29H,31H-phthalocyanine

Endpoint summary

Description of key information

- Skin sensitisation: not sensitising, no EC3 could be established, LLNA

- Respiratory sensitisation: not expected to be of concern for respiratory sensitisation

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Substance type: blue solid
- Analytical purity: no data given
- Lot/batch No.: KRON 200106
- Purity> 85%
- Expiration date of the lot/batch: 31-Mar-2006
- Stability under test conditions: stable under storage conditions
- Storage condition of test material: in the original container at room temperature (20 °C +- 3 °C), away from direct sunlight

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 16 g - 24 g (ordered)
- Housing: individually in Makrolon type-2 cages with standard softwood bedding
- Diet: Pelleted Standard Kliba 3433, batch no. 78/03 mouse maintainance diet (Provimi Kliba AG, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: under test conditions after haelth examination; only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +- 3 °C
- Humidity: 30 - 70 %
- Air changes per hr: 10 - 15
- Photoperiod: 12 hrs dark / 12 hrs light

Vehicle:

dimethyl sulphoxide

Concentration:

2.5, 5 and 10 %

No. of animals per dose:

4 animals per dose

Details on study design:

RANGE FINDING TESTS:
To determine the highest non-irritant and technically applicable test item concentration, a non-GLP test was conducted in 2 mice with concentrations of 1, 2.5, 5 and 10 %. In the main study 3 consecutive concentrations were assayed. The top dose was the highest achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.

MAIN STUDY:
Each test group was treated by topical application to the dorsal surface of each ear lobe (left and right) with different test item concentrations. The application volume, 25 µl, was spread over the entire dorsal surface (ca. 8 mm diameter) of each ear lobe once daily for 3 consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was passed briefly over the ear´s surface to prevent the loss of any of the test item applied.
Five days after the first topical application, all mice were administered with 250 µl of 79.6 µCi/ml 3HTdR (equal to 19.9 µCi 3HTdR) by intravenous injection via a tailvein.
Approx. 5 h after treatment with 3HTdR all mice were euthanized with dry ice (CO2). The draining lymph nodes were rapidly excised and pooled for each experimental group (8nodes/group). Single cell suspensions of pooled lymph node cells were prepared. Cells were resuspended in 5 % trichloroacetic acid ans incubated at 4 °C for at least 18 h to precipitate the macromolecules. The precipitates were resuspended in 5 % trichloroacetic acid and transferred to glass scintillation vialscontaining 10 ml scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was thenmeasured on a ß-scintillation counter. Similarly, background 3HTdR levels were also measured. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test group relative to the recorded control group (Stimulation Index S.I.). Before values were determined, mean scintillation background DPM was subtracted from data obtained.
In addition, mortality/viability (twice daily), body weights (priot to first application and prior to necropsy) and clinical signs (local/systemic, daily with particular attention to the treatment sites) were determined.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle was quantitatively added. The w/v dilutionswere prepared individually using a magnetic stirrer as homogenizer.
Test item solutions were made freshly before each dosing occasion and no more than 4 h prior to application.
Homogeneity was maintainedduring treatment with the magnetic stirrer.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

A test item was considered as sensitizer if the following criteria were fulfilled:
- Exposure to at least one concentration resulted in an incorporation of 3HTdR of at least 3-fold or greater than in controls as indicated by S.I.
- Data were compatible with a conventional dose-response, although allowance must be for either local toxicity or immunological suppression.

For statistical analysis, the mean values and standard deviations were calculated in the body weight tables.

Parameter:

SI

Value:

0.8

Test group / Remarks:

2.5%

Parameter:

SI

Value:

1.1

Test group / Remarks:

5%

Parameter:

SI

Value:

1

Test group / Remarks:

10%

Cellular proliferation data / Observations:

The background DPM was measured twice and was 3 or 10, respectively. The dpm, determined for the control group, was 4593, for the 2.5 % group it was 3672, for the 5.0 % group it was 5122 and for the 10.0 % group it was 4488.

No deaths occured during the study period.

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

The body weight of the aninals was within the normal range recorded for animals of this strain and age. One animal of the 10 % test item concentration group lost weight during the study, but this was considered to be incidental.

Test item concentration % (w/v)		dpm measurement	dpm - background	number of lymph nodes	dpm per lymph node	S.I.
	background	10
	background	3
	control group	4593	4586	8	573
2.5	test group	3679	3672	8	459	0.8
5.0	test group	5129	5122	8	640	1.1
10.0	test group	4495	4488	8	561	1.0

The positive control, alpha-hexylcinnamaldehyde, was tested in concentrations of 5.0, 10.0 and 25.0 % and produced S.I.s of 1.5, 2.3 and 8.4, respectively.

