,3-phenylene)diimino]bis[2,3-dihydro-1H-isoindol-1-one]

Test material form:

solid: nanoform

Details on test material:

- Analytical purity: 100 % UVCB
- Storage condition of test material: At room temperature, protected from light
- Physical state/appearance: yellow solid

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V. Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: first pretest: 9-10 weeks, second pretest: 8-9 weeks, main study: 8-9 weeks
- Weight at study initiation: mean: 20.1 g (18.5 - 22.4 g)
- Housing: group
- Diet: 2018C Teklad Global 18 % protein rodent diet, ad libitum
- Water: tap water ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2 °C
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

1, 2 and 5 % in DMSO

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Irritation: no visible erythema, redness was not determined due to inherent colour of test item

MAIN STUDY
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
- the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation).
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as a statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05).

Results and discussion

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1, v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in October 2013.

In vivo (LLNA)

Results

Results 1

Parameter:

other: disintegrations per minute (DPM)

Value:

2 198.2

Test group / Remarks:

control group / mean

Remarks on result:

other:

Remarks:

DPM values per animal (2 lymph nodes): control group: 4242, 1672, 2419, 1356, 1302

Results 2

Parameter:

other: disintegrations per minute (DPM)

Value:

1 828.8

Test group / Remarks:

test group 1 / mean

Remarks on result:

other:

Remarks:

DPM values per animal (2 lymph nodes): test group 1 (1 % test item concentration): 1331, 2032, 2411, 2057, 1313

Results 3

Parameter:

other: disintegrations per minute (DPM)

Value:

2 265.8

Test group / Remarks:

test group 2 / mean

Remarks on result:

other:

Remarks:

DPM values per animal (2 lymph nodes): test group 2 (2 % test item concentration): 1264, 2977, 1651, 2999, 2438

Results 4

Parameter:

other: disintegrations per minute (DPM)

Value:

2 119.4

Test group / Remarks:

test group 3 / mean

Remarks on result:

other:

Remarks:

DPM values per animal (2 lymph nodes): test group 3 (5 % test item concentration): 2242, 2389, 2028, 1719, 2219

Results 5

Parameter:

SI

Value:

0.62

Test group / Remarks:

test group 1 / mean

Remarks on result:

other:

Remarks:

test group 1 (1 % test item concentration): 0.6, 0.9, 1.1, 0.9, 0.6

Results 6

Parameter:

SI

Value:

1.06

Test group / Remarks:

test group 2 / mean

Remarks on result:

other:

Remarks:

test group 2 (2 % test item concentration): 0.6, 1.4, 0.8, 1.4, 1.1

Results 7

Parameter:

SI

Value:

0.96

Test group / Remarks:

test group 3 / mean

Remarks on result:

other:

Remarks:

test group 3 (5 % test item concentration): 1.0, 1.1, 0.9, 0.8, 1.0

Any other information on results incl. tables

A statistically significant or biologically relevant increase in DPM values, lymph node weights and lymph node cell counts was not observed in any treated group in comparison to the vehicle control group.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was not a skin sensitiser under the test conditions of this study

Executive summary:

In this study the test item was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. Test item suspension at different concentrations was prepared in the vehicle DMSO.

The local lymph node assay is recommended by international test guidelines (e.g., OECD) as an animal test for predicting skin sensitisation in humans and provides a rational basis for risk assessment. The basic principle underlying the LLNA is that sensitisers induce a primary proliferation of lymphocytes in the lymph node draining the application site. The ratio of proliferation in test item treated groups compared to that in vehicle controls is termed the Stimulation Index (S.I.). Radioactive labeling is used to measure cell proliferations.

For this purpose a local lymph node assay was performed using test item concentrations of 1, 2 and 5% (w/w). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation (as determined by two pre-experiments).

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. On day 6, all test item treated animals showed scabby ear skin. A possible erythema of the ear skin could not be evaluated due to the colour of the test item. A statistically significant increase in ear weights was observed in the high dose group in comparison to the vehicle control group (p<0.05). For BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. The index determined for the test item treated high dose group slightly exceeded this threshold (index 1.14). However, this was considered to be not biologically relevant, as the observed increase did not exceed the threshold value of 25% for excessive local skin irritation mentioned in OECD guideline 429. Nevertheless, the increase in ear weights indicates a slight irritant property of the test item.

A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices (S.I.) of 0.83, 1.03 and 0.96 were determined with the test item at concentrations of 1, 2 and 5% (w/w) in DMSO, respectively.

A statistically significant or biologically relevant increase in DPM values, lymph node weights and lymph node cell counts was not observed in any treated group in comparison to the vehicle control group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was not exceeded in any dose group.

Thus, the test item was not a skin sensitiser under the test conditions of this study.