Benzenamine, N-phenyl-, styrenated

Endpoint summary

Description of key information

Skin sensitisation: In vivo, Guinea Pig Maximisation Assay, OECD 406, GLP, Intradermal/epicutaneous, induction 0.5% and undiluted, challenge 50%/100%: 24h resp. 48h after challenge, none of the ten test animals showed dermal responses, not sensitising acc. to Regulation 1272/2008.

Skin sensitisation: In vivo, Bühler Assay, OECD 406, GLP, epicutaneous, undiluted: 24h resp. 48h after challenge, 2 out of 20 resp. 1 out of 20 animals showed erythematous responses, not sensitising acc. to Regulation 1272/2008.

Skin sensitisation: In vivo, Guinea Pig Maximisation Assay, OECD 406, GLP, Intradermal/epicutaneous, induction 7.5%, challenge 50%/100%: Dermal reactions were noted in four of the ten test animals at challenge, sensitising Cat 1B acc. to Regulation 1272/2008. 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-04-17 - 2001-05-18 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

EEC Methods for the determination of toxicity, Annex to Directive 96/54/EC (Official Journal No. L248, 30.9.96), Part B, Method B.6. Skin sensitization

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

OECD Guideline for Testing of Chemicals No. 406 "Skin Sensitization". Adopted 17 July 1992.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

EPA Health Effects Test Guidelines OPPTS 870.2600 "Skin Sensitization" EPA 712-C-98-197. August 1998.

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The present study was conducted for another purpose than REACH prior to the acceptance of OECD or comparable guidelines for a LLNA or in vitro methods.

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:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: D. Hall, Newchurch, Staffordshire, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: four to seven weeks of age on arrival
- Weight at study initiation: 315 - 450 g at the start of the study (Day 1)
- Housing: in groups of five in suspended metal cages with wire mesh floors. For environmental enrichment, autoclaved hay was given to the guinea-pigs three times weekly at irregular intervals and plastic tubular pipes were included in the cage. These procedures, which alleviate boredom and stereotype behaviours are standard practice at this laboratory and are not considered to have any influence on test results interpretation.
- Diet (e.g. ad libitum): vitamin C enriched guinea-pig diet (Harlan Teklad 9600 FD2 SQC) ad libitum
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: 5 days
- Indication of any skin lesions: none stated

ENVIRONMENTAL CONDITIONS
Animal room environmental controls were set to maintain:
- Temperature (°C): 21 ± 3°C
- Humidity (%): 30-70%
Any minor deviations from these ranges would not have had an adverse effect on the animals and would not affect the integrity or validity of the study. These environmental parameters were continuously recorded and the permanent record archived with other departmental raw data.
- Photoperiod (hrs dark / hrs light): Lighting was controlled by means of a time switch to give 12 hours of artificial light (0600 - 1800 hours GMT) in each 24 hour period.
- IN-LIFE DATES: From: To:

Route:

intradermal

Vehicle:

other: Alembicol D

Concentration / amount:

0.5% v/v

Day(s)/duration:

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:

a 2 x 4 cm patch of Whatman No. 3 paper was saturated with approximately 0.4 ml

Day(s)/duration:

on day 7 for 48 hours

Adequacy of induction:

highest technically applicable concentration used

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: undiluted and in Alembicol D

Concentration / amount:

undiluted and 50% v/v
A 2 x 2 cm patch of Whatman No. 3 paper was saturated with approximately 0.2 ml.

Day(s)/duration:

on day 22, two weeks after second induction, for 24h

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

10 test animals, 5 control animals

Details on study design:

RANGE FINDING TESTS:
TEST SUBSTANCE PREPARATION
A vehicle trial conducted with compound showed that it formed an amber coloured thick liquid at a concentration of 75% v/v in Alembicol D (a product of coconut oil, supplied by Alembic Products, Saltney, Chester, England). The maximum practical concentration was as supplied for topical application and the formulation passed through a needle at 10% v/v (as required for intradermal injections).
The test substance was prepared prior to each application on the day of dosing in Alembicol D. The concentrations used are described in the treatment procedure.
The absorption of the test substance was not determined.
The homogeneity, stability and purity of the test substance were the responsibility of the Sponsor.