Interpretation of results:

GHS criteria not met

Conclusions:

In this study stimulation indices of 0.8, 1.1 and 1.0 were determined with the test material at concentrations of 2.5, 5 and 10 % (w/v), respectively in DMSO.
Under the experimental conditions chosen, the test material was not found to be a sensitizer when tested at up to the highest achievable concentration of 10 % (w/v) in DMSO.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Green solid
Batch DEBF 015527
Stable under storage conditions
Expiry data 31-MAR-2006
Purity: >95%

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 16 g - 24 g (ordered)
- Housing: individually in Makrolon type-2 cages with standard softwood bedding
- Diet: Pelleted Standard Kliba 3433, batch no. 78/03 mouse maintainance diet (Provimi Kliba AG, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: under test conditions after haelth examination; only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +- 3 °C
- Humidity: 30 - 70 %
- Air changes per hr: 10 - 15
- Photoperiod: 12 hrs dark / 12 hrs light

Vehicle:

dimethyl sulphoxide

Concentration:

5, 10 and 25%

No. of animals per dose:

4 animals per dose

Details on study design:

RANGE FINDING TESTS:
To determine the highest non-irritant and technically applicable test item concentration, a non-GLP test was conducted in 2 mice with concentrations of 5, 10 and 25 %. The top dose is the highest technically achievable concentration.

MAIN STUDY:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 5 %, 10 % and 25 % (w/v) in dimethylsulfoxide (DMSO). The application volume, 25 µI, was spread over the entire dorsal sutiace (0 - 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was passed briefly over the ear's surface to prevent the loss of any of the test item applied. Five days after the first topical application, all mice were administered with 250 µI of
79.6 µCi/ml 3HTdR (equal to 19.9 µCi 3HTdR) by intravenous injection via a tailv ein.
Approximately five hours after treatment with 3HTdR all mice were euthanized with dry ice (C02).
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a ß-scintillation counter. Similarly, background 3 HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

A test item was considered as sensitizer if the following criteria were fulfilled:
- Exposure to at least one concentration resulted in an incorporation of 3HTdR of at least 3-fold or greater than in controls as indicated by S.I.
- Data were compatible with a conventional dose-response, although allowance must be for either local toxicity or immunological suppression.

For statistical analysis, the mean values and standard deviations were calculated in the body weight tables.

Positive control results:

Results with the latest postive control study are attached to the report (RCC Study Number 852080),

Parameter:

SI

Value:

1.4

Test group / Remarks:

5%

Parameter:

SI

Value:

1.3

Test group / Remarks:

10%

Parameter:

SI

Value:

1.3

Test group / Remarks:

25%

Cellular proliferation data / Observations:

No deaths occured during the study period.
No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.
The body weight of the aninals was within the normal range recorded for animals of this strain and age.

Test item concentration %( w/v)		Measurement dpm	Calculation			Result
			dpm - BG a)	number of lymph nodes	dpm per lymph node b)	S.I.
--	BG1	10	--	--	--	--
--	BG II	3	--	--	--	--
--	CG 1	5238	5231	8	654	--
5	TG 2	7418	7411	8	926	1.4
10	TG 3	6791	6784	8	848	1.3
25	TG 4	6816	6809	8	851	1.3

BG=Background (1 ml 5 % trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Group

S.I. = Stimulation Index

a)      = The mean value was taken from the figures BG I and BG II

b)      = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

The positive control, alpha-hexylcinnamaldehyde, was tested in concentrations of 5.0, 10.0 and 25.0 % and produced S.I.s of 1.5, 2.3 and 8.4, respectively.

Interpretation of results:

GHS criteria not met

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Substance type: blue powder
- Analytical purity: no data given
- Lot/batch No.: SF20927
- Storage condition of test material: room temperature in the dark

Species:

mouse

Strain:

other: CBA/CaBkl

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: B & K Universal Ltd, Hull, UK
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: individually in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet: Certified Rat and Mouse Diet, Code 5LF2 (BCM IPS Ltd., London, UK), ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C
- Humidity: 30 - 70 %
- Air changes: 15 changes per hr
- Photoperiod: 12 hrs dark / 12 hrs light

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

5 %, 10 % or 25 % (w/w)

No. of animals per dose:

four animals per dose

Details on study design:

Mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for 3 consecutive days. The test material was administered by using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of 4 animals received the vehicle anlone in the same manner.
Five days following the first topical application of the test material, all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR, 80 µCi/ml, specific activity 2.0 µCi/mmol; Amersham Biosciences UK Ltd.), giving a total of 20 µCi to each mouse.
Animals were observed post dose on day 1, twice daily on days 2 and 3 and on a daily basis on days 4, 5 and 6. Any signs of toxicity or ill health were recorded. The body weight of the animals was determined on day, 1 prior to dosing, and on day 6, prior to termination.
5 hours following the administration of 3HTdR all animals were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the mice were excised and pooled for each animal group. For each group, 1 ml of PBS was added to the pooled lymph nodes.
A single cell suspension of the pooled lymph node cells was prepared by gentle mechanic disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze and collected into a centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (ca. 190 g) for 10 min. The pellet was resuspended in PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspenden in 3 ml of 5 % Trichloroacetic acid (TCA).
After overnight incubation at 4 °C the precipitates were removed by centrifugation at 2100 rpm (ca. 450 g) for 10 min, resuspended in 1 ml TCA and transferred to 10 ml scintillation fluid (Optiphase, "Trisafe"). 3HTdR incorporation was measured by ß-scintillation counting. The number of radioactive disintegrations per minute was then measured using a Beckman LS6500 scintillation system.

The proliferation response of the lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into the lymph node cells of test nodes relative to that recorded for the control nodes (stimulation index). The test material will be regarded as sensitizer, if at least one of the tested concentrations results in threefold greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as non-sensitizer.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Key result

Parameter:

SI

Value:

>= 1.32 - <= 1.65

Test group / Remarks:

5%, 10%, and 25% test substance

Key result

Parameter:

other: disintegrations per minute (DPM)

Remarks:

dpm/node

Value:

683.38

Test group / Remarks:

vehicle

Key result

Parameter:

other: disintegrations per minute

Remarks:

dpm/node

Value:

>= 899.5 - <= 1 020.05

Test group / Remarks:

5%, 10%, and 25% test substance

Cellular proliferation data / Observations:

- Test group 1: vehicle (acetone/olive oil 4:1): not applicable Test group 2: 5 % test substance in vehicle: 1.32 Test group 3: 10 % test substance in vehicle: 1.65 Test group 4: 25 % test substance in vehicle: 1.49
- Test group 1: vehicle (acetone/olive oil 4:1): 683.38 dpm/node Test group 2: 5 % test substance in vehicle: 899.50 dpm/node Test group 3: 10 % test substance in vehicle: 1131.00 dpm/node Test group 4: 25 % test substance in vehicle: 1020.05 dpm/node

There were no deaths. No signs of systemic toxicity was noted in the test or in the control animals during the study. Blue-coloured staining on the ears, face, feet and fur was noted in all test animals during the study.

The body weight changes of the test animals during the study were comparable to those observed in the corresponding control animals over the same time period.

A stimulation index of less than 3 was recorded for the three concentrations of the test material.

The positive control alpha-hexylcinnamaldehyde provided positive results and was considered to be a sensitizer under the experimental conditions of the test.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of the study, the test material was considered not to be a sentisizer and will therefore not be classified according to GHS or EU criteria.

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 LLNA method was not available yet by the time the study was conducted.

Specific details on test material used for the study:

- Physical state: blue powder
- Analytical purity: > 98 %
- Impurities (identity and concentrations): no data given
- Lot/batch No.: 29122
- Storage condition of test material: room temperature in the dark

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: D. Hall, Newchurch, Staffordshire, UK
- Age at study initiation: four to seven weeks
- Weight at study initiation: 399 - 498 g
- Housing: 5 animals per suspended plastic cage with solid floor and sawdust bedding
- Diet: vitamin C enriched guinea pig diet (Harlan Teklad 9600 FD2 SQC), ad libitum
- Water: ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 +- 3 °C
- Humidity: 30 - 70 %
- Photoperiod: 12 hrs dark / 12 hrs light

Route:

intradermal and epicutaneous

Vehicle:

other: Alembicol D

Concentration / amount:

Based on the results of the preliminary investigations, the following concentrations of the test material were selected:
- Intradermal induction: 7.5 % (w/v) in Alembicol D
- Epicutaneous induction: 50 % (w/v) in Alembicol D
- Challenge: 25 % and 50 % (w/v) in Alembicol D

Route:

epicutaneous, occlusive

Vehicle:

other: Alembicol D

Concentration / amount:

Based on the results of the preliminary investigations, the following concentrations of the test material were selected:
- Intradermal induction: 7.5 % (w/v) in Alembicol D
- Epicutaneous induction: 50 % (w/v) in Alembicol D
- Challenge: 25 % and 50 % (w/v) in Alembicol D

No. of animals per dose:

5 control animals and 10 test animals

Details on study design:

RANGE FINDING TESTS:
The intradermal and topical irritancy of a range of dilutions of the test substance was investigated to identify the minimum irritant test substance concentration suitable for the induction phase and the maximum non-irritant concentration by the topical route of administration and a dilution of this for the challenge phase.

