Registration Dossier

Administrative data

Description of key information

Skin sensitisation (GPMT): Not sensitising (OECD 406, GLP)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25-7-16 to 26-8-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD 406 and GLP compliant
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The Murine Local Lymph Node Assay (LLNA) is the preferred method for new in vivo testing but there are certain limitations that may necessitate the use of TG 406, e.g. false positive findings with certain skin irritants.

The test substance Vinyltoluene is a skin irritant, for this reason we preferred the use of the guinea pig test.
Specific details on test material used for the study:
- Name of test material (as cited in study report): Vinyltoluene
- Physical state: liquid
- Analytical purity: ≥99.2%
- Isomers composition:
3-methylstyrene (CAS No. 100-80-1): 64.3 % w/w
4-methylstyrene (CAS No. 622-97-9): 35.7 % w/w
- Lot/batch No.:201605
- Expiration date of the lot/batch: May 2017
Species:
guinea pig
Strain:
other: BFA
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Luboš Sobota, U nádraží 901, 289 03 Městec Králové, Czech Republic, monitored breeding farm, RČH CZ 21760039
- Weight at study initiation: 321.973 ± 34.094 (test group); 331.831 ± 34.849 (control group)
- Housing: Bedding - Shavings of soft wood
- Diet: Pelleted standard diet for guinea pigs ad libitum (Altromin Spezialfutter GmbH & Co., KG, Im Seelenkamp 20, D-32791, Lage, Germany).
- Water: Drinking tap water ad libitum (supplemented by ascorbic acid) ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3C
- Humidity (%): 30 – 70 %
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle: 6am-6pm/6pm-6am
Route:
intradermal
Vehicle:
olive oil
Concentration / amount:
Induction - Intradermal injections: 20% in olive oil/1:1 mixture (v/v) FCA/physiological saline
Induction - Topical application: 100%
Challenge - Topical application : 70% in olive oil
Route:
epicutaneous, occlusive
Vehicle:
olive oil
Concentration / amount:
Induction - Intradermal injections: 20% in olive oil/1:1 mixture (v/v) FCA/physiological saline
Induction - Topical application: 100%
Challenge - Topical application : 70% in olive oil
No. of animals per dose:
20 animals (11 males and 9 females) were used in the treatment group
10 animals (6 males + 4 females) in the control group
Details on study design:
RANGE FINDING TESTS: Three animals (females) were used in the pilot experiment.
The water solubility of the test substance is 89 mg·L-1 (25 degree Celsius). At first the test substance concentrations were prepared in physiological saline for intradermal injections. This application form seemed to be visually inhomogeneous, for this reason the test substance concentrations were prepared in olive oil for all phases of the experiment. This application form seemed to be visually homogeneous.

Induction - Intradermal injections
50% test substance in olive oil: intense erythema of the skin, abscess
25% test substance in olive oil: moderate erythema of the skin, abscess
20% test substance in olive oil: moderate erythema of the skin
10% test substance in olive oil: discrete erythema
5% test substance in olive oil: non-irritant dose

Note: The 20% concentration of test substance in olive oil was chosen for the main study for Induction – Intradermal injections.

Induction - Topical application and Challenge - Topical application
100% test substance: discrete erythema
90% test substance in olive oil: discrete erythema
70% test substance in olive oil: non-irritant dose
50% test substance in olive oil: non-irritant dose
30% test substance in olive oil: non-irritant dose
20% test substance in olive oil: non-irritant dose
10% test substance in olive oil: non-irritant dose


MAIN STUDY:
Application -Induction: Intradermal Injections
Day 0 – treated group
Three pairs of intradermal injections of 0.1 mL volume were given in the shoulder region, which was cleared of hair so that one of each pair lies on eachside of the midline.
Injection 1: a 1:1 mixture (v/v) Freunds Complete Adjuvant (FCA)/ physiological saline
Injection 2: 20% test substance in olive oil
Injection 3: 20% test substance in a 1:1 mixture (v/v) FCA/physiological saline

In injection 3, the test substance was dissolved in FCA prior to combining with the aqueous phase (physiological saline).

Day 0 – control group
Three pairs of intradermal injections of 0.1 mL volume were given in the same sites as in the treated animals.
Injection 1: a 1:1 mixture (v/v) Freunds Complete Adjuvant (FCA)/physiological saline Injection 2: olive oil
Injection 3: a 50% formulation of the olive oil in a 1:1 mixture (v/v) FCA/physiological saline

Induction: Topical Application
Day 6 - treated group
Twenty four hours before application (day 5) the test area was cleared of hair. A filter paper (2 x 4 cm) with 100% test substance was applied to the test area and held in contact by an occlusive dressing for 48 hours.

Day 6 – control group
Twenty four hours before application (day 5) the test area was cleared of hair. A filter paper (2 x 4 cm) with olive oil only was applied in a similar manner to the test area and held in contact by an occlusive dressing for 48 hours.


Challenge: Topical Application
Day 20 - treated and control groups
The flanks of treated and control animals were cleared of hair before application. In treated and control animals a filter paper (2 x 2 cm) saturated with the 70% test substance in olive oil was applied to the left flank of the animals and a filter paper (2 x 2 cm) with olive oil only was applied to the right flank. The patches were hold in contact by an occlusive dressing for 24 hours.