TREATMENT PROCEDURE
Preliminary study
The intradermal and topical irritancy of a range of dilutions of the test substance was investigated to identify where possible (a) the minimum irritant test substance concentrations suitable for the induction phase of the main study and (b) a maximum non-irritant concentration by the topical route of administration and a dilution of this for the challenge phase.
The animals for the topical irritancy investigations were pre-treated with an intradermal injection of Freund's Complete Adjuvant, 50:50 with water for irrigation (Ph.Eur.), approximately one week prior to the start of the preliminary investigations.
The procedure employed for these investigations was as follows:
Intradermal injections - Intradermal injections (0.1 ml/site) were made into the clipped and shaved flank of two guinea-pigs, using a range of concentrations (0.1 to 10% v/v) of the test item in Alembicol D. The resulting dermal responses were assessed approximately 24 and 72 hours later.
Topical application - Patches of Whatman No. 3 paper (2 cm x 2 cm) were saturated (volume approximately 0.2 ml per patch) with a range of concentrations (25% v/v to as supplied) of the test item in Alembicol D and applied to the clipped and shaved flanks of each of four guinea-pigs. The patches were covered by a strip of "Blenderm" and firmly secured by "Elastoplast" wound round the trunk and fixed with an impervious plastic adhesive tape. The dressings were removed after an exposure period of approximately 24 hours and the reaction sites were assessed for erythema and oedema (reported as 0 hours). Further examination of the sites was carried out approximately 24 and 48 hours after removal of the dressings.
Selection of concentrations of test substance for the main study
Based on the results of the preliminary investigations, the following concentrations of the test item were selected:
Induction intradermal injection - 0.5% v/v in Alembicol D
This was the highest concentration that caused irritation but did not cause necrosis or give signs of toxicity.
Induction topical application - As supplied
Topical challenge - As supplied and 50% v/v in Alembicol D
From preliminary investigations the test material administered topically as supplied did not give rise to irritating effects.

MAIN STUDY
A. INDUCTION EXPOSURE
Induction intradermal injections - test animals
A 4 x 6 cm area of dorsal skin on the scapular region of the guinea-pig was clipped free of hair with electric clippers. On Day 1, three pairs of intradermal injections (0.1 ml/site) were made into a 2 x 4 cm area within the clipped area.
Injectables for the test animals were prepared as follows:
1. Freund's Complete Adjuvant was diluted with an equal volume of water for irrigation (Ph.Eur.).
2. Test item, 0.5% v/v in Alembicol D.
3. Test item, 0.5% v/v in a 50 : 50 mixture of Freund's Complete Adjuvant and Alembicol D.
Induction topical application - test animals
The preliminary investigations indicated the test substance applied topically as supplied did not produce skin irritation. Therefore, on Day 7 the same 4 x 6 cm 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% w/w sodium lauryl sulphate in petrolatum. On Day 85 a 2 x 4 cm patch of Whatman No. 3 paper was saturated with approximately 0.4 ml of the test item, as supplied. The patch was placed over the injection sites on the skin of the interscapular region of the test animals and covered by a length of impermeable plastic adhesive tape (5 cm width "Blenderm"). This in turn was firmly secured by elastic adhesive bandage (5 cm width "Elastoplast") wound round the torso of the animal and fixed with an impervious plastic adhesive tape. The dressing was left in place for approximately 48 hours.
Induction - control animals
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 for test and control animals to the intradermal injections were recorded 24 hours following the injections and the reactions to the induction topical application were recorded on removal of the bandages.

B. CHALLENGE EXPOSURE
Challenge - control and test animals
The control and test animals were challenged topically two weeks after the topical induction application (Day 22) 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 2 x 2 cm patch of Whatman No. 3 paper was saturated with approximately 0.2 ml of the test item, as supplied and applied to an anterior site on the flank. The test item, 50% v/v in Alembicol D was applied in a similar manner to the posterior site. The patches were sealed to the flank for 24 hours under strips of "Blenderm" (5 cm width) secured with "Elastoplast" (7.5 cm width) wound round the trunk and fixed with an impervious plastic adhesive tape.
The challenge sites were evaluated approximately 24 and 48 hours after removal of the patches.

Positive control substance(s):

yes

Remarks:

The sensitivity of the guinea-pig strain used is checked periodically at the laboratory with hexyl cinnamic aldehyde (HCA), a known moderate sensitizer.

Positive control results:

RESULTS
INDUCTION
Intradermal injections: Necrosis was recorded at all sites receiving Freund's Complete Adjuvant. Slight irritation was seen in test animals at sites receiving HCA, 10% v/v in Alembicol D and slight irritation was observed in control animals receiving Alembicol D.
Topical application: Slight to moderate erythema was observed in test animals following topical application with HCA, as supplied. No erythema was seen in the control animals receiving a dry patch.
CHALLENGE
Slight to well-defined dermal reactions were observed for all of the ten test animals compared to no dermal reactions in the control animals, therefore the reactions in the test animals represented hypersensitivity and all ten test animals gave positive sensitization responses.
CONCLUSION
In this study HCA produced evidence of skin sensitisation (delayed contact hypersensitivity) in all of the ten animals, thus confirming the sensitivity of the strain of animals and reliability of the experimental technique.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

as supplied and 50% v/v

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

There were no dermal reactions seen.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

as supplied and 50% v/v

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

There were no dermal reactions seen.

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

There were no dermal reactions seen.

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

There were no dermal reactions seen.