MAIN STUDY
A. INDUCTION EXPOSURE
- Site: 4 x 6 cm interscapular area
- Intradermal induction:
A clipped, 4 x 6 cm area of dorsal skin on the scapular region was prepared. 3 pairs of intradermal injections (0.1 ml/site) were conducted on day 1 into a 2 x 4 cm area within the clipped area. Injectables for the test animals were 1) Freund´s Complete Adjuvant, diluted with an equal volume of water for irrigation, 2) the test material 7.5 % (w/v) in Alembicol D and 3) the test material 7.5 % (w/v) in a 50 : 50 mixture of Freund´s Complete Adjuvant and Alembicol D.
- Epicutaneous induction:
On day 7 the same 4 x 6 cm area was clipped and the site was prepared by gentle rubbing with 0.5 ml per site of 7.5 % (w/w) sodium lauryl sulphate in petrolatum. On day 8, a 2 x 4 cm patch of Whatman No. 3 paper was saturated with ca. 0.4 ml of the test material (50 % in Alembicol D); the patch was placed over the injection sites and covered by an impermeable plastic adhesive tape which was secured by an elastic adhesive bandage, wound round the torso of the animal and fixed with an impervious plastic adhesive tape. The dressing was left in place for ca. 48 hours.
- Test groups: During the induction phase, the control animals were treated similarly to the test animals with the exception that the test substance was omitted from intradermal injections and topical applications.

B. CHALLENGE EXPOSURE
The control and test animals were challenged topically two weeks after the topical induction application (day 22), using the test material (25 and 50 % w/v in Alembicol D.
Hair were clipped on the left flank of each animal and a 2 x 2 cm patch of Whatman No. 3 paper was saturated with ca. 0.2 ml of the test material. TheWhatman papers containing 50 % w/v in Alembicol D were applied on the anterior site of the flank, the Whatman papers containing 20 % w/v in Alembicol D were applied in a similar manner on the posterior site. The patches were sealed on the flank for 24 hours under impermeable adhesive tape, secured by elastic adhesive bandage, wound round the torso of the animal and fixed with an impervious plastic adhesive tape. The challenged sites were evaluated ca. 24 and 48 h after removal of the patches.

All animals were observed daily for signs of ill health or toxicity. The body weight of each animal was recorded on day 1 of the study and following completion of the study prior to termination.
The dermal reactions resulting from challenge reaction were assessed using a numerical scoring system (Draize scores).
On completion of the study the animals were sacrificed by cervical dislocation.
The sensitivity of the guinea pig strain is checked periodically by the testing laboratory with hexyl cinnamic aldehyde (HCA), a known moderate sensitizer.

Positive control substance(s):

yes

Remarks:

hexyl cinnamic aldehyde (HCA), periodically checkings

Positive control results:

The following concentrations of HCA were administered: Intradermal injection 7.5 % v/v in Alembicol D; topical application as supplied (neat); challenge application as supplied (neat) and 50 % v/v in Alembicol D.
Slight to well defined dermal reactions were observed for all test animals after challenge reaction compared to no dermal reactions in the controls. The reactions in the test animals represented hypersensitivity and therefore all test animals gave positive sensitization responses.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25 % (w/v) in Alembicol D

No. with + reactions:

0

Total no. in group:

5

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50 % (w/v) in Alembicol D

No. with + reactions:

0

Total no. in group:

5

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

25 % (w/v) in Alembicol D

No. with + reactions:

0

Total no. in group:

5

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

50 % (w/v) in Alembicol D

No. with + reactions:

0

Total no. in group:

5

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25 % (w/v) in Alembicol D

No. with + reactions:

0

Total no. in group:

10

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 % (w/v) in Alembicol D

No. with + reactions:

0

Total no. in group:

10

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25 % (w/v) in Alembicol D

No. with + reactions:

0

Total no. in group:

10

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50 % (w/v) in Alembicol D

No. with + reactions:

0

Total no. in group:

10

No deaths and no signs of ill health or toxicity were observed. The body weight of the animals increased within the normal range over the period of the study.

During the induction period, necrosis was recorded at sites receiving FCA in test animals and also in control animals. No irritation was seen in test animals at sites receiving the test material in vehicle and in control animals receiving vehicle alone after intradermal injection as well as after topical application.

After challenge, no dermal reactions were seen in either animals, all tested animals gave negative responses.

Interpretation of results:

GHS criteria not met

Conclusions:

In this study, the tested material did not produce evidence of skin sensitization reactions in any of the tested animals under the experimental conditions chosen. Therefore the substance is not considered to have a potential to cause skin sensitization and will not be classified according to GHS or EU criteria.

Reference 5

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

When this study was performed, the LLNA did not yet exist.

