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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 May 2007 to 11 January 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
skin irritation: in vivo
Remarks:
skin irritation reported in LLNA study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
10 May 2007 to 11 January 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
This endpoint study record is part of a Weight of Evidence approach comprising a direct observation from RIPT studies, dermal irritation reported during developmental toxicity studies, irritation reported during an LLNA study and the results of an in vitro dermal adsorption study. The data sources are in agreement regarding dermal irritation and are sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
reference to other study
Reason / purpose:
reference to other study
Reason / purpose:
reference to other study
Reason / purpose:
reference to other study
Reason / purpose:
reference to other study
Reason / purpose:
reference to same study
Guideline:
other: equivalent to: OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Skin irritation observations reported
Principles of method if other than guideline:
A study equivalent to OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay) with no deviations was conducted. Application sight was observed for signs of irritation.
GLP compliance:
no
Remarks:
The study was not commissioned with compliance with REACH as a goal, rather the study was commissioned during early product development.
Specific details on test material used for the study:
Purity: 98%
Species:
mouse
Strain:
CBA
Remarks:
CBA:J
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: The Jackson Laboratory, Bar Harbor, ME.
- Females nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known: Animals were examined to ensure good health and suitability as test subjects for use in the study.
- Age at study initiation: appromixately 7-8 weeks
- Weight at study initiation: 17-21g
- Housing: housed individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week
- Indication of any skin lesions: Animals were examined to ensure good health and suitability as test subjects for use in the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25°C
- Humidity (%): 37-61%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 / 12

- IN-LIFE DATES: From: 07 June 2007 To: 11 June 2007
Type of coverage:
not specified
Preparation of test site:
not specified
Vehicle:
other: acetone/olive oil (4:1 v/v)
Amount / concentration applied:
11.3, 22.5, 33.8 and 45% in an acetone:olive oil (4:1) vehicle.
Highest concentration tested was 45% which was considered as the solubility limit in an acetone:olive oil 4:1 mixture.
Number of animals:
5 females/dose
Details on study design:
Five mice per group were dosed with the test substance at concentrations of 11.3, 22.5, 33.8 and 45% in an acetone:olive oil (4:1) vehicle. The mice were dosed once daily at approximately the same time for three consecutive days. All mice were observed twice daily on dosing days immediately prior to and approximately 4-6 hours after dosing, then once daily for moribundity, mortality, and any signs of toxicity and skin reactions.

Further study design relating to the investigation for skin sensitization is reported elsewhere within this dossier.
Irritation parameter:
other: irritation observation
Basis:
animal: all animals
Time point:
24 h
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Remarks on result:
not determinable because of methodological limitations
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Remarks on result:
not determinable because of methodological limitations
Irritant / corrosive response data:
There were no mortalities and no signs of systemic toxicity or irritation noted for the test and control animals.
Interpretation of results:
study cannot be used for classification
Remarks:
Weight of Evidence classification: GHS criteria not met
Conclusions:
No skin irritation was reported during the daily observations during a study equivalent to OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay).
Executive summary:

As part of a weight of evidence approach, the skin irritation observation reported during a study equivalent to OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay) with no deviations were considered. Five mice per group were dosed with the test substance at concentrations of 11.3, 22.5, 33.8 and 45% in an acetone:olive oil (4:1) vehicle following 1 week of acclimation period. The mice were dosed once daily at approximately the same time for three consecutive days. All mice were observed twice daily on dosing days immediately prior to and approximately 4-6 hours after dosing, then once daily for moribundity, mortality, and any signs of toxicity and skin reactions. The skin sensitization study continued to determine skin sensitization potential as described elsewhere within this dossier.

