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Administrative data

Description of key information

Not sensitising, OECD 429, EU Method B.42 and EPA OPPTS 870.2600, Huygevoort (2006)

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:
2 October 2006 - 13 December 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L 'Arbresle Cedex, France
- Age at study initiation: ca. 11 weeks
- Housing: animals were housed individually in labelled Macrolon cages furnished with sterilised sawdust as bedding material.
- Diet: pelleted rodent diet (SM R/M-Z from SNIFF® Spezialdiäten GmbH, Soets, Germany) ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 - 22.7 °C
- Humidity (%): 43 - 89 %
- Air changes (per hr): ca. 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark
Vehicle:
dimethylformamide
Concentration:
10, 25 and 50%
No. of animals per dose:
5
Details on study design:
In the main study, three groups of five experimental animals were treated with 25 µL test material concentrations of 10%, 25% or 50% on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (dimethyl formamide). Three days after the last exposure, all animals were injected with 0.25 mL 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index was subsequently calculated for each group.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The SI values calculated for the substance concentrations 5, 10 and 25% were 1.4, 2.2 and 6.2 respectively. An EC3 value of 13.1% was calculated using linear interpolation.

The calculated EC3 value was found to be in the acceptable range of 2 and 20%. The results of the 6 monthly HCA reliability checks of the recent years were 8.8, 5.5, 7.3 and 10.3%.

Based on the results, it was concluded that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.
Parameter:
SI
Remarks:
10%
Value:
1.4
Parameter:
SI
Remarks:
25%
Value:
2.2
Parameter:
SI
Remarks:
50%
Value:
6.2
Parameter:
EC3
Value:
13.1
Test group / Remarks:
Extrapolated
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 408, 727 and 582 respectively. The mean DPM/animal value for the vehicle control group was 290.

Skin reaction/ irritation
No skin reactions were observed in any of the animals examined.

Macroscopy of the auricular lymph nodes and surrounding area

All nodes of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted.

Body weights

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Toxicity and mortality

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.


Positive control

The six monthly reliability check with Hexylcinnamic aldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the study the test material was found not to be a skin sensitiser.
Executive summary:

The potential of the test material to cause skin sensitisation was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 429, EU Method B.42 and EPA OPPTS 870.2600.

Test material concentrations selected for the main study were based on the results of a preliminary study.

In the main study, three groups of five experimental animals were treated with test material concentrations of 10%, 25% or 50% on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (dimethyl formamide). Three days after the last exposure, all animals were injected with 0.25 mL 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index was subsequently calculated for each group.

No skin reactions were observed in any of the animals examined.

All nodes of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 408, 727 and 582 respectively.

The mean DPM/animal value for the vehicle control group was 290.

The SI values calculated for the substance concentrations 10%, 25% and 50% were 1.4, 2.5 and 2.0 respectively.

Since there was no indication that the test material could elicit an SI ≥ 3 when tested up to 50%, it was established that the EC3 value (if any) exceeds 50%.

The six monthly reliability check with Hexylcinnamic aldehyde indicates that the Local Lymph Node Assay as performed at the test laboratory is an appropriate model for testing for contact hypersensitivity.

Based on these results, the test material was not considered to be a skin sensitiser.


Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In the key study, the potential of the test material to cause skin sensitisation was investigated under GLP conditions and in accordance with the standardised guidelines OECD 429, EU Method B.42 and EPA OPPTS 870.2600.

Test material concentrations selected for the main study were based on the results of a preliminary study.

In the main study, three groups of five experimental animals were treated with test material concentrations of 10%, 25% or 50% on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (dimethyl formamide). Three days after the last exposure, all animals were injected with 0.25 mL 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index was subsequently calculated for each group.

No skin reactions were observed in any of the animals examined.

All nodes of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 408, 727 and 582 respectively.

The mean DPM/animal value for the vehicle control group was 290.

The SI values calculated for the substance concentrations 10%, 25% and 50% were 1.4, 2.5 and 2.0 respectively.

Since there was no indication that the test material could elicit an SI≥ 3 when tested up to 50%, it was established that the EC3 value (if any) exceeds 50%.

The six monthly reliability check with Hexylcinnamic aldehyde indicates that the Local Lymph Node Assay as performed at the test laboratory is an appropriate model for testing for contact hypersensitivity.

Based on these results, the test material was not considered to be a skin sensitiser.

In the supporting study, the potential of the test material to cause skin sensitisation was investigated in accordance with the standardised guideline OECD 406.

The dosage of test material selected for the main study was based on the results of a preliminary study.

In the main study, Hartley Guinea pigs were treated with a topical application containing 0.2 mL test material at 500 mg/mL on days 0, 7 and 14 for induction. The challenge procedure was performed on the untreated flank for 6 hours at a concentration of 500 mg/mL after a 14 day rest period. Reading were made approximately 24 and 48 hours after removal of the challenge patch. A negative control group was run concurrently, treated in the same manner with 0.2 mL test materuial a 500 mg/mL concentration, except that during the induction pahse the test material was omitted. 2,4 -dinitrochlorobenzene served as positive control.

Erythema and swelling were not observed in nagative ad treated animals after challenge. Various erythema and swelling in the skin of positive animals was observed at 24 and 48 hours after patch removal at challenge exposure.

Under the conditions of the study the proportion of animals sensitised to the positive control material was 100%, the proportion of animals sentised to the test material was 0%. The test material therefore does not fulfil the criteria for classification as a skin sensitiser according to European Regulation (EC) No 1272/2008.


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The substance does not fulfil the classification criteria for classification as a skin sensitiser according to European Regulation (EC) No 1272/2008.