Specific details on test material used for the study:

- Lot INCU 002025
- blue powder
- Purity: >95%
- Homogeneity: homogeneous
- Storage conditions: Room temperature

Species:

guinea pig

Strain:

other: Mol:DH (Moellegaard)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: M & B A/S, DK-8680 Ry Denmark
- Microbiological status of animals, when known: no data
- Weight at study initiation: mean 318 g
- Housing: 5 animals per cage
- Diet ad libitum
- Water ad libitum
- Acclimation period: at least five days
- Indication of any skin lesions: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C (except short lasting deviations due to disturbances of air condition)
- Humidity (%): 50 ± 20 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light / dark cycle

Route:

intradermal

Vehicle:

other: sesame oil

Concentration / amount:

5%

Day(s)/duration:

single

Adequacy of induction:

highest technically applicable concentration used

Route:

intradermal

Vehicle:

other: 50 % Freund 's Complete Adjuvant emulsion

Concentration / amount:

5%

Day(s)/duration:

single

Adequacy of induction:

highest technically applicable concentration used

Route:

epicutaneous, occlusive

Vehicle:

other: sesame oil

Concentration / amount:

25 %

Day(s)/duration:

2

Adequacy of induction:

highest technically applicable concentration used

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: sesame oil

Concentration / amount:

25%

Day(s)/duration:

24h

Adequacy of challenge:

other: highest technically achievable concentration

No. of animals per dose:

10 (Treatment group)
5 (control group)
3 (range-finder for non-irritant concentration)

Details on study design:

DETERMINATION OF THE PRIMARY NON-IRRITATING CONCENTRATION
Prior to the determination of the primary non-irritation concentration in a dermal-occlusive test three animals each received 4 intradermal injections of a 50% Freund's Complete Adjuvant emulsion (4 x 0.1 mL) into the dorsal area, since Freund' s Complete Adjuvant may lower the threshold of primary irritation. Thereafter, the test substance was administered to the right or left flank of the guinea pigs according to the following procedure:

Table 2: Distribution of the test concentrations (dermal)

Animal No. Left flank Right flank
1 25 % in sesame oil 5 % in sesame oil
2 25 % in sesame oil 1 % in sesame oil
3 5 % in sesame oil 1 % in sesame oil

The hair on the flanks of the animals was removed mechanically. 0.5 mL of the test substance preparation was administered to a 2 x 2 cm cellulose patch, which was fixed to the flank and covered occlusively for 24 hours with a bandage and film. 24 hours after removal of the patches, the treated skin areas were examined for erythema and edema

DETERMINATION OF THE TOLERANCE OF THE INTRADERMAL INJECTIONS
To determine the tolerance of intradermal injections, each of the following preparations was administered twice by intradermal injection to 2 guinea pigs. The injection sites (sites 1, 2 and 3) were all within a dorsal area measuring 2 x 4 cm in the vicinity of the shoulders.

The injection sites (sites 1, 2 and 3) were all within a dorsal area measuring 2 x 4 cm in the vicinity of the shoulders

Table : Test concentrations
site appl. vol. conc. vehicle
1 2 x 0.1 ml 5.0 % sesame oil
2 2 x 0.1 ml 1.0 % sesame oil
3 2 X 0.1 ml 0.2 % sesame oil

24, 48, 72 and 96 hours after administration the injection sites were examined for local tolerance.


Intradermal injections with Freund's Adjuvant (with and without test substance) caused clear edema as well as indurations. The administration sites treated with the test substance in sesame oil showed clear edema. Intradermal injections of the vehicle alone exhibited two animals showing slight erythema. Due to the blue color of the test substance the treated skin of the animals could not be assessed for erythema.

Due to these strong irritation reactions of the skin, 10% sodium dodecylsulfate was not administered at day 7.

Challenge controls:

Animals with induction treatment of 50 % Freund's Adjuvants or sesame oil

Positive control substance(s):

yes

Remarks:

Alpha-Hexylcinnamaldehyde

Positive control results:

After the challenge treatment 9 animals of the treatment group (90 %) showed a positive reaction during the observation period.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

No skin reactions were observed in the control and the treatment group 24 and 48 hours after removal of the occlusive bandage. Additionally, the treated skin was discolored light blue which did not influence the assessment of skin reactions.

Interpretation of results:

GHS criteria not met

Reference 6

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

When this study was performed, the OECD guideline for the LLNA did not yet exist.

Specific details on test material used for the study:

- Physical state: blue powder
- Analytical purity: > 98 %
- Impurities (identity and concentrations): no data given
- Lot/batch No.: 29122
- Storage condition of test material: room temperature in the dark

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: D. Hall, Newchurch, Staffordshire, UK
- Age at study initiation: four to seven weeks
- Weight at study initiation: 399 - 498 g
- Housing: 5 animals per suspended plastic cage with solid floor and sawdust bedding
- Diet: vitamin C enriched guinea pig diet (Harlan Teklad 9600 FD2 SQC), ad libitum
- Water: ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 +- 3 °C
- Humidity: 30 - 70 %
- Photoperiod: 12 hrs dark / 12 hrs light

Route:

epicutaneous, occlusive

Vehicle:

other: Alembicol D (coconut oil product)