There were no mortalities and no signs of systemic toxicity or irritation noted for the test and control animals.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Deviations:
no
GLP compliance:
no
Remarks:
The study was not commissioned with compliance with REACH as a goal, rather the study was commissioned during early product development.
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot No. 08230AB
- Purity: 99.8%

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: The Jackson Laboratory, Bar Harbor, ME.
- Females nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known: Animals were examined to ensure good health and suitability as test subjects for use in the study.
- Age at study initiation: appromixately 7-8 weeks
- Weight at study initiation: 17-21g
- Housing: housed individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week
- Indication of any skin lesions: Animals were examined to ensure good health and suitability as test subjects for use in the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25°C
- Humidity (%): 37-61%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 / 12

- IN-LIFE DATES: From: 07 June 2007 To: 11 June 2007

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
11.3, 22.5, 33.8 and 45% in an acetone:olive oil (4:1) vehicle.
Highest concentration tested was 45% which was considered as the solubility limit in an acetone:olive oil 4:1 mixture.
No. of animals per dose:
5 females/dose
Details on study design:
Five mice per group were dosed with the test substance at concentrations of 11.3, 22.5, 33.8 and 45% in an acetone:olive oil (4:1) vehicle. The mice were dosed once daily at approximately the same time for three consecutive days. All mice were observed twice daily on dosing days immediately prior to and approximately 4-6 hours after dosing, then once daily for moribundity, mortality, and any signs of toxicity and skin reactions. Approximately 2 days after the last dose, mice were injected with the radio-label ([3H]-thymidine) and approximately 5 hours later, the auricular nodes were excised and single-cell suspensions were prepared. The cells were washed with phosphate-buffered saline to remove unbound radiolabel and then precipitated with 5% trichloroacetic acid. The total radiolabel incorporation in these precipitates was subsequently quantitated by liquid scintillation spectrometry. A single animal was dosed with less than the required volume of [3H]-thymidine (50% dose group) and this animal was not included in the analysis.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
other: 1-chloro-2,4-dinitrobenzene (DNCB)
Statistics:
The observed values of disintegrations per minutes (DPM) of acid-precipitated auricular lymph node cells were analysed using analysis of variance (ANOVA). Statistical analyses were performed using SigmaStat for windows software.

Results and discussion

Positive control results:
2.5 µg/mL of DNCB: Proliferative response - 13024.53 DPM/lymph node; Stimulation index – 19.39
42.5% of HCA: Proliferative response - 4260.59 DPM/lymph node; Stimulation index – 6.34

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1
Test group / Remarks:
vehicle control
Parameter:
SI
Value:
1.66
Test group / Remarks:
11.3%
Parameter:
SI
Value:
1.66
Test group / Remarks:
22.5%
Parameter:
SI
Value:
1.31
Test group / Remarks:
33.8%
Key result
Parameter:
SI
Value:
1.46
Test group / Remarks:
45%
Cellular proliferation data / Observations:
Evidence of induction of T-cell proliferation was not observed with the test substance, as the stimulation index was less than three at each of the test concentrations

There were no mortalities and no signs of systemic toxicity or irritation noted for the test and control animals.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
There was no evidence of induction of a lymphocyte proliferative response indicative of skin sensitisation to the test substance.
Executive summary:

Five mice per group were dosed with the test substance at concentrations of 11.3, 22.5, 33.8 and 45% in an acetone:olive oil (4:1) vehicle. The mice were dosed once daily at approximately the same time for three consecutive days. All mice were observed twice daily on dosing days immediately prior to and approximately 4-6 hours after dosing, then once daily for moribundity, mortality, and any signs of toxicity and skin reactions. Approximately 2 days after the last dose, mice were injected with the radio-label ([3H]-thymidine) and approximately 5 hours later, the auricular nodes were excised and single-cell suspensions were prepared. The cells were washed with phosphate-buffered saline to remove unbound radiolabel and then precipitated with 5% trichloroacetic acid. The total radiolabel incorporation in these precipitates was subsequently quantitated by liquid scintillation spectrometry. 

 

There were no mortalities and no signs of systemic toxicity or irritation noted for the test and control animals. Evidence of induction of T-cell proliferation was not observed with the test substance, as the stimulation index was less than three at each of the test concentrations.

The positive control groups had significantly increased DPM values and a mean stimulation index >3.

There was no evidence of induction of a lymphocyte proliferative response indicative of skin sensitisation to the test substance.