[No public or meaningful name is available]

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
According to the later amendment of REACH, i.e. Commission Regulation (EU) 2016/863 of 31 May 2016, testing by the dermal route does not need to be conducted if:
— the substance does not meet the criteria for classification as acute toxicity or STOT SE by the oral route and
— no systemic effects have been observed in in vivo studies with dermal exposure (e.g. skin irritation, skin sensitisation) or, in the absence of an in vivo study by the oral route, no systemic effects after dermal exposure are predicted on the basis of non-testing approaches (e.g. read across, QSAR studies).
The oral LD50 in rats is >2000 mg/kg, one of ten animals died at a dose of 2000 mg/kg after 3h, clinical signs were i.a. apathy and diarrhea. Recovery was complete in the 2000 mg/kg group by Day 2. In the available OECD 404 skin irritation study, there were no signs of toxicity or ill health in any rabbit during the observation period. Further, in the two available GPMTs, also no signs of ill health or toxicity were recorded. Hence, testing can be waived.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Remarks:

OECD 401

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994-12-06 - 1995-04-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

adopted February 24, 1987

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

by Umweltministerium Baden-Württemberg

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Interfauna, Suddeutsche Versuchstierfarm, Oberer Bann 37, D-78532 Tuttlingen
- Females (if applicable) nulliparous and non-pregnant: yes
- Weight at study initiation: 202 - 245g (females), 207 - 245 g (males)
- Fasting period before study: yes, overnight
- Housing: The rats housed individually in Macrolon cages (area 800 cm², height 17 cm)
- Diet (e.g. ad libitum): Haltungsdiat "ALMA 0801 H 1003", twice 8 g daily
- Water (e.g. ad libitum): Free access by daily changing of the watering bottles
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 22 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12:12

Route of administration:

oral: capsule

Vehicle:

other: sesame oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 2000, 2800, or 3920 mg test item in each 1000µl pure sesame oil
- Amount of vehicle (if gavage): 1000µl
- Purity: pure

MAXIMUM DOSE VOLUME APPLIED: no exceeding the limit volume of 1 ml/100 g body weight

Doses:

2000, 2800, or 3920 mg/kg

No. of animals per sex per dose:

Group I and II (2000 resp. 2800 mg/kg, each group consists of 5 male and 5 female Sprague-Dawley rats)
Group III (3920 mg/kg, the group consists of 5 male Sprague-Dawley rats)
Control group (sesame oil): (the group consists of 2 male and 2 female Sprague-Dawley rats)

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: daily
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

The following times of death after administration of the test item are recorded:
2000 mg/kg:
rat No. 636 3 hours
2800 mg/kg:
rat No. 653 and 654 10 minutes
rat No. 651 1.5 hours
rat No. 644 2 hours
rat No. 642 3 hours
3920 mg/kg:
rat No. 656 2.5 hours
The LD50 value of the test item turned out to be unquantifiable in this toxicity study because of the non-linear dose relationship of the mortality rate observed. However, it can be deduced from the mortality rate almost certainly that the LD50 for rats of both sexes is greater than 2000 mg/kg body weight.

Clinical signs:

other: Acute toxicological symptoms attributed to the exposure to the test item could be observed in five rats of group I (2000 mg/kg) and in all rats of group II (2800 mg/kg) and of group III (3920 mg/kg).

Gross pathology:

The post-mortem findings of all 25 rats of the test show no macroscopical organ changes in the pathological examination.

Interpretation of results:

GHS criteria not met

Remarks:

EU implementation

Conclusions:

The study was performed on the registered substance itself according to OECD 401 under GLP, the report is well documented. Hence, the results can be considered as sufficiently reliable to assess the acute toxicity of the test item to rats via the oral route. The LD50 was determined to be > 2000 mg/kg, hence, the substance does not need to be classified as acutely toxic acc. Regulation 1272/2008.

Executive summary:

The acute oral toxicity of the test item was determined acc. OECD TG 401 under GLP.