Concentration / amount:

50 % (w/v)

Day(s)/duration:

48h

Adequacy of induction:

highest technically applicable concentration used

Route:

intradermal

Vehicle:

other: Alembicol D (coconut product)

Concentration / amount:

7.5%

Day(s)/duration:

Single injection

Adequacy of induction:

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

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: Alembicol D (coconut oil product)

Concentration / amount:

25 % (w/v)

Day(s)/duration:

24h and 48h

Adequacy of challenge:

other: 50% of the highest technically achievable concentration

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

other: Alembicol D (coconut oil product)

Concentration / amount:

50%

Day(s)/duration:

24h and 48h

Adequacy of challenge:

other: highest technically achievable concentration

No. of animals per dose:

5 control animals and 10 test animals

Details on study design:

RANGE FINDING TESTS:
The intradermal and topical irritancy of a range of dilutions of the test substance was investigated to identify the minimum irritant test substance concentration suitable for the induction phase and the maximum non-irritant concentration by the topical route of administration and a dilution of this for the challenge phase.

MAIN STUDY
A. INDUCTION EXPOSURE
- Site: 4 x 6 cm interscapular area
- Intradermal induction:
A clipped, 4 x 6 cm area of dorsal skin on the scapular region was prepared. 3 pairs of intradermal injections (0.1 ml/site) were conducted on day 1 into a 2 x 4 cm area within the clipped area. Injectables for the test animals were 1) Freund´s Complete Adjuvant, diluted with an equal volume of water for irrigation, 2) the test material 7.5 % (w/v) in Alembicol D and 3) the test material 7.5 % (w/v) in a 50 : 50 mixture of Freund´s Complete Adjuvant and Alembicol D.
- Epicutaneous induction:
On day 7 the same 4 x 6 cm area was clipped and the site was prepared by gentle rubbing with 0.5 ml per site of 7.5 % (w/w) sodium lauryl sulphate in petrolatum. On day 8, a 2 x 4 cm patch of Whatman No. 3 paper was saturated with ca. 0.4 ml of the test material (50 % in Alembicol D); the patch was placed over the injection sites and covered by an impermeable plastic adhesive tape which was secured by an elastic adhesive bandage, wound round the torso of the animal and fixed with an impervious plastic adhesive tape. The dressing was left in place for ca. 48 hours.
- Test groups: During the induction phase, the control animals were treated similarly to the test animals with the exception that the test substance was omitted from intradermal injections and topical applications.

B. CHALLENGE EXPOSURE
The control and test animals were challenged topically two weeks after the topical induction application (day 22), using the test material (25 and 50 % w/v in Alembicol D.
Hair were clipped on the left flank of each animal and a 2 x 2 cm patch of Whatman No. 3 paper was saturated with ca. 0.2 ml of the test material. TheWhatman papers containing 50 % w/v in Alembicol D were applied on the anterior site of the flank, the Whatman papers containing 20 % w/v in Alembicol D were applied in a similar manner on the posterior site. The patches were sealed on the flank for 24 hours under impermeable adhesive tape, secured by elastic adhesive bandage, wound round the torso of the animal and fixed with an impervious plastic adhesive tape. The challenged sites were evaluated ca. 24 and 48 h after removal of the patches.

All animals were observed daily for signs of ill health or toxicity. The body weight of each animal was recorded on day 1 of the study and following completion of the study prior to termination.
The dermal reactions resulting from challenge reaction were assessed using a numerical scoring system (Draize scores).
On completion of the study the animals were sacrificed by cervical dislocation.
The sensitivity of the guinea pig strain is checked periodically by the testing laboratory with hexyl cinnamic aldehyde (HCA), a known moderate sensitizer.

Positive control substance(s):

yes

Remarks:

hexyl cinnamic aldehyde (HCA), periodically checkings

Positive control results:

The following concentrations of HCA were administered: Intradermal injection 7.5 % v/v in Alembicol D; topical application as supplied (neat); challenge application as supplied (neat) and 50 % v/v in Alembicol D.
Slight to well defined dermal reactions were observed for all test animals after challenge reaction compared to no dermal reactions in the controls. The reactions in the test animals represented hypersensitivity and therefore all test animals gave positive sensitization responses.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25 % (w/v) in Alembicol D

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50 % (w/v) in Alembicol D

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

25 % (w/v) in Alembicol D

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

50 % (w/v) in Alembicol D

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25 % (w/v) in Alembicol D

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 % (w/v) in Alembicol D

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25 % (w/v) in Alembicol D

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50 % (w/v) in Alembicol D

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

No deaths and no signs of ill health or toxicity were observed. The body weight of the animals increased within the normal range over the period of the study.

During the induction period, necrosis was recorded at sites receiving FCA in test animals and also in control animals. No irritation was seen in test animals at sites receiving the test material in vehicle and in control animals receiving vehicle alone after intradermal injection as well as after topical application.