Observation of skin reactions
Day 22-23 - treated and control groups
Approximately 48 hours from the start of the challenge application the skin reaction were observed and recorded according to the “Magnusson and Kligman grading scale”.
Approximately 24 hours after this observation a second observation (72 hours) were made and skin reaction was recorded again.

Magnusson and Kligman grading scale for the evaluation of challenge patch test reactions
0 = no visible change
1 = discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling
Positive control substance(s):
yes
Remarks:
Alfa-hexylcinnamaldehyde (CAS No.:101-86-0) Sigma-Aldrich, Germany
Positive control results:
Reliability of the experimental technique is checked periodically in about six-month intervals in an experiment with the known sensitiser α-hexylcinnamaldehyde.

The result of the last experiment (March 2016): positive skin reaction – 3 animals (total number of animals in exposed group = 10), i.e. 30 %. According to the guideline the minimal number of animals with positive skin reaction should be 30 %. The result is satisfactory.

The results of reference study are described in Study No. 123/12/6MK- Ref/03/16: Alfa-hexylcinnamaldehyd - Senzibilizace kůže, VUOS-CETA Report No. 16-302.
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
70%
No. with + reactions:
1
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 70%. No with. + reactions: 1.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
70%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 70%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0.

Body weight

The body weight of animals increased throughout the study. The exposure to the test substance had no influence on the body weight of the animals. The individual values of body weight are presented in Tables 1 and 2.

Clinical observation

During the course of the experiment the animals did not show any clinical symptoms of intoxication. At the end of experiment the animals were sacrificed (ether narcosis).

Evaluation of skin reactions

Evaluation of skin reactions after induction

Intradermal injections

The skin reaction evaluation of the test area of all animals from the exposed group showed intense erythema around the first and third injection sites and moderate erythema around the second injection sites. These changes faded away 96 hours after evaluation. The skin reaction evaluation of the test area of all animals from the control group showed intense erythema around the first injection sites and moderate erythema around the third injection sites. There were no visible changes around the second injection sites. These changes faded away after 72 hours after evaluation.

Topical application

The evaluation of skin reactions of the exposed group showed in all animals discrete erythema in the test area which faded away 24 hours after evaluation.

The evaluation of skin reactions in the control group with olive oil showed no visible changes in the skin of all animals.

Evaluation of skin reactions after challenge

Exposed group at 48 hours after the challenge

The evaluation of skin reactions in the exposed group carried out 48 hours after the start of the challenge phase of the study showed discrete erythema in the skin of 1 animal on the left flank where the test substance was applied (Table 3).

Exposed group at 72 hours after the challenge

The evaluation of skin reactions in the exposed group carried out 72 hours after the start of the challenge phase of study did not show any changes in the skin of the animals on either flank (Table 3) .

Control group at 48 and 72 hours after the challenge

The evaluation of skin reactions in the control group carried out 48 and 72 hours after the start of the challenge phase of study did not show any changes in the skin of the animals on either flank (Table 3).

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test substance, Vinyltoluene, was not shown to be a contact allergen in guinea pigs under the given test conditions.
Executive summary:

In a dermal sensitization study (16-564) with Vinyltoluene (≥99.2%; 3-methylstyrene (CAS No. 100-80-1): 64.3 % w/w

4-methylstyrene (CAS No. 622-97-9): 35.7 % w/w) in olive oil, young adult BFA albino guinea pigs were tested using the guinea pig maximisation test. For induction, 20% vinyltoluene in olive oil (intradermal injections) and 100% vinyltoluene (topical application) was used. For challenge, 70% vinyltoluene in olive oil was used for topical application. The evaluation of skin reactions after challenge was carried out at 48 and 72 hrs. The positive control was α-hexylcinnamaldehyde.

Vinyltoluene caused a positive skin reaction in 1 of 20 animals, i.e. in 5 % of animals which were exposed to the test substance. There were no reactions in the control group. The exposed animals showed no other negative clinical symptoms throughout the experiment. The body weight of animals increased during the study and it was not affected by the treatment. In this study, Vinyltoluene is not a dermal sensitizer.

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

A guinea pig maximisaiton test (GPMT) was available and was chosen as the key study. It was of acceptable quality and reliability with a Klimisch score of 1.

In a dermal sensitization study (16-564) with Vinyltoluene (≥99.2%; 3-methylstyrene (CAS No. 100-80-1): 64.3 % w/w 4-methylstyrene (CAS No. 622-97-9): 35.7 % w/w) in olive oil, young adult BFA albino guinea pigs were tested using the guinea pig maximisation test. For induction, 20% vinyltoluene in olive oil (intradermal injections) and 100% vinyltoluene (topical application) was used. For challenge, 70% vinyltoluene in olive oil was used for topical application. The evaluation of skin reactions after challenge was carried out at 48 and 72 hrs. The positive control was α-hexylcinnamaldehyde.

Vinyltoluene caused a positive skin reaction in 1 of 20 animals, i.e. in 5 % of animals which were exposed to the test substance. There were no reactions in the control group. The exposed animals showed no other negative clinical symptoms throughout the experiment. The body weight of animals increased during the study and it was not affected by the treatment. In this study, Vinyltoluene is not a dermal sensitizer in this assay.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available information in the dossier, the substance vinyl toluene (CAS No. 25013-15-4) does not need to classified

for skin sensitisation when the criteria outlined in Annex I of 1272/2008/EC and Annex I of 286/2011/EC are applied.