Registration Dossier

Administrative data

Description of key information

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:
From February 08, 2018 to March 10, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
Buehler test
Justification for non-LLNA method:
An LLNA study was not conducted as the substance is a surfactant. Surfactants have been shown to often give false-positive results in LLNA studies. Therefore GPMT studies are more adapted to this type of chemistry.
Species:
guinea pig
Strain:
other: LAL/HA/BR
Sex:
male
Details on test animals and environmental conditions:
Species and strain: Guinea pigs, LAL/HA/BR
Source: LAB-ÁLL Bt., 1174. Budapest, Hunyadi u. 7, Hungary
Justification of species: the guinea pig is a standard species used for skin sensitisation studies, recognised by the international guidelines as recommended test system.
Body weight at randomisation (Day-1): 306 – 409 g
Age of animals at first dosing (Day 1): young adult, ~ 6 weeks old
Acclimation time: at least 15 days before start of treatment under laboratory conditions

Husbandry

Animal health: Health inspection was performed at arrival of the animals. Only healthy animals were used for the test. The health status was certified by the staff Veterinarian.
Housing/Enrichment: Animals were housed in Macrolon cages size IV, with up to 3 animals/cage to allow socialisation in the main study and 4 animals/cage in the preliminary study.
Bedding: Laboratory bedding, Lignocel 3/4-S was available to animals during the study.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 17.4–27.4 °C
Relative humidity: 21–60 %
Ventilation: 15-20 air exchange/hour

Food and Feeding: Animals received Cunigra Diet for Rabbits (= Bonafarm-Bábolna Takarmány Ltd., Hungary), ad libitum.

Water Supply: Animals received tap water from municipal supply as for human consumption, containing at least 50 mg/100 mL ascorbic acid, ad libitum. The drinking water is routinely analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Identification: The animals were individually marked using ear punching. The cages were marked with individual identity cards with information about study code, sex, cage number, dose group and individual animal number.

Randomisation: All animals were sorted according to weight on the day of the randomisation prior to the start of the treatment period. After this, the animals were allocated to the test groups. The result of the randomisation was checked according to the actual body weights assuring an acceptable homogeneity and variability among the groups.

Measurement of body weight: Body weight was recorded with a precision of 1 g at randomisation (Day -1), then at least weekly, including Day-31 or preterminal euthanasia (Day-22) prior to euthanasia. The mean values and the standard deviations were calculated and reported.

Observations: Mortality/Clinical signs: Daily during the test. As part of this observation, detailed clinical observation was made weekly during the test

Skin reactions were observed and recorded as follows:
• Preliminary test: 1 (±5 min), 24 (±2), 48 (±2) and 72 (±2) hours after patch removal.
• Dermal induction exposure: 24 (±2) hours after the patch removal.
• Challenge exposure: 24 (±2) and 48 (±2) hours after the patch removal.
Route:
epicutaneous, occlusive
Vehicle:
other: 80% ethanol/distilled water
Concentration / amount:
50% w/v
Day(s)/duration:
6 hours
Adequacy of induction:
highest technically applicable concentration used
Route:
epicutaneous, occlusive
Vehicle:
other: 80% ethanol/distilled water
Concentration / amount:
25% w/v
Day(s)/duration:
6 hours
Adequacy of induction:
not specified
No.:
#20
Route:
epicutaneous, occlusive
Vehicle:
other: acetone
Concentration / amount:
50% (w/v)
Day(s)/duration:
6 hours
Adequacy of challenge:
highest non-irritant concentration
Route:
epicutaneous, occlusive
Vehicle:
other: acetone
Concentration / amount:
25% (w/v)
Day(s)/duration:
6 hours
Adequacy of challenge:
other: safeguard dose
No. of animals per dose:
Preliminary test: 4 animals
Main test: 20 in the test group, 10 in the control group
Details on study design:
During the vehicle selection, it was only possible to dissolve the test substance in 80% ethanol/water and acetone, and for both limited at a maximum concentration of 50% (w/v). According to OECD 406, Buehler method, the 80% ethanol/water is the preferred vehicle for the induction phase, whereas and acetone is more suitable for the challenge phase. OECD 406, Buehler method, therefore both vehicles were tested in the preliminary study.

