2-Chloro-5-chloromethylpyridine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

Deviations:

yes

Remarks:

modifications: 1. non-radioactive alternative, measuring lymph node cell counts; 2. in addition, measurement of ear swelling and ear weight to discriminate the irritating potential from the sensitizing potential of the test substance

Principles of method if other than guideline:

Modified LLNA (IMDS: Integrated Model for the Differentiation of Skin Reactions). Modifications are authorised in the OECD TG 429. Information on validation of IMDS and scientific justification is given in: Vohr HW et al. Arch. Toxicol., 73, 501-509, (2000): Ehling G et al., Toxicology, 212, 60-68 and 69-75 (2005); Gamer AO et al., Regul. Toxicol. Pharmacol., 52, 290-298 (2008).

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:

-

EC Number:

454-800-3

EC Name:

-

Cas Number:

70258-18-3

Molecular formula:

C6H5Cl2N

IUPAC Name:

2-chloro-5-(chloromethyl)pyridine

Test material form:

solid

In vivo test system

Test animals

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 27-35 g
- Housing: 1 animal per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 40-70
- Air changes (per hr): ca. 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

0, 1, 3, 10 %

No. of animals per dose:

6

Details on study design:

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was formulated immediately before each administration in dimethyl sulfoxide (DMSO). The formulations were visually described as solutions. The stability of the test item in the vehicle was analytically verified for up to 2 hours. The test item in the formulation or the vehicle were applied epicutaneously onto the dorsal part of both ears of the animals. This  treatment was repeated on three consecutive days (d1, d2 and d3). The volume administered was 25 µL/ear/day. The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (d4). The lymphatic organs (the auricular lymph nodes) were then removed and transferred into physiological saline (PBS).

INVESTIGATIONS:
- weight of the lymph nodes (given as stimulation index compared to vehicle treated control group)
- cell counts of lymph nodes (given as stimulation index compared to vehicle treated control group; positive if greater or equal as 1.4 stimulation index )
Stimulation indices were calculated by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones.
- ear swelling (given in 0.01 mm and as index; positive, if 1.10 was exceeded)
- ear weight (given in mg / 8 mm diameter piece and as index)

Positive control substance(s):

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

Statistics:

When it was statistically reasonable, the values from treated groups were compared with those from the control groups by the Mann-Whitney or the Wilcoxon significance test (Rank Sum Test or One Way ANOVA or Kruskal-Wallis ANOVA) at significance levels of 5% (one-tailed for LLNA/IMDS or PNLA (larger)). Outlying values in the LN weights were eliminated at a probability level of 99% by Nalimov's method. In addition, for the LLNA/IMDS the smallest significant differences in the means were calculated
by Scheffe's method, which according to Sachs can be used for both equal and unequal sample sizes.

Results and discussion

Positive control results:

Alpha hexyl cinnamic aldehyde, checked in regular intervals, showed a clear sensitizing potential in the local lymph node assay (IMDS).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks:

cell count index (%)

Value:

1.3

Test group / Remarks:

1 %

Remarks on result:

other: The “positive level”, which is 1.4 for cell count indices, was not exceeded in the low dose group (= NOEL).

Results 2

Parameter:

SI

Remarks:

cell count index (%)

Value:

1.6

Test group / Remarks:

3 %

Remarks on result:

other: The “positive level”, which is 1.4 for cell count indices, has been exceeded in the mid dose group. This increase is of statistical significance.

Results 3

Parameter:

SI

Remarks:

cell count index (%)

Value:

1.74

Test group / Remarks:

10 %

Remarks on result:

other: The “positive level”, which is 1.4 for cell count indices, has been exceeded in the high dose group. This increase is of statistical significance.

Any other information on results incl. tables

Table 1: Summary of the LLNA results (means of 6 animals per group)

Parameter investigated	Vehicle control	Test item 1 %	Test item 3 %	Test item 10 %
Stimulation index: weight of draining lymph nodes	1.00	1.23	1.50 *	1.54 *
Stimulation index: cell count in draining lymph nodes	1.00	1.30	1.60 *	1.74 *
Ear swelling in 0.01 mm on day 4 (index)	19.92 (1.00)	20.33 (1.02)	20.42 (1.03)	22.67 (1.14) *
Ear weight in mg / 8 mm diameter punch on day 4 (index)	14.59 (1.00)	14.40 (0.99)	14.21 (0.97)	14.93 (1.02)

* statistically significant increase (p ≤ 0.05)

The results show that the test item has a sensitising potential in mice after dermal application. Compared to vehicle treated animals there was a clear increase regarding the weights of the draining lymph nodes and a clear increase in the cell counts in the mid and highest dose group. These changes were of statistical significance and the "positive level" of index 1.4 was exceeded.

The "positive level" of ear swelling which is 2 x 10 -2 mm increase, i.e. about 10% of the control values, has been exceeded in the highest dose group. An increase in this parameter would point to an acute irritating (inflammatory) response. However, such an irritating property can also be combined with a moderate skin sensitizing potential of a test compound.

The body weights of the animals were not affected by any treatment.

Taken together, a specific activation of the cells of the immune system via dermal route was determined after application of 3 and 10 % 2-chlor- 5-chlormethylpyridin by the method used. Thus, 2-chlor-5-chlormethylpyridin has a moderate sensitising potential in mice. Therefore, the concentration of 1 % turned out to be the NOEL for the parameters investigated in this study.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

Migrated information

Executive summary:

2-Chlor-5-chlormethylpyridin was investigated in the modified local lymph node assay (LLNA-IMDS) on female mice according to OECD TG 429. Concentrations of 0 (vehicle control), 1, 3 and 10 % formulated in dimethyl sulphoxide (DMSO) were tested. Compared to vehicle treated animals there were increases regarding the cell counts (indices of 1.30, 1.60 and 1.74 %, resp.) and the weights of the draining lymph nodes (indices of 1.23, 1.50 and 1.54 %, resp.) in all dose groups. These changes were of statistical significance in the mid and highest dose group and the "positive level" of index 1.4 was exceeded. The "positive level" of ear swelling which is 2 x 10 -2 mm increase, i.e. about 10 % of the control values, has been exceeded in the highest dose group. An increase in this parameter would point to an acute irritating (inflammatory) response. However, such an irritating property can also be combined with a moderate skin sensitizing potential of a test compound.

Taken together, a specific activation of the cells of the immune system via dermal route was determined after application of 3 and 10 % 2-chlor- 5-chlormethylpyridin by the method used. Thus, 2-chlor-5-chlormethylpyridin has a moderate sensitising potential in mice. Therefore, the concentration of 1 % turned out to be the NOEL for the parameters investigated in this study.