2-Thiophenecarboxylic acid, 5-chloro-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-10-24 to 2005-12-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

24 April 2002

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

480-060-6

EC Name:

-

Cas Number:

24065-33-6

Molecular formula:

Hill formula: C5H3ClO2S CAS formula: C5H3ClO2S

IUPAC Name:

5-chlorothiophene-2-carboxylic acid

In vivo test system

Test animals

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkehnann/Nederland, Kreuzelweg 53, N1.-5960AD Horst, Netherland
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation: 25-34 g
- Housing: During the adaptation period the animals were housed in conventional Makrolon® type II cages, up to 8 mice and during the study period in type II cages, one animal being kept in each cage. Low-dust wood granulate from J. Rettenmaier & Söhne Füllstoff-Fabriken, 73494 Rosenberg, Germany, was used as bedding.
- Diet (e.g. ad libitum): ad libitum, PROVIMI KLIBA SA 3883 maintenance diet for rats and mice (from Provimi Kliba SA, CH-4303 Kaiseraugst)
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: at least 7 days


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

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 3, 10, and 30%

No. of animals per dose:

6

Details on study design:

MAIN STUDY:

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The so-called stimulation (or LLN-) index is calculated by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones. Thus, in case of no stimulating effect the index is always about 1.00 (+/- standard deviation), and the indices of vehicle treated animals are set to 1.00 (+/- standard deviation).

TREATMENT PREPARATION AND ADMINISTRATION: The test item was formulated once at day 1 of the study in DMF. The formulations were visually described as solutions. The test item in the formulation and 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.
Based on our experiences with this test system and the known properties of the test item the following concentrations were used: 0%, 3%, 10% and 30%.

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 group (s; vehicle) by a one-way analysis ofvariance (ANOVA) when the variances are considered homogeneous according to a homogeneity testing like Cochran‘s test. Alternatively, if the variances are considered to be heterogenous (p ≤ 0.05), a non-parametric Kruskal-Wallis test has been used (Kruskal-Wallis ANOVA) at significance levels of 5% . Two sided multiple test procedures were done according to Dunnett or Bonferroni-Holm, respectively. 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. In this method of statistical processing of measurements a large number of comparisons is made, and as a result of the multiple tests the overall probability of error is considerably greater than the p values suggest (increased number of false-positive results). On the other hand, the known methods of adjusting p values lead to an excessive increase in the number of false negatives. In view of these problems the biological and toxicological relevance is also taken into consideration in the evaluation ofstatistical significance. For this reason, in the case of indices only the standard deviations between groups and difference analysis of the mean values (Scheffe‘s method) were used in the evaluation of the biological relevance.

Results and discussion

Positive control results:

The Local Lymph Node Assay Test methodology was checked for reliability in a test on female NMRI mice using Alpha Hexyl Cinnamic Aldehyde formulated in different vehicles (PEG 400, DAE 433, DMF, MEK, Aceton/Olive Oil (4:1) and Cremophor EL/ physiological saline solution 2% v/v) at the concentrations of 3%, 10% and 30%.
The sensitivity as well as the reliability ofthe experimental technique is thus confirmed by this study. A similar check is done in regular intervals using one of the above vehicles confirming the reliability of the method.
The Local Lymph Node Assay Test methodology was checked for reliability in a test on female NMRI mice using Alpha Hexyl Cimramic Aldehyde formulated in the vehicle Acetone/Olive Oil (4:1) at the concentrations of 3%, 10% and 30%.
The results show that the test item has a clear sensitizing potential.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks:

cell count

Value:

1.18

Variability:

+/- 41.31

Test group / Remarks:

30%

Results 2

Key result

Parameter:

SI

Remarks:

cell count

Value:

1.11

Variability:

+/- 41.40

Test group / Remarks:

10%

Results 3

Key result

Parameter:

SI

Remarks:

cell count

Value:

0.9

Variability:

+/- 30.78

Test group / Remarks:

3%

Results 4

Key result

Parameter:

SI

Remarks:

ear weight

Value:

1.21

Variability:

+/- 31.57

Test group / Remarks:

30%

Results 5

Key result

Parameter:

SI

Remarks:

ear weight

Value:

1.18

Variability:

+/- 35.47

Test group / Remarks:

10%

Results 6

Key result

Parameter:

SI

Remarks:

ear weight

Value:

0.82

Variability:

+/- 29.01

Test group / Remarks:

3%

Cellular proliferation data / Observations:

CLINICAL OBSERVATIONS: None

BODY WEIGHTS: None

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness).
A significant increase compared to vehicle treated animals regarding ear swelling and ear weight was detected in the mid dose group.
It could be concluded that the test item has an irritating potential after application of a 10% concentration in DMF.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The study indicates that the LLNA/IMDS does point to a non-specific (irritating) immuno-stimulating potential of the test item. A significant increase compared to vehicle treated animals regarding ear swelling and ear weight was detected in the mid dose group. It could be concluded that the test item has an irritating potential after application of a 10% concentration in DMF. Taken together, no antigen specific activation ofthe cells ofthe immune system via dermal route was determined after application ofup to and including 30% 5-Chlorthiophen-2-carbonsäure by the method used. Therefore, the concentration of 30% turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization. By this a skin sensitizing potential has not been detected.

Executive summary:

In a local lymph node assay according to OECD guideline 429 (2002) 5-Chlorthiophen-2-carbonsäure dissolved in dimethylformamide was assessed for its possible contact allergenic potential using test item concentrations of 3, 10 and 30%.

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) between 0.82 and 1.21  were determined with the test item at concentrations of 3, 10, and 30 % in dimethylformamide, respectively. These results indicate that the test substance could not elicit an SI ≥ 3 and 5-Chlorthiophen-2-carbonsäure is therefore not regarded as skin sensitiser under the conditions of this study.

Based on these results 5-Chlorthiophen-2-carbonsäure is not classified as a sensitizer based on CLP, EU GHS (Regulation (EC) No 1272/2008).