Preliminary study:
 
Concentrations of 50% w/v (highest feasible concentration) and 25% w/v diluted in 80% ethanol/distilled water or acetone were tested to identify the primary irritation by dermal application, therefore two concentrations per vehicle were used. For the dermal application, approximately 0.5 mL per concentration was applied onto the clipped and shaved skin of the animals. A closed patch exposure was performed..
Two animals were used per concentration. The time of exposure was 6 hours. Local effects were examined and scored 1, 24 , 48 and 72 h after the patch removal. Skin effects were scored for erythema and oedema. Any additional observations were recorded if present.
 
It was found that none of the tested concentrations produced skin reactions. However, the formulations stuck to the hair around the treatment area and made the removal of the hair (for observation) difficult.
 
Main study:
 
Dermal Induction Exposure (Week 1, 2 and 3)
 
Approximately 24 hours before the first treatment an area of approximately 8 x 8 cm size on the back region of all animals was carefully shaven.
 
The test animals were treated with approximately 0.5 mL of test substance. A 5 x 5 cm patch of sterile gauze patch was applied. An approximately 2.5 x 2.5 cm area of this gauze patch was saturated with the test substance at the selected 50% (w/v) concentration in 80% ethanol/distilled water and placed on the back of the animals. These gauze patches were kept in contact with the skin by a patch with a surrounding adhesive hypoallergenic plaster (Closed Patch Test). The animals of the control group were treated with 0.5 mL vehicle only (80% ethanol/distilled water), using similar treatment procedures as in the test group.
The duration of induction exposure was approximately 6 hours. After the patch removal any remaining test substance was removed, a gauze swab and ethanol.
The animals of the test and vehicle control groups were treated once on the back region 3 times (once each week). The same application as on Day-1 was carried out on the same test area on Day-8 and again on Day-15. If hair growth became excessive, the exposed surface was shaved one day before the next treatment.
Challenge controls:
Challenge Exposure (Day 29)
 
Two weeks after the last dermal induction treatment, the animals were exposed to challenge dose, dermally on the untreated left flank. The left shaved side of all animals (both the test and the control) were treated with 50% (w/v) test substance formulated in acetone.
The right shaved side of all animals were treated with 50% (w/v) dilution of the maximum dermal challenge dose (25% (w/v) test substance formulated in acetone). Application was as described previously (Closed Patch Test).
After the patch removal any remaining test substance was removed, a gauze swab and acetone.
Positive control substance(s):
not required
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
50% (w/v)
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Group:
positive control
Remarks on result:
other: not reported

- Due to unknown reasons, the general condition of the animals (control and treatment group) become weak with slight or moderate activity decrease from Day-19 (one animal from Day-15, animal no. 4) up to the end of the experiment (Day-31). After a consultation with the Veterinarian the animals received higher C-vitamin level with the drinking water. Despite of the precautions, the worsening condition of one animal lead to moribund status from the treatment group (animal no.: 27). This animal was preterminally euthanized on Day-22 because of ethical reasons. The rest of the animal’s condition improved to the end of the experiment. Twenty-three animals become symptom-free and 6 animals’ condition improved to only slight ‘activity decrease’ towards the end of the experiment. These clinical signs could not be connected to the treatment as control and treatment group were affected equally. Therefore, signs of systemic toxicity that were observed in any animal, are considered not test substance-related. According to the Veterinarian consultation these incidentthese findings have no effect on the outcome of the observations for sensitisation. Marked body weight loss was observed between Day-14 and Day-21 in the control and treatment groups. The body weight was back to normal or increased to the end of the experiment except one animal (animal no.: 27), which was preterminally euthanized on Day-22 due to moribund state.