After challenge, no dermal reactions were seen in either animals, all tested animals gave negative responses.

Interpretation of results:

GHS criteria not met

Reference 7

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Substance type: blue powder
- Analytical purity: >99%
- Lot/batch No.: SF20927
- Storage condition of test material: room temperature in the dark

Species:

mouse

Strain:

other: CBA/CaBkl

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: B & K Universal Ltd, Hull, UK
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: individually in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet: Certified Rat and Mouse Diet, Code 5LF2 (BCM IPS Ltd., London, UK), ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C
- Humidity: 30 - 70 %
- Air changes: 15 changes per hr
- Photoperiod: 12 hrs dark / 12 hrs light

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

5 %, 10 % or 25 % (w/w)

No. of animals per dose:

four animals per dose

Details on study design:

Mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for 3 consecutive days. The test material was administered by using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of 4 animals received the vehicle alone in the same manner.
Five days following the first topical application of the test material, all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR, 80 µCi/ml, specific activity 2.0 µCi/mmol; Amersham Biosciences UK Ltd.), giving a total of 20 µCi to each mouse.
Animals were observed post dose on day 1, twice daily on days 2 and 3 and on a daily basis on days 4, 5 and 6. Any signs of toxicity or ill health were recorded. The body weight of the animals was determined on day 1 prior to dosing, and on day 6, prior to termination.
5 hours following the administration of 3HTdR all animals were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the mice were excised and pooled for each animal group. For each group, 1 ml of PBS was added to the pooled lymph nodes.
A single cell suspension of the pooled lymph node cells was prepared by gentle mechanic disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze and collected into a centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (ca. 190 g) for 10 min. The pellet was resuspended in PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspenden in 3 ml of 5 % Trichloroacetic acid (TCA).
After overnight incubation at 4 °C the precipitates were removed by centrifugation at 2100 rpm (ca. 450 g) for 10 min, resuspended in 1 ml TCA and transferred to 10 ml scintillation fluid (Optiphase, "Trisafe"). 3HTdR incorporation was measured by ß-scintillation counting. The number of radioactive disintegrations per minute was then measured using a Beckman LS6500 scintillation system.

The proliferation response of the lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into the lymph node cells of test nodes relative to that recorded for the control nodes (stimulation index). The test material will be regarded as sensitizer, if at least one of the tested concentrations results in threefold greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as non-sensitizer.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Parameter:

SI

Value:

1.32

Test group / Remarks:

5%

Parameter:

SI

Value:

1.65

Test group / Remarks:

10%

Parameter:

SI

Value:

1.49

Test group / Remarks:

25%

Cellular proliferation data / Observations:

DPM data:
Test group 1: vehicle (acetone/olive oil 4:1): 683.38 dpm/node
Test group 2: 5 % test substance in vehicle: 899.50 dpm/node
Test group 3: 10 % test substance in vehicle: 1131.00 dpm/node
Test group 4: 25 % test substance in vehicle: 1020.05 dpm/node

There were no deaths. No signs of systemic toxicity was noted in the test or in the control animals during the study. Blue-colored staining on the ears, face, feet and fur was noted in all test animals during the study.

The body weight changes of the test animals during the study were comparable to those observed in the corresponding control animals over the same time period.

A stimulation index of less than 3 was recorded for the three concentrations of the test material.

The positive control alpha-hexylcinnamaldehyde provided positive results and was considered to be a sensitizer under the experimental conditions of the test.

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

All substances in this category share a similar structure with a molecular weight > 500 g/mol. Based on the high molecular weight and insolubility, the substances in this category will not be able to penetrate biological membranes. Thus, no systemic exposure is expected. Skin sensitisation studies are available for four substances in this category: CAS 147-14-8, CAS 1328-53-6, CAS 574-93-6 and CAS CAS27614-71-7. The skin sensitisation data can be used for read-across to all substances in this category.

 

CAS No. 147-14-8: 

An in vivo study with 10 female guinea pigs in the test group and 5 female guinea pigs as control group was conducted, according to the method described by Magnusson and Kligman. The intradermal induction (6 injections in groups of two per animal) was conducted with FCA, the test material 7.5 % (w/v) in Alembicol D and the test material 7.5 % (w/v) in a mixture of FCA and Alembicol D. Epicutaneous induction (one week after intradermal induction) was conducted with 50 % of the test material in Alembicol D. The challenge (14 days after epicutaneous induction) was performed with 25 % and with 50 % of the test material in Alembicol D (OECD TG 406, GLP; Huntingdon 2001, K1). No animal in the control group and no animal in the treated group exhibited positive reactions 24 or 48 hours after the challenge procedure.