The test substance was administered orally in a single dose to three groups of Sprague-Dawley rats:

Group I (10 rats, 5 male and 5 female): 2000 mg/kg applied

Group II (10 rats, 5 male and 5 female): 2800 mg/kg applied

Group III (5 rats, male): 3920 mg/kg applied

 

Mortality

group	I	II	III
	2000	2800	3920
	mg/kg	mg/kg	mg/kg
mortality
number
male	1 outof 5	2 out of 5	1 out of 5
female	0 outof 5	3 out of 5	Not tested

 

Acute toxicological symptoms attributed totheexposure to the test item could be observed in five rats of group I (2000 mg/kg) and in all rats of group II (2800 mg/kg) and of group III (3920 mg/kg). The post-mortem findings of all 25 rats of the test show no macroscopical organ changes inthepathological examination. The LD 50 value of the test item turned out to be unquantifiable in this toxicity study because ofthenon-linear dose relationship of the mortality rate observed. However, it can be deduced from the mortality rate almost certainlythatthe LD50 for rats of both sexes is greater than 2000 mg/kg body weight. Testing of further groups was omitted due to animal welfare.

Based on the obtained results, the substance does not need to be classified as acutely toxic acc. Regulation 1272/2008.

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Remarks:

OECD 404

Reference

Endpoint:

skin irritation: in vivo

Remarks:

in vivo study was conducted prior to REACH implementation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994-12-08 - 1994-12-19 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

OECD-Guideline for Testing of Chemicals, Section 4, 404 "Acute Dermal Irritation / Corrosion", adopted July 17, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

other: Albino

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. Zimmermann, Schweizer Hof, D-73453 Untergröningen/Aalen
- Age at study initiation: young
- Housing: The rabbits housed individually in wire grating cages (area 2450 cm², height 42 cm) without bedding.
- Diet (e.g. ad libitum): Raiffeisen-Ringfutter, Kaninchen Trockenfutter 52/P, Raiffeisen Kraftfutterwerk Kehl, ad libitum.
- Water (e.g. ad libitum): Community tap water from Karlsruhe; free access for the animals by daily changing of the watering-bottles.
- Acclimation period: The animals were acclimated to the test conditions for 5 days prior to the administration.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23 ° C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES:
Acclimatization: December 08 1994 - December 12, 1994
Administration: December 13 1994
Observation: December 13,1994 - December 19 1994

Type of coverage:

occlusive

Preparation of test site:

shaved

Remarks:

On the fifth day the dorsal area of the trunk was shaved in an area of 3 x 5 cm².

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 ml / animal
- Concentration (if solution): undiluted

Duration of treatment / exposure:

4h

Observation period:

1 week

Number of animals:

3 animals, Identification numbers: 009 (male), 015 and 022 (female)

Details on study design:

TEST SITE
- Area of exposure: The test substance was dermally applied in a single dose of 0.5 ml/animal onto the dorso-lumbar region of 3 albino rabbits.
- Type of wrap if used: The region treated was protected with the 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). It was held in place for a period of 4 hours 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).

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After removal of the bandage at the end of the exposure period the residual test substance was removed by gently washing with water.
- Time after start of exposure: 4h

OBSERVATION TIME POINTS
1 hour after bandage removal, then daily

SCORING SYSTEM:
- Method of calculation: Evaluation of the Skin Reaction according to table 1 in OECD-Guideline 404

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0.7

Max. score:

2

Reversibility:

fully reversible within: day 4 or 5

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Remarks on result:

other:

Remarks:

no animal showed any signs of edema

Irritation parameter:

erythema score

Basis:

animal #1

Remarks:

Rabbit 015 female

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: day 4 or 5

Remarks on result:

other:

Remarks:

Erythema score was 0 after 1h, each 1 at day 1, 2, and 3, and 0 at day 5 and 6

Irritation parameter:

erythema score

Basis:

animal #2

Remarks:

Rabbit 022 female

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal #3

Remarks:

Rabbit 009, male

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

fully reversible within: day 4 or 5

Remarks on result:

other:

Remarks:

Erythema score was 0 after 1h, each 1 at day 1, 2, and 3, and 0 at day 5 and 6

Irritant / corrosive response data:

After removing the occlusive bandage after a period of 4 hours, the exposure area was washed and cleaned with water. In the following observation period a dermal irritation of the exposure area in the form of an erythema (two rabbits) was noticed.
The control area did not show any pathological skin reaction. According to table 1 of OECD-Guideline 404 at all times of evaluation the value was classified as 0.

