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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:
From February 20, 2017 to March 22, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): Phosphoric acid, mono- and bis (C16-20 branched and linear alkyl) esters
- Batch: CH 200529/001
- Composition: UVCB
- Appearance: Lightly yellow liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
Source: Janvier, Le Genest-Saint-Isle, France.
Number of animals: 20 females (nulliparous and non-pregnant), five females per group (main study only).
Age and body weight: young adult animals (approx. 8-9 weeks old) were selected.
Body weight variation was within +/- 20% of the sex mean.
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle.
Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Water: free access to tap water.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 5, 10 and 25% w/w
No. of animals per dose:
5
Details on study design:
The test substance concentrations selected for the main study were based on the results of a pre-screen test. In the main study, three experimental groups of five female CBA/J mice were treated with test substances concentrations of 5, 10 or 25% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. 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 (SI) was subsequently calculated for each group.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
SI and EC3 values

Results and discussion

Positive control results:
The SI values calculated for the positive control concentrations 5, 10 and 25% were 1.4, 2.4 and 4.3, respectively. An EC3 value of 14.7% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 13.4, 14.1, 17.3, 9.8, 17.8% and 18.0%.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
>= 1.4 - <= 6.1
Test group / Remarks:
test substance concentrations of 5, 10 and 25% w/w
Key result
Parameter:
EC3
Value:
9.7
Test group / Remarks:
test substance groups
Cellular proliferation data / Observations:
- No erythema was observed in any of the animals, scaliness was noted for all animals treated at 25% and some animals treated at 10% between Days 4 and 6.
- The majority of auricular lymph nodes were considered normal in size, except for one node in one animal treated at 25%, which was considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.
- Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 644, 1406 and 2811 DPM, respectively. The mean DPM/animal value for the vehicle control group was 460 DPM. The SI values calculated for the test substance concentrations 5, 10 and 25% were 1.4, 3.1 and 6.1, respectively. These results indicate that the test substance could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 9.7% was calculated.

- No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Based on these results:
- according to the recommendations made in the test guidelines (including all amendments), the test substance would be regarded as skin sensitizer.
- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), the test substance should be classified as skin sensitizer (Category 1B).
- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), the test substance should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.

Applicant's summary and conclusion

Interpretation of results:
other: CLP criteria met
Remarks:
Category 1B
Conclusions:
Under the study conditions, the test substance was considered to a skin sensitizer (LLNA).
Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance according to OECD Guideline 429, EU Method B42 and US EPA OPPTS 870.2600 (LLNA), in compliance with GLP. The test substance concentrations selected for the main study were based on the results of a pre-screen test. In the main study, three experimental groups of five female CBA/J mice were treated with test substances concentrations of 5, 10 or 25% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v)). A positive control group (alpha-hexylcinnamaldehyde) was also included in the experiment. Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. 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 (SI) was subsequently calculated for each group. Mortality/viability, body weights, clinical signs, and irritation (and other local effects) were recorded as well. No erythema was observed in any of the animals, scaliness was noted for all animals treated at 25% and some animals treated at 10% between Days 4 and 6. The majority of auricular lymph nodes were considered normal in size, except for one node in one animal treated at 25%, which was considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 644, 1406 and 2811 DPM, respectively. The mean DPM/animal value for the vehicle control group was 460 DPM. The SI values calculated for the test substance concentrations 5, 10 and 25% were 1.4, 3.1 and 6.1, respectively. These results indicate that the test substance could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 9.7% was calculated. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Under the study conditions and based on the EC3 value, the test substance was considered to be a moderate skin sensitizer (van Sas, 2017).