Remarks on result:

no indication of skin sensitisation

Reading:

other: not stated

Group:

positive control

Dose level:

Intradermal injection: 10% v/v in Alembicol D; Topical application: As supplied (neat); Challenge application: As supplied (neat) and 50% v/v in Alembicol D

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

Slight to well-defined dermal reactions were observed for all of the ten test animals compared to no dermal reactions in the control animals, therefore the reactions in the test animals represented hypersensitivity.

Remarks on result:

positive indication of skin sensitisation

Interpretation of results:

GHS criteria not met

Conclusions:

The study was conducted under GLP according to OECD guideline 406 (GPMT) on the registered substance itself without deviations. The method is considered to be scientifically reasonable with no deficiencies in documentation. Negative and positive controls gave the appropriate response. Hence, the results can be considered as reliable to assess the skin sensitizing potential of the test item. In this study the test item did not produce evidence of skin sensitization (delayed contact hypersensitivity) in any of the ten test animals. The test item is not considered to have the potential to cause skin sensitization.
As all of the animals gave negative responses, the test item does not require labelling as skin sensitizer.

Executive summary:

This study was performed to assess the skin sensitization potential of the test item using the guinea-pig. The method followed was that described in:

EEC Methods for the determination of toxicity, Annex to Directive 96/54/EC (Official Journal No. L248, 30.9.96), Part B，Method B.6. Skin sensitization.

OECD Guideline for Testing of Chemicals No. 406 "Skin Sensitization". Adopted 17 July 1992.

EPA Health Effects Test Guidelines OPPTS 870.2600 "Skin Sensitization" EPA 712-C-98-197. August 1998.

MAGNUSSON, B. and KLIGMAN, A.M. (1970) Allergic Contact Dermatitis in the Guinea-pig: Identification of contact allergens, Thomas, C.C" Springfield, Illinois, U.S.A.

The guinea-pigs were dosed by intradermal injection and topical application, as these are the routes of exposure required by the test guidelines and method.

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

Intradermal injection: 0.5% v/v in Alembicol D

Topical application: As supplied

Challenge application: As supplied and 50% v/v in Alembicol D

Ten test and five control guinea-pigs were used in this study.

In this study the test item did not produce evidence of skin sensitization (delayed contact hypersensitivity) in any of the ten test animals. The test item is not considered to have the potential to cause skin sensitization.

As all of the animals gave negative responses, the test item does not require labelling with the risk phrase R43 "May cause sensitization by skin contact" in accordance with Commission Directive 93/21/EEC. The same applies for labelling as skin sensitizer according to Regulation 1272/2008 and amendments.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2000-08-22 - 2000-09-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

EEC Methods for the determination of toxicity, Annex to Directive 96/54/EC (Official Journal No. L248, 30.9.96), Part B, Method B.6. Skin sensitization.

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

OECD Guideline for Testing of Chemicals No. 406 "Skin Sensitization". Adopted 17 July 1992.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

EPA Health Effects Test Guidelines OPPTS 870.2600 "Skin Sensitization" EPA 712-C-98-197. August 1998.

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The present study was conducted for another purpose than REACH prior to the acceptance of OECD or comparable guidelines for a LLNA or in vitro methods.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: D. Hall, Newchurch, Staffs, UK.
- Age at study initiation: approx. 5 - 8 weeks
- Weight at study initiation: 362 - 425 g
- Housing: The guinea-pigs were housed in groups of five in suspended metal cages with wire mesh floors. For environmental enrichment, autoclaved hay was given to the guinea-pigs three times weekly at irregular intervals and plastic tubular pipes were included in the cage. These procedures, which alleviate boredom and stereotype behaviours are standard practice at this laboratory and are not considered to have any influence on test results interpretation.
- Diet (e.g. ad libitum): vitamin C enriched guinea-pig diet (Harlan Teklad 9600 FD2 SQC) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C
- Humidity (%): 30 to 70%.
- Photoperiod (hrs dark / hrs light): Lighting was controlled by means of a time switch to give 12 hours of artificial light (0600 - 1800 hours GMT) in each 24 hour period.

Route:

intradermal and epicutaneous

Vehicle:

other: Alembicol D (product of coconut oil, supplied by Alembic Products, Saltney, Chester, England)

Concentration / amount:

7.5% v/v in Alembicol D (induction)
50% / 100% v/v in Alembicol D (challenge)

Route:

epicutaneous, occlusive

Vehicle:

other: Alembicol D (product of coconut oil, supplied by Alembic Products, Saltney, Chester, England)

Concentration / amount:

7.5% v/v in Alembicol D (induction)
50% / 100% v/v in Alembicol D (challenge)

No. of animals per dose:

10 females (test group)
5 females (control group)

Details on study design:

RANGE FINDING TESTS:
Preliminary study
The intradermal and topical irritancy of a range of dilutions of the test substance was investigated to identify where possible (a) the minimum irritant test substance concentrations suitable for the induction phase of the main study and (b) a maximum non-irritant concentration by the topical route of administration and a dilution of this for the challenge phase.
The animals for the topical irritancy investigations were pre-treated with an intradermal injection of Freund's Complete Adjuvant, 50: 50 with water for irrigation (Ph.Eur., sterile water), approximately one week prior to the start of the preliminary investigations.
The procedure employed for these investigations was as follows;
Intradermal injections - Intradermal injections (0.1 ml/site) were made into the clipped and shaved flank of two guinea-pigs, using a range of concentrations (0.1 to 10% v/v) of the test item in a suitable vehicle (Alembicol D). The resulting dermal responses were assessed approximately 24 and 72 hours later.
Topical application - Patches of Whatman No. 3 paper (2 cm x 2 cm) were saturated (volume approximately 0.2 ml per patch) with a range of concentrations (25 % v/v to as supplied) of the test item in a suitable vehicle (Alembicol D) and applied to the clipped and shaved flanks of each of four guinea-pigs. The patches were covered by a strip of "Blenderm" and firmly secured by "Elastoplast" wound round the trunk and fixed with an impervious plastic adhesive tape. The dressings were removed after an exposure period of approximately 24 hours and the reaction sites were assessed for erythema and oedema. Further examination of the sites was carried out approximately 24 and 48 hours after removal of the dressings.
Selection of concentrations of test substance for the main study
Based on the results of the preliminary investigations, the following concentrations of the test item were selected:
Induction intradermal injection - 7.5% v/v in Alembicol D
This was the highest concentration that caused irritation but did not adversely affect the animals.
Induction topical application - As supplied
Topical challenge - As supplied and 50% v/v in Alembicol D
The test material applied topically as supplied did not give rise to irritating effects.


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Test groups:
Induction
Induction intradermal injections - test animals
A 4 x 6 cm area of dorsal skin on the scapular region of the guinea-pig was clipped free of hair with electric clippers. Three pairs of intradermal injections (0.1 ml/site) were made into a 2 x 4 cm area within the clipped area as shown in Figure 1.
Injectables for the test animals were prepared as follows:
1. Freund's Complete Adjuvant was diluted with an equal volume of water for irrigation (Ph.Eur.).
2. The test item, 7.5% v/v in Alembicol D.
3. The test item, 7.5% v/v in a 50 : 50 mixture of Freund's Complete Adjuvant and Alembicol D.

Induction topical application - test animals
The preliminary investigations indicated that the neat material applied topically did not produce skin irritation. Therefore, the same 4 x 6cm interscapular area was clipped and shaved free of hair on Day 5 and the site was pre-treated by gentle rubbing with 0.5 ml per site of 10% w/w sodium lauryl sulphate in petrolatum on Day 6. On Day 7, a 2 x 4 cm patch of Whatman No. 3 paper was saturated with approximately 0,4 ml of The test item, as supplied. The patch was placed over the injection sites on the skin of the interscapular region of the test animals and covered by a length of impermeable plastic adhesive tape (5 cm width "Blenderm"). This in turn was firmly secured by elastic adhesive bandage (5 cm width "Elastoplast") wound round the torso of the animal and fixed with an impervious plastic adhesive tape. The dressing was left in place for approximately 48 hours.

- Control group:
Induction - control animals
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 to the intradermal injections were recorded 24 hours following the injections and the reactions to the induction topical application were recorded on removal of the bandages.

- Site: dorsal skin on the scapular region
- Frequency of applications: day 1 and 7
- Duration: 48h (topical induction)
- Concentrations: 7.5%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 2 weeks after topical induction
- Exposure period: 24h
- Test groups, Control group:
Challenge - control and test animals
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 2 x 2 cm patch of Whatman No. 3 paper was saturated with approximately 0.2 ml of the test item, as supplied and applied to an anterior site on the flank. The test item, 50% v/v in Alembicol D was applied in a similar manner to the posterior site. The patches were sealed to the flank for 24 hours under strips of "Blenderm" (5 cm width) secured with "Elastoplast" (7.5 cm width) wound round the trunk and fixed with an impervious plastic adhesive tape.

- Site: left flank
- Concentrations: 50% v/v in Alembicol D
- Evaluation (hr after challenge): 24, 48, 72h after removal of challenge patches

Positive control substance(s):

yes

Remarks:

Hexyl cinnamic aldehyde (HCA), the sensitivity of the guinea-pig strain used is checked periodically at Huntingdon Life Sciences.

Positive control results:

INDUCTION
Intradermal injections
Necrosis was recorded at all sites receiving Freund's Complete Adjuvant.
Well-defined irritation was seen in test animals at sites receiving HCA, 10% v/v in Alembicol D and slight irritation was observed m control animals receiving Alembicol D.
Topical application
Slight erythema was observed in test animals following topical application with HCA, as supplied. No erythema was seen in the control animals receiving Alembicol D.

