[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Remarks:

in vivo study was conducted prior to REACH implementation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1995-01-10 - 1995-02-17 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

OECD-Guideline for Testing of Chemicals Section 4, 406 "Skin Sensitisation", adopted July 17, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Study was performed prior to REACH implementation and adoption of LLNA OECD TG.

Test material

Test material information

Constituent 1

Reference substance name:

Amines, C11-13 (branched) alkyl, salts with O,O-bis(2-ethylhexyl) hydrogen dithiophosphate

Molecular formula:

C27H60NPS2O2 C28H62NPS2O2 C29H64NPS2O2 (main component)

IUPAC Name:

Amines, C11-13 (branched) alkyl, salts with O,O-bis(2-ethylhexyl) hydrogen dithiophosphate

Test material form:

liquid

Details on test material:

Before Substance ID change known as CAS 71888-91-0 EC 276-159-7

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Pirbright White

Remarks:

Albino

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Interfauna, Süddeutsche Versuchstierfarm, Oberer Bann 37, D-78532 Tuttlingen
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: younger than one year
- Housing: The animals housed individually in Macrolon cages (area 780 cm²).
- Diet (e.g. ad libitum): Raiffeisen Ringfutter, Trockenfutter 52 P, Raiffeisen Kraftfutterwerk Kehl
- Water (e.g. ad libitum): tap water of Karlsruhe, additive:20 mg ascorbic acid and 100 mg citric acid per 100 ml water, free access for the animals by daily changing of the watering-bottles
- Acclimation period: five days
- Indication of any skin lesions: none stated; animals were without morbid signs

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23°C
- Humidity (%):30-70%
- Photoperiod (hrs dark / hrs light): 12:12
- IN-LIFE DATES:
From: 1995-01-20 (start of acclimatization)
To: 1995-02-17 (reading of challenge reactions)

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal and epicutaneous

Vehicle:

other: Freund's complete adjuvant & water or sesame oil

Concentration / amount:

- Induction, day 0, intradermal: Three intradermal injections were made in a row:
1) Freund's Complete Adjuvant (FCA) (No. F-5881, Sigma Chemical Company, P.O. Box 14508, St. Louis, USA) diluted with aqua ad iniectabilia in the ratio 1+1 and shaken up. The volume of each injection was 0.1 ml.
2) Test item, diluted with sesame oil in the ratio 1 + 3. The volume of each injection was 0.1 ml. The solutions administered were not older than 1 hour.
3) First, the test item is emulsified in FCA in the ratio 2 + 3. This emulsion is further diluted with H2O in the ratio 5 + 3. The resulting mixture containing 25 % of the test item is then injected. The volume of each injection was 0.1 ml
- Induction, dermal (day 8): A filter paper (2x4 cm²) was fully-loaded with approx. 1 ml undiluted test item. It was then applied with the loaded side down to the test area

Day(s)/duration:

intradermal: single application / epicutaneous: 48 h

Adequacy of induction:

highest technically applicable concentration used

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: sesame oil

Concentration / amount:

a linen patch loaded with the test substance was applied

Day(s)/duration:

for 24 h, on day 21, 2 weeks after topical induction; evaluation 24&48h after removal of the bandage

Adequacy of challenge:

other: undiluted; highest concentration possible

No. of animals per dose:

10 (test item)
5 (control)
group I: (test substance) (animals No. 220-229)
group II: (control) (animals No. 230-234)
male animals: No. 225-229; 232-234
female animals: No. 220-224; 230-231

Details on study design:

RANGE FINDING TESTS:
The concentration of the test substance for each induction exposure should be the highest to cause mild irritation.
In order to check the animal tolerability of the test concentration applied, a small-scale pilot study with two albino guinea pigs was performed. After clipping away their hair the following intradermal injections were made in each dorsal side:
above: 100 µl test item diluted with sesame oil in the ratio 1:3
below: 100 µl test item diluted with sesame oil in the ratio 1:7
During the next 3 days the skin area around each prick spot was checked for skin irritation effects. No erythema or oedema could be observed. Further testing with more concentrated test item solution was omitted in consequence of the high viscosity of the test substance.

MAIN STUDY
After randomization and assignment to both groups the animals were acclimatized to the test conditions for 5 days prior to the administration.
The study is chronologically subdivided into the acclimatization period (day -5 to -1), the induction phase (day 0 to 8) with an intradermal administration of the test substance on day 0 and a dermal one on day 8, followed by a phase free from treatment (day 9 to day 19) during which a hypersensitive state is developed in both phases. The challenge phase (dermal administration on day 20) follows with subsequent valuation of potential sensitisation reactions on the days 22 and 23.

