2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

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:

2018

Report date:

2018

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:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-3,6-dichloropyridine-2-carboxylic acid

EC Number:

604-721-7

Cas Number:

150114-71-9

Molecular formula:

C6H4Cl2N2O2

IUPAC Name:

4-amino-3,6-dichloropyridine-2-carboxylic acid

Test material form:

solid: particulate/powder

Details on test material:

TSN314667

Specific details on test material used for the study:

TSN314667
Batch: ENBK-169021-016
Purity: 95.9%

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Animal Breeding Facility, Jai Research Foundation
- Females (if applicable) nulliparous and non-pregnant: [yes/no/not specified] yes
- Age at study initiation: 10 to 12 weeks old at the initiation of treatment
- Weight at study initiation: Minimum: 19.9 g, Maximum: 25.2 g(for main study)
- Housing: Solid floor polypropylene mice cages (size: 290 mm x 220 mm x 140 mm). Each cage is fitted with a top grill having provision for keeping rodent pellet feed and water bottles. The bottom of the cages is layered with clean sterilized rice husk as the bedding material. Animals were group-housed during acclimatisation. On the days of test item application (Days 0, 1 and 2), the animals were housed in individual cages. From day 3 the animals were group-housed 5 mice/cage. On day 5 post administration of the radiolabelled material the animals were transferred to the metabolic cages. Tunnels were provided as enrichment material.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): at 22 ± 3 °C
- Humidity (%): 30-70%
- Air changes (per hr): minimum 15/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark, light hours being 06:00 - 18:00 hours

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Three groups were treated with aminopyralid TGAI at the concentrations of 10%, 25% and 75% (w/v) in dimethylformamide (DMF) for three consecutive days (days 0, 1 and 2) on the dorsum of both ears (25 µL/ear). One group served as vehicle control and was treated with dimethylformamide (DMF) and another group was treated with the positive control, HCA (α-hexylcinnamaldehyde) at a concentration of 25% (v/v) in dimethylformamide (DMF).

No. of animals per dose:

5/group

Details on study design:

A preliminary assay was carried out to assess the irritant potential of the test item through ear thickness measurement and erythema. In the preliminary assay, two groups of mice comprising 2 females per group were treated with aminopyralid TGAI (25 µL/ear) at 75% and 100% (w/v) aminopyralid TGAI in dimethylformamide (DMF) for three consecutive days (days 0, 1 and 2). Clinical observations were recorded daily during the experiment. Ear thickness was measured on days 0, 2 and 5 (at 72 hours post last application). Erythema was scored daily according to the procedure described in section 2.20. Body weight was recorded on days 0 and 5.

Main study
Animals were observed for local irritation and clinical reactions, and weighed on day 0 and day 5 prior treatment. On day 5, the uptake of 3H-methyl thymidine into the auricular (local) lymph nodes draining the site of chemical application was measured (animals sacrificed five hours post-administration) to assess the lymph node proliferative response.

Positive control substance(s):

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

Statistics:

The proliferative response of lymph node cells from the pooled lymph node of each mouse will be expressed as the number of radioactive disintegrations per minute (DPM) per mouse, subtracting out any background DPM. Final results will be expressed as the Stimulation Index (SI) which is calculated as a ratio (i.e., SI = mean DPM of test group divided by mean DPM of solvent/vehicle control group).

If SI found to be ≥ 3, a statistical analysis will be carried out for the assessment of the dose response relationship and pair-wise comparison between treatment and solvent/vehicle control group. All the parameters characterised by continuous data such as body weight and radioactive disintegrations per minute (DPM) will be subjected to appropriate statistical techniques.

Test item is regarded as a skin sensitiser in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-TdR incorporation in the lymph node cells of the test group lymph nodes relative to that recorded for solvent/vehicle control lymph nodes, as indicated by the SI. However, the magnitude of SI will not be the sole factor used in determining the biological significance of a skin sensitisation response. Factors that will be considered in evaluating the biological significance of a response include the results of the stimulation index, statistical analysis, dose-response relationship, chemical toxicity, solubility and consistency of the vehicle

Results and discussion

Positive control results:

In all mice treated with 25% HCA (v/v) in dimethylformamide (DMF), a local reaction consisting of erythema (score of 1) was observed from days 1 to 4.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.7

Test group / Remarks:

10%

Results 2

Key result

Parameter:

SI

Value:

0.97

Test group / Remarks:

25%

Results 3

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

75%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Not classified as skin sensitiser

Executive summary:

The Local Lymph Node Assay was conducted to evaluate the skin sensitisation potential of aminopyralid TGAI in CBA/J mice.

 

A preliminary assay was conducted to identify the appropriate test concentrations for the Local Lymph Node Assay main study. Two groups (2 female/group) were treated with aminopyralid TGAI (25 µL/ear) at 75% and 100% (w/v) aminopyralid TGAI in dimethylformamide (DMF). Ear thickness measurement revealed less than 25% increase and no erythema was observed at 75% (w/v) while greater than 25% increase and very slight erythema was observed at 100% (w/v) concentration in dimethylformamide.

 

In the main study, five groups of mice comprising 5 females per group were selected. Three groups were treated with aminopyralid TGAI at the concentrations of 10%, 25% and 75% (w/v) in dimethylformamide (DMF) for three consecutive days (days 0, 1 and 2) on the dorsum of both ears (25 µL/ear). One group served as vehicle control and was treated with dimethylformamide (DMF) and another group was treated with the positive control, HCA (α-hexylcinnamaldehyde) at a concentration of 25% (v/v) in dimethylformamide (DMF). Animals were observed for local irritation and clinical reactions, and weighed on day 0 and day 5 prior treatment. On day 5, the uptake of³H-methyl thymidine into the auricular (local) lymph nodes draining the site of chemical application was measured (animals sacrificed five hours post-administration) to assess the lymph node proliferative response.

 

There were no indications of systemic toxicity in aminopyralid TGAI treated animals. No erythema was observed at the site of application at all the tested concentrations. In all mice treated with 25% HCA (v/v) in dimethylformamide (DMF), a local reaction consisting of erythema (score of 1) was observed from days 1 to 4.

 

Stimulation indices (SI) for the groups treated with aminopyralid TGAI at the concentrations of 10%, 25% and 75% (w/v) in dimethylformamide (DMF) were0.70, 0.97 and 1.20, respectively. The positive response for HCA (SI = 6.34) was within the historical control data of the laboratory, confirming the reliability of the test procedure.

 

In conclusion, the SI obtained for aminopyralid TGAI at all tested concentrations showed a less than threefold increase when compared to the vehicle control value, indicating no skin sensitisation potential in the LLNA assay. Based on this study results, aminopyralid TGAI is not a dermal sensitiser.

 

Based on the results of this study, an indication of the classification foraminopyralid TGAI is as follows:

 

Globally Harmonized System of

Classification and Labelling of Chemicals (GHS 2017)       :   Not classified as skin sensitiser