CHALLENGE
Slight to well-defined dermal reactions were observed for all of the ten test animals compared to no dermal reactions in the control animals, therefore all ten test animals gave positive sensitization responses.

CONCLUSION
In this study HCA produced evidence of skin sensitization (delayed contact hypersensitivity) in all of the ten animals, thus confirming the sensitivity and reliability of the experimental technique.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

4

Total no. in group:

10

Clinical observations:

No signs of ill health or toxicity were observed.

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 4.0. Total no. in groups: 10.0. Clinical observations: No signs of ill health or toxicity were observed..

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

2

Total no. in group:

10

Clinical observations:

No signs of ill health or toxicity were observed

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: No signs of ill health or toxicity were observed.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

4

Total no. in group:

10

Clinical observations:

No signs of ill health or toxicity were observed

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 4.0. Total no. in groups: 10.0. Clinical observations: No signs of ill health or toxicity were observed.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

No signs of ill health or toxicity were observed

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: No signs of ill health or toxicity were observed.

Reading:

other: 3rd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

100%

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

No signs of ill health or toxicity were observed

Remarks on result:

other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 100%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: No signs of ill health or toxicity were observed.

Reading:

other: 3rd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

50%

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

No signs of ill health or toxicity were observed

Remarks on result:

other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 50%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: No signs of ill health or toxicity were observed.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100% + 50%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No signs of ill health or toxicity were observed

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 100% + 50%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No signs of ill health or toxicity were observed.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100% + 50%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No signs of ill health or toxicity were observed

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 100% + 50%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No signs of ill health or toxicity were observed.

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

100% + 50% HCA

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

Slight to well-defined dermal reactions were observed for all of the ten test animals.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 100% + 50% HCA. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: Slight to well-defined dermal reactions were observed for all of the ten test animals..

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

100% + 50% HCA

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

Slight to well-defined dermal reactions were observed for all of the ten test animals

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 100% + 50% HCA. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: Slight to well-defined dermal reactions were observed for all of the ten test animals.

Interpretation of results:

other: potential skin sensitizer

Remarks:

Criteria used for interpretation of results: EU GHS

Conclusions:

The study was conducted under GLP according to OECD guideline 406 (GPMT) on the registered substance itself without deviations. The method is in general to be considered scientifically reasonable with no deficiencies in documentation. Negative and positive controls gave the appropriate response. Hence, the results can be considered in general as reliable to assess the skin sensitizing potential of the test item. At challenge, dermal reactions were noted in four of the ten test animals compared to none in controls, therefore these four test animals gave positive responses. The remaining six animals gave negative responses.
According to Regulation 1272/2008 and amendments, in the GPMT, the following percentages of animals should show positive responses to be classified as skin sensitizer Category 1:
- ≥ 30 % responding at ≤ 0.1 % intradermal induction dose or ≥ 60 % responding at > 0.1 % to ≤ 1 % intradermal induction dose (sub-category 1A)
- ≥ 30 % to < 60 % responding at > 0.1 % to ≤ 1 % intradermal induction dose or ≥ 30 % responding at > 1 % intradermal induction dose (sub-category 1B)
In the present study, 40% responded to an induction with 7.5% of the test item. These values are hence within limit values ≥ 30 % responding at > 1 % intradermal induction dose for classification as skin sensitization Cat. 1B. Therefore, the test item may be considered to be a skin sensitiser under the test conditions of this study. However, the test conditions were chosen harsher, i.e. way above the limit values as given by the C&L regulation.

Executive summary:

In a dermal sensitization study (OECD 406) with the test item in Alembicol D, young adult Dunkin-Hartley guinea pigs (15 females) were tested using the method of Magnuson-Kligman. Hexyl cinnamic aldehyde (HCA) served as positive control.

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

During induction with intradermal injections, necrosis was recorded at sites receiving Freund's Complete Adjuvant in test and control animals. Slight irritation was seen in test animals at sites receiving the test item, 7.5% v/v in Alembicol D and slight irritation was also observed in control animals receiving Alembicol D. With topical application, no erythema was observed in test animals following topical application with the test item, as supplied. No erythema was seen in the control guinea-pigs.

At challenge, dermal reactions were noted in four of the ten test animals compared to none in controls, therefore these four test animals gave positive responses. The remaining six animals gave negative responses. The test item is considered to have the potential to cause skin sensitization.

Positive and negative controls gave the appropriate results.

In this study, the test item is a dermal sensitizer and may be classified as skin sensitizing Cat. 1B according to Regulation 1272/2008.

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study without relevant deviations on the registered substance itself. The prolongation of the challenge exposure is not expected to lead to an underestimation of the sensitizing potential of the test item.