A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: day 0, day 8

- Test groups:
- Induction, day 0, intradermal: An area of 4 x 6 cm² on the dorsum was clipped with an electric clipper. Three intradermal injections were made in a row:
1) Freund's Complete Adjuvant (FCA) (No. F-5881, Sigma Chemical Company, P.O. Box 14508, St. Louis, USA) diluted with aqua ad iniectabilia in the ratio 1+1 and shaken up. The volume of each injection was 0.1 ml.
2) Test item, diluted with sesame oil in the ratio 1 + 3. The volume of each injection was 0.1 ml. The solutions administered were not older than 1 hour.
3) First, the test item is emulsified in FCA in the ratio 2 + 3. This emulsion is further diluted with H2O in the ratio 5 + 3. The resulting mixture containing 25 % of the test item is then injected. The volume of each injection was 0.1 ml
- Causing of a local irritation (day 7): As expected no dermal irritations were noticed in both groups. Therefore, the fur of all guinea pigs was clipped in the dorsal test area with an electric clipper once again. Twenty-four hours before topical induction application the test area was painted with 0.5 ml of a mixture of 10 % sodium lauryl sulfate (No. 20763, anal. grade, Serva Feinbiochemica GmbH & Co. KG, Heidelberg, Germany) in vaseline (DAB 10, Carl Roth GmbH + Co., Karlsruhe, Germany) in order to create a local dermal irritation.
- Induction, dermal (day 8): Eight days after intradermal induction the topical dermal induction was performed. A filter paper (2x4 cm2) was fully-loaded with approx. 1 ml undiluted Additin M 10306. It was then applied with the loaded side down to the test area marked with a rectangular outline and held in contact by an occlusive dressing for a period of 48 hours. In detail, the filter paper was covered with the impermeable non-stripped, non-irritating plastic material of the reverse of a patch (width 6 cm) (Werovil, No. 15 00 20, WERO-MEDICAL, D-65232 Taunusstein, Germany). It was held in place by an occlusive bandage made from a dressing (width 8 cm, with selvages, coated completely with cellophane) (No. 09 00 22, WERO-MEDICAL). The bandage was fixed with 1.25 cm broad strips of Adhesive Tape ST (WERO-MEDICAL).

- Control group:
- Induction, day 0, intradermal: An area of 4 x 6 cm² on the dorsum was clipped with an electric clipper. Three intradermal injections were made in a row: Volume of each injection: 0.1 ml.
1) Freund's Complete Adjuvant (FCA) (No. F-5881, Sigma Chemical Company, P.O. Box 14508, St. Louis, USA) diluted with aqua ad iniectabilia in the ratio 1+1 and shaken up. The volume of each injection was 0.1 ml.
2) Sesame oil; the volume of each injection was 0.1ml.
3) Sesame oil emulsified in FCA m the ratio 2+1. This emulsion is further diluted with H2O in the ratio 3 + 1. The resulting mixture containing 50 % sesame oil is the injected. The volume of each injection was 0.1 ml.
- Causing of a local irritation (day 7): As expected no dermal irritations were noticed in both groups. Therefore, the fur of all guinea pigs was clipped in the dorsal test area with an electric clipper once again. Twenty-four hours before topical induction application the test area was painted with 0.5 ml of a mixture of 10 % sodium lauryl sulfate (No. 20763, anal. grade, Serva Feinbiochemica GmbH & Co. KG, Heidelberg, Germany) in vaseline (DAB 10, Carl Roth GmbH + Co., Karlsruhe, Germany) in order to create a local dermal irritation.
- Induction, dermal (day 8): The animals of the control group were treated in the same way as the test item group. However, instead of the test substance the pure vehicle sesame oil was applied dermally under the filter paper. The exposure area was protected with an occlusive bandage, too.

- Site: dorsum
- Frequency of applications: single application per day
- Duration: single application (day 0), 48h (day 8)

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: The challenge phase (dermal administration on day 20, 2 weeks after last induction) follows with subsequent valuation of potential sensitisation reactions on the days 22 and 23.
- Exposure period: 24h
- Test groups / Control group:
Challenge (day 21): The animals were challenged two weeks after the topical dermal induction.
The hair was removed from two 3x5 cm2 areas on both flanks by clipping with an electric clipper. To both groups a linen patch loaded with the test substance was applied to the left flank and a linen patch loaded with the vehicle (sesame oil) was applied to the right flank.
An occlusive dressing made from the materials specified above protected the exposure areas during a period of 24 hours.

- Site: flanks
- Concentrations: undiluted
- Evaluation (hr after challenge): Reading of challenge reactions (days 23 + 24): The challenge sites were evaluated 24 and 48 hours after removal of the bandage.