 

Skin sensitisation testing was performed in female guinea pigs according to the method of MAGNUSSON & KLIGMAN (OECD TG 406) and under GLP. Intradermal induction was performed using 5 % Pigment Blue 15 in sesam oil (Aventis 2002, K1). Dermal induction and challenge treatment were carried out with 25 % Pigment Blue 15 in sesam oil. The validity of the test system is confirmed by the periodically conducted positive control test using alpha-hexylcinnamaldehyde for the maximization test. Based on the results of this study Pigment Blue 15 showed no evidence for sensitising properties according to the classification criteria of Directive 2001/59/EC.

 

In another study, a local lymph node assay was conducted according to OECD TG 429 and under GLP conditions (Safepharm 2004, K1). Four female CBA/CaBkl mice per dose (5 %, 10 % or 25 % w/w of the test material in acetone/olive oil 4:1 v/v) were treated by daily application of 25 µl of the appropriate concentration of either the test material or with vehicle alone to the dorsal surface of each ear for 3 consecutive days. Five days following the first topical application, all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 20 µCi 3H-methyl thymidine (3HTdR) to each mouse. Animals were observed post dose, any signs of toxicity or ill health were recorded. The body weight of the animals was determined prior to dosing and prior to termination. 5 hours following administration of 3HTdR all animals were killed. The draining auricular lymph nodes were excised, pooled for each group and a single cell suspension of the cells was prepared. 3HTdR incorporation was measured by ß-scintillation counting. The proliferation response of the lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into the lymph node cells of test nodes relative to that recorded for the control nodes (stimulation index). There were no early deaths during the study. No signs of systemic toxicity was noted in the test or in the control animals during the study. Blue-coloured staining on the ears, face, feet and fur was noted in all test animals during the study. The body weight changes of the test animals during the study were comparable to those observed in the corresponding control animals over the same time period. A stimulation index of less than 3 was recorded for the three concentrations of the test material, indicating no sensitizing potential of the tested material.

 

CAS No. 1328-53-6:

A local lymph node assay was conducted according to OECD TG 429 and under GLP conditions (RCC Ltd. 2004, K1). Three groups each of four female mice were treated daily with the test item at concentrations of 5 %, 10 % and 25 % (w/v) in dimethylsulfoxide (DMSO) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 25 % was the highest technically achievable concentrationinthe vehicle. A control group of four mice was treated with the vehicle (dimethylsulfoxide (DMSO)) only. Five days after the first topicalapplicationthe mice were injected intravenouslyintoa tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymp node cells was determined by the incorporation of 3H-methyl thymidine measured in aß-scintillation counter. No clinical signs were observed.All treated animals survived the scheduled study period. The results obtained (STIMULATION INDEX (S.I.)) were 1.4, 1.3 and 1.3 for all three test groups.

 

CAS No. 574-93-6:

In an OECD TG 406 and GLP complaint study (Huntingdon Life Sciences 2001, K1), the tested material did not produce evidence of skin sensitization reactions in any of the tested animals under the experimental conditions chosen. Therefore the substance is not considered to have a potential to cause skin sensitization.

 

In a second study which was conducted according to OECD TG 429 and following GLP (SafePharm Laboratories 2004, K1), the test material was considered not to be a sentisizer.

 

CAS No. 27614-71-7:

A local lymph node assay was conducted according to OECD TG 429 and under GLP conditions (RCC Ltd. 2004, K1). Groups of four female CBA mice per dose (5 %, 10 % or 25 % w/w of the test material in DMSO) were treated by daily application of 25 µl of the appropriate concentration of either the test material or with vehicle alone to the dorsal surface of each ear for 3 consecutive days. Five days following the first topical application, all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 19.9 µCi 3H-methyl thymidine (3HTdR) to each mouse. Animals were observed post dose, any signs of toxicity or ill health were recorded. The body weight of the animals was determined prior to dosing and prior to termination. 5 hours following administration of 3HTdR all animals were killed. The draining auricular lymph nodes were excised, pooled for each group and a single cell suspension of the cells was prepared. 3HTdR incorporation was measured by ß-scintillation counting. The proliferation response of the lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into the lymph node cells of test nodes relative to that recorded for the control nodes (stimulation index). There were no early deaths during the study. No signs of systemic toxicity was noted in the test or in the control animals during the study. The body weight changes of the test animals during the study were comparable to those observed in the corresponding control animals over the same time period (except for one animalof the 10 % test item concentration group which lost weight during the study, but this was considered to be incidental). No dose-response relation was observed and a stimulation index of less than 3 was recorded for the three concentrations of the test material. The test item concentration of 2.5 % produced a S.I. of 0.8, a concentration of 5.0 % produced a S.I. of 1.1 and a concentration of 10.0 % produced a S.I. of 1.0.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data on skin sensitisation, classification for skin sensitisation is not warranted in accordance with the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.