2,4-dichloro-1-(trichloromethyl)benzene

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)

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)

Deviations:

no

Test guideline 2

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:

2,4-dichloro-1-(trichloromethyl)benzene

EC Number:

235-868-1

EC Name:

2,4-dichloro-1-(trichloromethyl)benzene

Cas Number:

13014-18-1

Molecular formula:

C7H3Cl5

IUPAC Name:

2,4-dichloro-1-(trichloromethyl)benzene

Test material form:

solid: crystalline

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca Ola Hsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90., Hungary
- Age at study initiation: young adult mice, 8-12 weeks (at start of the experiment)
- Weight at study initiation: 19.2 -24.2 g
- Housing: during the test: grouped caging (4 animals/cage)
- Cage type: Type II. polypropylene/polycarbonate
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice ad libitum
- Water: tap water ad libitum
- Acclimation period: 14 or 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity (%): 30 – 70
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

First Pre-test: 100 % (i.e. 1 g/mL), 50 % and 25 % (w/v) prepared with DMF
Second Pre-test: 10 %, 5 %, 2.5 % or 1 % (w/v) formulations in DMF

Main experiment: 10 %, 5 %, 2.5 % or 1 % (w/v) formulations in DMF

No. of animals per dose:

Pre-tests: 2 animals per test concentration
Main experiment: 4 animals per dose group

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: pre-experiments on solubility of the test item were performed following the recommendation of the relevant guidelines
- Irritation: Two dose range finding test were performed at concentrations of 100 % , 50 %, 25 % 10 %, 5 %, 2.5 % or 1 % (w/v) formulations in DMF. Eventual signs of local irritation were documented and the ear thickness was determined.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Proliferative response of the lymph node cells
- Criteria used to consider a positive response:
incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice (stimulation index SI ≥ 3 )


TREATMENT PREPARATION AND ADMINISTRATION:
- each mouse was topically treated with 25 µL of the appropriate formulations of the test item, of the positive control substance and of the vehicles
- the formulations were applied with a pipette, on the dorsal surface of each ear
- each animal was dosed once a day for three consecutive days (Days 1, 2 and 3)

Positive control substance(s):

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

Statistics:

Significance of the dose-response was evaluated by linear regression and regression curve analysis was also used for calculation of EC3 values.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks:

10 %

Value:

12.4

Test group / Remarks:

2 animals

Results 2

Parameter:

SI

Remarks:

5 %

Value:

8.1

Test group / Remarks:

2 animals

Results 3

Parameter:

SI

Remarks:

1%

Value:

2.9

Test group / Remarks:

2 animals

Any other information on results incl. tables

Pre-tests

In the first preliminary test mortality was observed in the 100 % (w/v) dose group (1/2 animals) on Day 5. Due to the significant systemic effect (sever decreased activity, hunched back posture, piloerection, tremor, lying on cage floor were observed) the other animal was considered moribund and humanely killed on the same day. In the 50 % (w/v) dose group the following systemic effects were observed: decreased activity (2/2 animals, from Day 4 to Day 6 for one animal or on Day 6 for the other animal) and piloerection (1/2 animals, on Days 4 and 5). Additionally the mean body weight decreased by 7 % in this dose group. In the 25 % (w/v) dose group no significant loss of body weight, but decreased activity (2/2 animals, on Day 6) was observed. In the 50 % and 25 % (w/v) dose groups scab (observed at the base of ears, 2/2 animals in both groups) and significantly (≥ 25 %) increased ear thickness (1/2 animals and 2/2 animals in the 50 % and 25 % (w/v) dose groups, respectively) with a maximum value of 55 % were also observed. These effects were considered as indication of an irritation effect although no significant erythema (scored as ≥ 3) was observed at the treatment site (ears) at the tested concentrations. Due to the observed significant adverse effects a subsequent preliminary test was performed with lower test concentrations.

In the second preliminary test no mortality was observed during the second preliminary test. No significant, treatment related effect on the body weights was observed in any treatment groups. No other signs of systemic toxicity were observed in any treatment group. As a local effect exfoliation (2/2 animals) and scab (1/2 animals) were observed in the 10 % (w/v) dose group, although no significant erythema (scored as ≥ 3) or significantly (≥ 25 %) increased ear thickness were observed at the treatment site (ears) at the tested concentrations. The observed effects at 100 %, 50 %, 25 % (w/v) concentrations were considered excessive.

Although the observations at 10 % (w/v) concentration (exfoliation and scab) could indicate a possible irritation, it was considered not excessive since no other obvious signs of irritation (erythema and/or increased ear thickness) were observed. Based on this 10 % (w/v) concentration was selected as the maximum concentration in the main test in order to test the highest concentration possible.

Main test

Body weight measurements:

No significant, treatment related effect on the body weights was observed in any treatment group. Clinical observations and signs of irritation: No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score 3) or any other local effect was observed in any treatment group.

Proliferation assay:

The lymph nodes in the positive control group were larger than the relevant control (AOO). Visually larger lymph nodes compared to the relevant control (DMF) were observed in the 10 % and 5 % (w/v) test item treated groups. Visual appearance of the lymph nodes was normal in the negative (vehicle) control groups (both AOO and DMF) and in the other test item treated groups (2.5 % and 1 %, w/v). Significant lymphoproliferation (SI ≥ 3) was observed for the test item at concentrations of 10 %, 5 % and 2.5 % (w/v). The corresponding stimulation index values were 12.4, 8.1, 3.6 and 2.9 at treatment concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively. The calculated EC3 value was 1.2 % (w/v) in this LLNA.

The positive control substance induced the appropriate stimulation compared to the control (SI = 22.2).

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the present Local Lymph Node Assay, the test item tested up to the maximum applicable (non-toxic, non-irritant) concentration of 10 % (w/v) was shown to have skin sensitization potential.

Based on the results obtained from testing the test item has to be classified as Skin Sens 1A, H317 according to Regulation (EC) No 1272/2008 (CLP).