Challenge controls:

none

Positive control substance(s):

yes

Remarks:

The sensitivity of the guinea pig strain used and the reliability of the experimental technique were assessed by use of p-phenylenediamine which is known to have mild-to-moderate skin sensitization properties in a separate study.

Results and discussion

Positive control results:

Mortality / toxicological symptoms
Group III (reference substance): Guinea pig No. 187 was found dead on day 21 of the reference substance study. None of the surviving animals of group III showed signs of toxicological symptoms.
Group IV (control): Guinea pig No. 192 was found dead on day 20 of the reference substance study. Guinea pig No. 194 was found dead on day 21 of the reference substance study. None of the surviving animals of group IV showed signs of toxicological symptoms.

Body weight
At the end of the experiment all surviving animals had an increase in weight compared to the beginning of the study.

Classification of the findings caused by the reference substance p-phenylenediamine
As stated in "Criteria of judgement" the relevance of the observations is based upon the irritation score and the number of animals reacting as compared with the negative control group. Four of the surviving 9 animals showed symptoms of allergic reactions. Therefore, the potency of p-phenylenediamine to cause allergic symptoms is shown as well as the sensitivity of the guinea pig strain used.

allergic reactions animal challenge reaction
No. day 23 day 24
180 1 1
181 0 0
182 0 0
183 1 0
erythemata noted
184 1 0
185 1 1
186 0 0
187 + a)
188 0 0
189 0 0

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

4

Total no. in group:

10

Clinical observations:

None of the guinea pigs of group I died during the experiment or showed signs of toxicological symptoms.

Remarks on result:

positive indication of skin sensitisation

Remarks:

Four of the animals of the test group showed symptoms of allergic reactions. Therefore, the test substance shows skin sensitisation potential under the experimental conditions.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

2

Total no. in group:

10

Clinical observations:

None of the guinea pigs of group I died during the experiment or showed signs of toxicological symptoms.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None of the guinea pig of group II died during the experiment or showed signs of toxicological symptoms.

Remarks on result:

no indication of skin sensitisation

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None of the guinea pig of group II died during the experiment or showed signs of toxicological symptoms.

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.5% (intradermal, epicutaneus induction & challenge)

No. with + reactions:

4

Total no. in group:

9

Clinical observations:

death of the 10th animal

Remarks on result:

positive indication of skin sensitisation

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.5% (intradermal, epicutaneus induction & challenge)

No. with + reactions:

2

Total no. in group:

9

Clinical observations:

death of the 10th animal

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

EU implementation

Conclusions:

This study was performed in compliance with OECD-Guideline 406 under GLP, is well-documented and with sufficient information for classification, the results can hence be considered as reliable. Positive and negative controls gave the appropriate results. Under the conditions of this experiment the test substance produced skin reactions in 4 out of 10 animals of the treatment group.
According to Regulation 1272/2008, when an adjuvant type guinea pig test method for skin sensitisation is used, a response of at least 30 % of the animals is considered as positive.
According to the guideline, a minimum of 10 animals is used in the treatment group and at least 5 animals in the control group. When fewer than 20 test and 10 control guinea pigs have been used, and it is not possible to conclude that the test substance is a sensitiser, testing in additional animals to give a total of at least 20 test and 10 control animals is strongly recommended.
The present test is an adjuvant test, and 40% of the animals showed reactions in the form of discrete or patchy erythema. Hence, the substance gave a positive reaction acc. Regulation 1272/2008, and so also the number of animals is sufficient. According to Commission Regulation (EU) No 286/2011 of 10 March 2011, Table 3.4.4, Animal test results for sub-category 1B, a substance must be classified as skin sensitizer Cat. 1B, if in a GPMT ≥ 30 % of the animals are responding at > 1 % intradermal induction dose. Intradermal induction dose was 25%, and 40% responded, so a subcategorisation into Skin Sens. Cat. 1B ist indicated.

Executive summary:

The skin sensitisation study was performed according to the method of Magnusson & Kligman in compliance with OECD-Guideline 406. Two groups (test substance and control) with 10 guinea pigs (test substance) and 5 guinea pigs (control) were employed, both male and female animals contained.

Under the conditions of this experiment, i.e. after the intradermal and dermal applications of the test item with subsequent challenge four animals showed allergic reactions. As expected, the animals of the control group showed no allergic reactions.

During the experiment the body weight of all animals had a normal physiological growth. Resulting from this study the test substance has skin sensitisation potential. A histopathological examination was not performed.

The substance must be regarded as a potential skin sensitizer.