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

Bühler test

Deviations:

yes

Remarks:

challenge exposure was 24h instead of 6h

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Type of study:

Buehler test

Justification for non-LLNA method:

The present study was conducted for another purpose than REACH prior to the acceptance of OECD or comparable guidelines for a LLNA or in vitro methods.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Porcellus
- Age at study initiation: 5 to 9 weeks at the time of receipt plus at least nine days for acclimatization, i.e. 6 to 11 weeks at initiation
- Weight at study initiation: 266 and 367 g at the time of receipt, study initiation: 319 - 415 g
- Housing: The animals were housed in single sex groups of up to five animals. Hanging stainless steel cages with wire-mesh floors were used, each measuring 62 cm x 62 cm x 25 cm. Trays for excreta were placed beneath each cage and the absorbent pad provided as a tray-liner was changed at least once weekly.
- Diet (e.g. ad libitum): Pelleted diet (FD1, S.Q.C., Special Diets Services Ud.) ad libitum
- Water (e.g. ad libitum): water from the public supply ad libitum
- Acclimation period: at least 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19° to 23°C
- Humidity (%): 30% to 70% R.H.
- Photoperiod (hrs dark / hrs light): 12 hour day and 12 hour night

The guinea pig was selected for this study as it is the preferred species nominated in the OECD guidelines for skin sensitisation testing. The Dunkin-Hartley strain supplied by Harlan Porcellus has proven susceptibility to known skin sensitisers at this laboratory.

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100%

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100%

No. of animals per dose:

10 (test item), 5 (control)

Details on study design:

RANGE FINDING TESTS:
The flanks of each animal in groups of two male and two female guinea pigs (7 to 12 weeks old), were closely shorn. On the following day, doses (0.3 ml) of several dilutions of the test material were absorbed onto 16 cm² filter paper patches. The patches were applied to skin on the shorn flanks, covered by occlusive tape and retained by an elastic adhesive bandage for 24 hours. The dermal test sites were washed with corn oil after removal of the dressings. On the day after removal of the patches and bandages the dermal test sites were examined for signs of irritation which were scored using a four point scale. The undiluted test material was selected for topical induction and challenge, as that formulation proved to be non-irritant in the dose range-finding tests.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 15 days
- Test groups: 10 males, 10 females, undiluted test item
- Control group: 5 males, 5 females, corn oil
- Site: denuded left flank
- Frequency of applications: Days 1, 8 and 15
- Duration: each 24h
- Concentrations: undiluted

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: day 28
- Exposure period: 24h
- Test groups: undiluted test item
- Control group: undiluted test item
- Site: denuded right flank
- Concentrations: undiluted
- Evaluation (hr after challenge): 0, 24, 48h

OTHER:

Main test
The main test was conducted using a group of ten male and ten female guinea pigs together with a control group of five males and five females (6 to 11 weeks old). Individual bodyweights were recorded at the beginning of the main study and before challenge. The test procedure was divided into two stages:

a) Induction
Each guinea pig was subject to induction by occluded topical application of corn oil (control group) or undiluted test item (test group) on Days 1, 8 and 15.
The left flank of each animal was closely shorn on the day before each of the induction applications using electric clippers followed by an electric razor. A 16 cm2 patch of filter paper was moistened with 0.3 ml of undiluted test item and placed onto the denuded left flank of each test group animal. The patches were covered with occlusive tape and held in place by an elastic adhesive bandage for 24 hours. Similar patches of filter paper moistened with corn oil were applied to the control guinea pigs. The sites of induction were washed with corn oil after removal of the bandages. Dermal reactions to the induction procedure were recorded on the day after removal of the patches.

b) Challenge
Challenge was carried out four weeks after the first phase of induction.
Hair was removed from the right flank of each test and control animal by clipping and shaving on the day before challenge. On Day 28 a 4 cm2 patch of filter paper, moistened with 0.1 ml of undiluted test item was placed onto the shaven area. The test patch was covered by occlusive tape and held in position by elastic adhesive bandage. Control animals were treated with the same formulation of test material that was applied to test animals. The patches were removed after 24 hours and the challenge sites were washed with corn oil. Dermal reactions to challenge were assessed shortly after removal of the challenge patches, 24 hours after challenge and 48 hours after challenge. Each response was scored using a four point scale.
The result of the test is expressed as the number of positive responses shown by the test animals at 24 and/or 48 hours after rem oval of the challenge patches. The frequency of positive responses rather than their intensity is regarded as the important statistic in this test.

Challenge controls:

5/5 m/f guinea pigs treated with corn oil during induction were exposed to the undiluted test item.