Under the conditions of this experiment the test substance produced skin lesions in the form of an erythema. Two rabbits showed this effect estimated at grade 1 (maximum possible 4). The erythemata were observed from day 1 to day 3. No oedema could be observed during the observation period. The effects observed turned out to be reversible.

Other effects:

Mortality: After the dermal administration of 0.5 ml test item per animal for a period of 4 hours none of the 3 rabbits died during the experiment.
Toxicological symptoms: During the observation period of 6 days none of the animals showed signs of toxicological symptoms.
Behaviour: The reaction of the rabbits treated was quiet and watchful. The behaviour pattern was not different to that of other rabbits which were not in the test.
Body weight: During the experiment the body weight of all three rabbits had an increase.

Interpretation of results:

GHS criteria not met

Conclusions:

This study was performed in compliance with OECD-Guideline 404 under GLP, is well-documented and with sufficient information for classification, the results can hence be considered as reliable. Under the conditions of this experiment the test substance produced skin lesions in the form of an erythema. Two rabbits showed this effect estimated at grade 1 (maximum possible 4). The erythemata were observed from day 1 to day 3. No oedema could be observed during the observation period. The effects observed turned out to be reversible. According to Regulation 1272/2008, a substance must be classified as skin irritant Cat. 2, if i.a.
(1) Mean value of ≥ 2,3 - ≤ 4,0 for erythema/eschar or for oedema in at least 2 of 3 tested animals from gradings at 24, 48 and 72 hours after patch removal or, if reactions are delayed, from grades on 3 consecutive days after the onset of skin reactions; or
(2) Inflammation that persists to the end of the observation period normally 14 days in at least 2 animals, particularly taking into account alopecia (limited area), hyperkeratosis, hyperplasia, and scaling;
Those criteria are not met here, and so the substance does not need to be classified as irritating to the skin.

Executive summary:

This study was performed in compliance with OECD-Guideline 404 "Acute Dermal Irritation/Corrosion" with three albino rabbits under GLP.

Under the conditions of this experiment the test substance produced skin lesions in the form of an erythema. Two rabbits showed this effect estimated at grade 1 (maximum possible 4). The erythemata were observed from day 1 to day 3. No oedema could be observed during the observation period. The effects observed turned out to be reversible.

During the experiment all three rabbits had an increase in weight.

Visible toxicological signs could not be observed; none of the animals died during the observation period.

GHS criteria are not met, the substance does not need to be classified as irritating to the skin.

Cross-reference 3

Reason / purpose for cross-reference:

data waiving: supporting information

Remarks:

GPMT

Reference

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

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.

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)

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

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.

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

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.

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.

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

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.

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

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

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.

Cross-reference 4

Reason / purpose for cross-reference:

data waiving: supporting information

Remarks:

GPMT

Reference

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:

1995-05-31 - 1995-07-15 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

Adopted: 17 July 1992

Deviations:

no

GLP compliance:

yes

Remarks:

self-declaration

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.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: D. Hall, Newchurch, Staffordshire, England
- Microbiological status of animals, when known:
- Age at study initiation: 4-5 weeks
- Weight at study initiation: 307 to 367 g
- Housing: in groups of five in suspended metal cages with wire mesh floors
- Diet (e.g. ad libitum): vitamin C enriched guinea pig diet FD2 ad libitum
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: 5 days
- Indication of any skin lesions: none stated, animals were healthy

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approx. 21°C
- Humidity (%): 30-70%
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12:12

Route:

intradermal

Vehicle:

other: Alembicol D

Remarks:

a product of coconut oil

Concentration / amount:

2.5%

Day(s)/duration:

single application on day 1

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100% as supplied

Day(s)/duration:

starting on day 7, for 48h

Adequacy of induction:

other: undiluted, non-irritating substance

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: Alembicol D

Remarks:

a product of coconut oil

Concentration / amount:

50%

Day(s)/duration:

on day 21, for 24h

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

other: Alembicol D

Remarks:

a product of coconut oil

Concentration / amount:

30% and 15%

Day(s)/duration:

on day 28, for 24h

No. of animals per dose:

6 (pre-study)
10 (control)
20 (test group)

Details on study design:

RANGE FINDING TESTS:
Animals for these investigations were pre-treated with an intradermal injection of Freund's complete adjuvant, 50:50 with water for irrigation, approx. 1 week prior to the start of the preliminary investigations.
The intradermal and topical irritancy of a range of dilutions of the test substance was investigated to identify where possible (a) concentrations of the test substance that would produce irritation suitable for the induction phase of the main study and (b) a maximum non-irritant concentration by the topical route of administration for the challenge phase.
Based on the reults of the preliminary investigations, the following concentrations of the test item were selected:
Induction intradermal injection: 2.5% v/v in Alembicol D: This was the highest concentration intradermally that caused some irritation but did not adversely affect the animals.
Induction topical application: as supplied
Topical challenge: as supplied and 50% v/v in Alembicol D

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: day 1 and day 7 for 48h
- Test groups:
Induction intradermal injections: A 40*60 mm area of the dorsal skin on the scapular region of the guinea pig was clipped free of hair with electric clippers. Three pairs of intradermal injections were made into a 20*40 mm area within the clipped area in a row.
Injectables for the test animals were prepared as follows:
1. Freund’s complete adjuvant was diluted with an equal volume of water for irrigation.
2. Test item, 2.5% v/v in Alembicol D
3. Test item, 2.5% v/v in Alembicol D in a 50:50 mixture of Freund’s complete adjuvant and Alembicol D
Induction topical applications: Six days after the injections, the same 40*60 mm interscapular area was clipped and shaved free of hair and the site was pre-treated by gentle rubbing with 0.5 ml per site of 10% v/v sodium lauryl sulphate in petrolatum. 24 h later a 20*40 mm patch of Whatman No. 3 paper was saturated with approx. 0.4 ml of the test item as supplied. The patch was placed on the skin of the test animals and covered by a length of impermeable plastic adhesive tape. This was firmly secured by elastic adhesive bandage and fixed with plastic adhesive tape. The dressing was left in place for 48h.
The dermal reactions were observed after each induction phase in both control and test animals by group.
- Control group:
During the induction phase, the control animals were treated similarly to the test animals with the exception that the test substance was omitted from the intradermal injections and topical application. The dermal reactions were observed after each induction phase in both control and test animals by group.
- Site: dorsal skin
- Frequency of applications: Day 1 and 6
- Duration: single injection / 48h
- Concentrations: 2.5% / undiluted

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: 2 weeks after induction, and 1 week thereafter
- Exposure period: each for 24h
- Test groups / Control group:
The control and test animals were challenged topically two weeks after the topical induction application using the test item as supplied and 50% v/v in Alembicol D. Hair was removed by clipping and then shaving from an area on the left flank of each guinea pig. A 20*20 patch of Whatman No. 3 paper was saturated with approx. 0.2 ml of the test item as supplied and applied to the flank. The test item 50% v/v in Alembicol D was applied in a similar manner to a posterior site. The patches were sealed to the flank for 24h under strips of “Blenderm” covered by “Elatoplast” wound round the trunk and secured with “Sleek”.
A second challenge application was made one week later. The method employed was similar to that described above, with the exception that on this occasion the test item, 30% and 15%, was applied to the right flank of the control and test animals.
- Site: flank
- Concentrations: as supplied and 50% v/v in Alembicol D / 30% and 15%
- Evaluation (hr after challenge): The challenge sites were evaluated 24, 48, and 72 h after removal of the pathes.

