Benzoic acid, 2,3,4,5-tetrachloro-6-cyano-, methyl ester, reaction products with 2-methyl-1,3-benzenediamine and sodium methoxide

Endpoint summary

Description of key information

study according to OECD TG 429 (GLP), mouse: not sensitising

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:

2014-02-19 to 2014-04-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

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)

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

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).

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.

Parameter:

SI

Value:

0.83

Test group / Remarks:

1% test item concentration

Parameter:

SI

Value:

1.03

Test group / Remarks:

2% test item concentration

Parameter:

SI

Value:

0.96

Test group / Remarks:

5% test item concentration

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.

Table 1:

Test item concentration	Group Calculation
	Mean DPM per animal (2 lymph nodes)*	SD	S.I.
Vehicle Control Group (DMSO)	2198.2	1226.3	1.00
1% test substance	1828.8	486.4	0.83
2% test substance	2265.8	783.4	1.03
5% test substance	2119.4	258.0	0.96

* Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals).

The EC3 value could not be calculated, since all S.I.´s were below the threshold value of 3.

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

To assess the skin sensitizing potential of the test substance, a Local Lymph Node Assay (LLNA) according to OECD TG 429 and GLP was conducted in mice (CBA/CaOlaHsd). Test item suspensions at different concentrations (1, 2 and 5 % w/w) were prepared using DMSO as a vehicle. 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). 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. 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%, 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.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008, as amended for the 13th time in Regulation (EU) 2018/1480.