Positive control substance(s):

not required

Positive control results:

not applicable

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

2

Total no. in group:

20

Clinical observations:

erythematous responses

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 2.0. Total no. in groups: 20.0. Clinical observations: erythematous responses.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

erythematous responses, one of the same animals showing reactions after 24h

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: erythematous responses, one of the same animals showing reactions after 24h.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

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:

10

Remarks on result:

no indication of skin sensitisation

Group:

positive control

Remarks on result:

not measured/tested

Interpretation of results:

GHS criteria not met

Conclusions:

The study was conducted under GLP according to OECD guideline 406 (Bühler test) on the registered substance itself with one deviations which does not affect the potential of the study to detect sensitising substances. The method is to be considered scientifically reasonable with no deficiencies in documentation. Negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the skin sensitising potential of the test item. 2 out of 20 resp. 1 out of 20 animals showed erythematous responses 24h resp. 48h after the removal of the challenge patches. According to Regulation 1272/2008, in the Bühler assay, the following percentages of animals should show positive responses to be classified as skin sensitiser Category 1:
- ≥ 15 % responding at ≤ 0.2 % topical induction dose or ≥ 60 % responding at > 0.2 % to ≤ 20 % topical induction dose (sub-category 1A)
- ≥ 15 % to < 60 % responding at > 0.2 % to ≤ 20 % topical induction dose or ≥ 15 % responding at > 20 % topical induction dose (sub-category 1B)
In the present study, only 10% resp. 5% responded to an induction with the undiluted test item. These values are above the limit values for classification.
Therefore, the test item is considered to not be a skin sensitiser under the test conditions of this study.

Executive summary:

In a dermal sensitisation study (OECD 406) with the undiluted test item, young adult Dunkin-Hartley guinea pigs (10/sex) were tested using the method of Bühler.

24h resp. 48h after challenge, 2 out of 20 resp. 1 out of 20 animals showed erythematous responses. This result does not meet the criteria according to Regulation 1272/2008 for classification as skin sensitiser Cat. 1A or 1B.

In this study, the test item is not a dermal sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

There are three reliable Klimisch 1 OECD 406 GLP guideline studies in vivo with guinea pigs on the registered substance itself available. One study was performed according to Bühler, the other two according to Magnusson-Kligman.

 

The Guinea Pig Maximisation Test (GPMT) of Magnusson and Kligman involves i.a. intradermal injections during induction and employs Freund’s adjuvant. The latter serves as an enhancer of the immune system as it contains proteins which stimulate unspecific the immune system. Also, intradermal injections present the test item to the immune system to a greater extent than epicutaneous induction. Basically, the test was designed to identify securely all possible sensitizers, which leads to a higher rate of false-positive results. This design hence overestimates the possible hazard of a substance for humans, as usually the immune systems is not unspecific induced and exposure is mostly limited to epidermal contact to the substance.

The first available GPMT did not show any indication of the skin sensitizing potential of the substance. 24h and 48h after challenge, none of the animals showed any skin reactions, gradings were consistently zero in all animals. Test concentrations (intradermal inductions) were chosen properly, positive and negative controls gave the appropriate responses, and clearly no classification as skin sensitizer is triggered.

The second GPMT lead to a classification as skin sensitizer cat. 1B according to Regulation 1272/2008, the Bühler assay further did not. The need for classification according to Regulation 1272/2008 depends on the exceeding of certain limit values. The relevant values (percentage of animals showing responses) for classification as Cat. 1B are given here:

Guinea pig maximisation test: ≥ 30 % to < 60 % responding at > 0,1 % to ≤ 1 % intradermal induction dose or ≥ 30 % responding at > 1 % intradermal induction dose

Buehler assay: ≥ 15 % to < 60 % responding at > 0,2 % to ≤ 20 % topical induction dose or ≥ 15 % responding at > 20 % topical induction dose

After 24h at challenge, 40% (4/10) of the guinea pigs responded to an induction with 7.5% of the test item in the GPMT. 40% are to a minor extend above the limit value of 30%, and 7.5% are way higher than the stipulated 1%. The test conditions were chosen harsher, i.e. way above the limit values as given by the C&L regulation. So, lesser concentration could possible have led to a negative result, as shown in the selected key study. Basically, the overestimating test design leads marginally to a positive result. Hence this substance may be considered as a borderline case for classification as skin sensitizer Cat. 1B.

Further, the GPMT was conducted on a previous version of the recent product which is basically similar to the recent version but has a slightly differing impurity profile. It is definitively known that the test item in the GPMT has a content of diphenylamine (DPA) of up to 2.5%, and further 1.5% unknown impurities. It is possible that those impurities may be aminic UV-stabilizers, which are not present any more in the recent product, i.e. the registered substance. DPA is not a dermal sensitizer, but the unknown impurities may have led to the positive result.

 

The Bühler assay involves no adjuvant and exposure to the test item is limited to epicutaneous contact. This may lead to a diminished sensitivity compared to the GPMT, but the Bühler assay has a higher relevance for human exposure as it mimics it more precisely. Further, the test item in the Bühler assay matches with regard to the impurity profile better with the registered substance compared to the GPMT (borderline case) test item.