OTHER:
Observations:
Clinical signs: All animals were observed daily for signs of ill health or toxicity
Bodyweight: The body weight of each guinea pig on the main study was recorded on day 1 (day of intradermal injections) and on the last day observations were made of dermal responses to the challenge applications.
Dermal responses: The dermal reactions resulting from intradermal injection and topical application on the preliminary study, and topical application at the challenge were assessed using the following numerical system:

Erythema and Eschar Formation
No erythema 0
Very slight erythema (barely perceptible) 1
Well defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to eschar formation preventing grading of erythema 4

Oedema Formation
No oedema 0
Very slight oedema (barely perceptible) 1
Slight oedema (edges of area well defined by definite raising) 2
Moderate oedema (raised approximately 1 mm) 3
Severe oedema (raised more than 1 mm and extending beyond area of exposure) 4

The diameter (mm) of the dermal response at the intradermal injection sites was recorded in the preliminary study only to assist in the choice of concentrations for the main study.
Any other lesion not covered in this scoring system was described.
The challenge sites were evaluated 24, 48, 72 h after removal of the patches.

Positive control substance(s):

yes

Remarks:

hexyl cinnamic aldehyde

Positive control results:

Results of 7 independent trials:
Positive Inconclusive Negative
10/10 0/10 0/10
9/10 0/10 1/10
10/10 0/10 0/10
9/10 0/10 1/10
10/10 0/10 0/10
10/10 0/10 0/10
8/10 1/10 1/10

Reading:

1st reading

Group:

other: negative control and test group

Dose level:

undiluted / 50%

Clinical observations:

No signs of ill health or toxicity were recorded. Bodyweight increases were recorded for all guinea pigs over the period of the study.

Remarks on result:

not determinable

Remarks:

Following the first challenge application, dermal responses (generally slight erythema and oedema) were seen for the majority of the test and control animals. The degree of irritation seen precluded the precise evaluation of the test animals response. Therefore a second challenge was carried out, one week later, using lower concentrations of the test substance.

Key result

Reading:

2nd reading

Group:

test chemical

Dose level:

30% / 15%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No signs of ill health or toxicity were recorded. Bodyweight increases were recorded for all guinea pigs over the period of the study.

Remarks on result:

no indication of skin sensitisation

Remarks:

No dermal reactions were seen in either test or control animals following the second challenge application.

Reading:

2nd reading

Group:

negative control

Dose level:

30% / 15%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No signs of ill health or toxicity were recorded. Bodyweight increases were recorded for all guinea pigs over the period of the study.

Reading:

other: not stated

Group:

positive control

Dose level:

10% intradermal induction, undiluted epicutaneous induction, 50% and undiluted challenge

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Remarks:

in 4 of 7 studies

Reading:

other: not stated

Group:

positive control

Dose level:

10% intradermal induction, undiluted epicutaneous induction, 50% and undiluted challenge

No. with + reactions:

9

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Remarks:

in 3 of 7 studies

Interpretation of results:

GHS criteria not met

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.
Following the first application challenge application, dermal responses (generally slight erythema and oedema) were seen for the majority of the test and control animals. The degree of irritation seen precluded the precise evaluation of the test animals response. Therefore a second challenge was carried out, one week later, using lower concentrations of the test substance. No dermal reactions were seen in either test or control animals following the second challenge application. Based on the results of the second challenge application there was no evidence of skin sensitization in any of the twenty test animals.
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 sensitizer, 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 none of the animals showed reactions over control. Hence, the substance gave a negative reaction acc. Regulation 1272/2008, and does not need to be regarded as skin sensitizer. Also, the number of animals is sufficient.

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 20 male guinea pigs (test substance) and 10 male guinea pigs (control) were employed.

Based on the results of a preliminary study and in compliance with the guideline, the following dose levels were selected:

Intradermal injection: 2.5% v/v in Alembicol D

Topical application: as supplied

First challenge application: as supplied and 50% v/v in Alembicol D

Second challenge application: 30% and 15% v/v in Alembicol D

Based on the results of the second challenge the test item did not produce evidence of skin sensitization (delayed contact hypersensitivity) in any of the twenty test animals. The test item does not need to be regarded as skin sensitizer acc. Regulation 1272/2008.