- Marked body weight loss was observed between Day-14 and Day-21 in the control and treatment group. The body weight was back to normal or increased to the end of the experiment except one animal (animal no.: 27), which was preterminally euthanized on Day-22 due to moribund state. There were no notable differences between the test animal group and the control group.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the study conditions, no signs of contact sensitisation were detected in any of the animals after challenge exposure in surviving guinea pigs previously exposed to the test substance.
Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance according to OECD Guideline 406, EU Method B.6 and EPA OPPTS 870.2600, in compliance with GLP. The study was performed in the guinea pig (Buehler method), using 3 inductions. Twenty test animals were subjected to sensitisation procedures in a three-stage process. Based on the results of a preliminary test, the substance was used at a concentration of 50% (w/v) formulated in 80% ethanol/distilled water for topical sensitisation induction treatment. Ten control guinea pigs were simultaneously exposed to 80% ethanol/distilled water only during the sensitisation induction phase. Two weeks after the last induction exposure, a challenge dose with 50% (w/v) of the test substance in acetone was administered topically on the left side of all animals. The right side of the animals were treated at concentration of 25% (w/v) formulated in acetone as a safeguard dose. Challenge was performed by dermal application of the test substance for approximately 24 h with a fully occlusive foil (Closed Patch Test). Skin reactions were measured 24 and 48 h after patch removal. Due to unknown reasons, the animals (control and treatment group) became weak with slight or moderate activity decrease from Day 19 (one animal and from Day 15 (one animal) up to the end of the experiment (Day 31). After a consultation with the Veterinarian the animals received higher Vitamin C levels with the drinking water. Despite the precautions, the worsening condition of one animal led to a moribund status. The animal was pre-terminally euthanized on Day 22 for ethical reasons. The rest of the animal’s condition improved to the end of the experiment. Twenty-three animals became symptom-free and 6 animal’s condition improved to only slight ‘activity decrease’ towards the end of the experiment. These clinical signs could not be connected to the treatment as control and treatment group were affected equally. Therefore, signs of systemic toxicity that were observed in any animal were considered not test substance-related. The bodyweight was back to normal or increased to the end of the experiment except in one animal. Under the study conditions, no signs of contact sensitisation were detected in any of the animals after challenge exposure in surviving guinea pigs previously exposed to the test substance (Zsolt, 2018).

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

A study was conducted to determine the skin sensitisation potential of the test substance according to OECD Guideline 406, EU Method B.6 and EPA OPPTS 870.2600, in compliance with GLP. The study was performed in the guinea pig (Buehler method), using 3 inductions. Twenty test animals were subjected to sensitisation procedures in a three-stage process. Based on the results of a preliminary test, the substance was used at a concentration of 50% (w/v) formulated in 80% ethanol/distilled water for topical sensitisation induction treatment. Ten control guinea pigs were simultaneously exposed to 80% ethanol/distilled water only during the sensitisation induction phase. Two weeks after the last induction exposure, a challenge dose with 50% (w/v) of the test substance in acetone was administered topically on the left side of all animals. The right side of the animals were treated at concentration of 25% (w/v) formulated in acetone as a safeguard dose. Challenge was performed by dermal application of the test substance for approximately 24 h with a fully occlusive foil (Closed Patch Test). Skin reactions were measured 24 and 48 h after patch removal. Due to unknown reasons, the animals (control and treatment group) became weak with slight or moderate activity decrease from Day 19 (one animal and from Day 15 (one animal) up to the end of the experiment (Day 31). After a consultation with the Veterinarian the animals received higher Vitamin C levels with the drinking water. Despite the precautions, the worsening condition of one animal led to a moribund status. The animal was pre-terminally euthanized on Day 22 for ethical reasons. The rest of the animal’s condition improved to the end of the experiment. Twenty-three animals became symptom-free and 6 animal’s condition improved to only slight ‘activity decrease’ towards the end of the experiment. These clinical signs could not be connected to the treatment as control and treatment group were affected equally. Therefore, signs of systemic toxicity that were observed in any animal were considered not test substance-related. The bodyweight was back to normal or increased to the end of the experiment except in one animal. Under the study conditions, no signs of contact sensitisation were detected in any of the animals after challenge exposure in surviving guinea pigs previously exposed to the test substance (Zsolt, 2018).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available in vivo study indicates that the test substance is not a skin sensitiser. The substance is therefore not classified for this endpoint according to EU CLP (EC 1272/2008) criteria.