 

In general, both test designs are relevant for human risk assessment; one diminishes the risk for a false-negative result, the other is more relevant for real human exposure. Ideally, different test designs should not lead to a different outcome with regard to the necessity for classification of a substance as skin sensitizer. However, the GPMT lead to a classification as skin sensitizer cat. 1B according to Regulation 1272/2008, the Bühler assay did not. Here, the test item’s ID needs to be regarded, and it must be evaluated which one meets better the impurity profile of the registered substance. This is clearly the one used in the Bühler assay, which is hence more relevant here, and which was confirmed with the negative GPMT on a suitable test item.

 

Further, considering the properties of Substituted Diphenylamines (The American Chemical Councils RAPA Panel, has derived a “Substituted Diphenylamines” category of chemicals for this substance) in general, a negative outcome of a skin sensitization test is more realistic, too. For the various members of the group, several data on REACH-relevant endpoint, i.a. skin sensitization, is available. SDPAs are widely used lipophilic antioxidants. They are made up of a diphenylamine core and one to four alkyl or phenyl side chains. The common synthetic pathway for the production of SDPAs is through an electrophilic aromatic substitution reaction between an olefin and diphenylamine (DPA) through reductive alkylation. For full details, please refer to the respective publication: ENV/JM/MONO(2016)50),www.oecd.org/document/23/0,3343,en_2649_34379_33957015_1_1_1_1,00.html. They all bear similarities in structures as well as in physicochemical and toxicological properties. An excerpt of the properties of various members is displayed in the following table:

 

CAS	EC	Name	In vivo & in vitro skin irritation	In vivo & in vitro eye irritation	Genetic toxicity (gene and chromosome mutations),	Skin sensitisation evaluation or local lymph node assay
10081-67-1	233-215-5	Benzenamine, 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]-	Not irritating	Not irritating	Negative ± S9	sensitising (rare allergen)
101-67-7	202-965-5	Benzenamine, 4-octyl-N-(4-octylphenyl)	No data	No data	Negative ± S9	No data
15721-78-5	239-816-9	Benzenamine, 4-(1,1,3,3-tetramethylbutyl)-N-[4-(1,1,3,3-tetramethylbutyl)phenyl]-	Not irritating	Not irritating	Negative ± S9	not sensitising
36878-20-3	253-249-4	Benzenamine, ar-nonyl-N-(nonylphenyl)-	Not irritating	Not irritating	Negative ± S9	not sensitising
68411-46-1	270-128-1	Benzenamine, N-phenyl-, reaction products with 2,4,4-trimethylpentene	Not irritating	Not irritating	Negative ± S9	not sensitising
68921-45-9	272-940-1	Benzenamine, N-phenyl-, reaction products with styrene and 2,4,4-trimethylpentene	Not irritating	Not irritating	Negative ± S9	not sensitising
68442-68-2	270-485-3	Registered substance: Benzenamine, N-phenyl-, styrenated	Not irritating	Not irritating	Negative ± S9	2 contradicting studies

 

As shown in the table above, all members of the category “Substituted Diphenylamines” consistently showed no irritating effects, neither to the skin nor eye, were consistently negative in various genotoxicity tests (gene mutations in bacteria and mammalian cells, chromosome aberrations), and are considered to be not sensitizing. Hence, it can be reasonably assumed that this applies also to the registered substance. Further, non-irritating effects may be seen indicative for a certain non-reactivity with skin components, i.e. proteins, and negative results in genotoxicity tests are indicative that no DNA-binding has occurred. Last but not least, ToxTree modelling [Ideaconsult Ltd (2004-2013). Estimation of Toxic Hazard – A decision Tree approach, version 2.6.6,http://toxtree.sourceforge.net/] did not identify anyskin sensitisation reactivity domains alertsfor the registered substance. So it can be reasonably concluded that the registered substance is incapable to react with proteins in order to form antigens, which is the initial step in skin sensitization. In consequence, it can be reasonably concluded from read-across and SAR data that the registered substance does not need to be regarded as skin sensitizer.

 

Summarizing, both the negative Bühler assay and GPMT on a proper test item, the borderline-positive GPMT on a test item with an unsuitable impurity profile, negative read-across data from the category “Substituted Diphenylamines” and SAR estimations allow the conclusion that the registered substance does not need to be classified as skin sensitizer. There is no indication given that the obtained results are not relevant for human risk assessment. The tonnage-driven data requirements under REACH are fully met, and no data gaps were hence identified.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin Sensitisation:

For more details, see above. There is various data on skin sensitisation available. Regulation 1272/2008 explicitly foresees weight of evidence approaches, so the given data is suitable to assess the necessity for classification of the registered substance. Both the negative Bühler and GPMT assay on a proper test item, the borderline-positive GPMT on a test item with an unsuitable impurity profile, negative read-across data from the category “Substituted Diphenylamines” and SAR estimations allow the conclusion that the registered substance does not need to be classified as skin sensitiser.