4-Ethoxy-4'-(trans-4-ethylcyclohexyl)-2,3-difluor-1,1'-biphenyl

Endpoint summary

Description of key information

Under the given experimental conditions of an OECD 406 study, a read-across source substance induced no reactions.

A read-across source substance was concluded to be no skin sensitiser in an OECD 429 study.

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:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

For this endpoint one-to-one read across was performed to chemical similar compounds of the same chemical class with a comparable phys. chem. profile and similar responses in biological assays. The relevant studies were performed according to GLP and the methods applied are fully compliant with OECD TG. Please see chapter 13 for a detailed justificaton report.

Reason / purpose for cross-reference:

read-across source

Key result

Parameter:

SI

Value:

1.12

Test group / Remarks:

Test Group: 10 %

Key result

Parameter:

SI

Value:

2.02

Test group / Remarks:

Test Group: 5 %

Key result

Parameter:

SI

Value:

1.6

Test group / Remarks:

Test Group 2.5 %

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jul 02 - Sep 26, 2007

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)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands B.V. Postbus 6174 NL - 5960 AD Horst
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: 19.4 +/- 0.8 g
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-85
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: day 1 To: day 5

Vehicle:

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

Concentration:

pre test for signs of irritation: 1, 2.5, 5, and 10 % (v/v)
main test: 2.5, 5, and 10 % (v/v)

No. of animals per dose:

pre test: 2
main test: 5 (f) per group (3 test groups, 1 control group) total 20 (f)

Details on study design:

RANGE FINDING TESTS:
To determine the highest non-irritant test concentration or the highest technically applicable concentration, a pretest was performed in two mice on three consecutive days. The data of a solubility experiment showed that the highest test item concentration, which could be technically used was a 10 % solution. At concentrations of 1 and 2.5 % the treated mouse did not show any signs of irritation. At concentrations of 5 and 10 % the treated mouse showed reddening of the ear skin only after the third application of the test item. The test item in the main study was, therefore, assayed at 2.5, 5, and 10 %.


MAIN STUDYANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD 429
- Criteria used to consider a positive response: 3-fold greater response at one concentration in SI than control animals

TREATMENT PREPARATION AND ADMINISTRATION:
a) Topical application of 25 µL test item preparation (test group) or vehicle (control group)
b) five days after topical application: iv application of 3H-methyl thymidin
c) prior each treatment (a and b) ear thickness measurement
d) five hours after treatment (b) necropsy and analysis of the 3H thymidin incorporation in draining lymph nodes
e) method: pooled per animal

Positive control substance(s):

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

Statistics:

Dunnets Test

Positive control results:

Conc. SI
5 %: 2.43
10 % 4.07
25 % 4.88

Key result

Parameter:

SI

Value:

1.6

Test group / Remarks:

Test Group 2.5 %

Key result

Parameter:

SI

Value:

2.02

Test group / Remarks:

Test Group: 5 %

Key result

Parameter:

SI

Value:

1.12

Test group / Remarks:

Test Group: 10 %

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
DPM per lymph node
Control: 793.6
2.5 %: 1267.1
5 %: 1599.8
10 %: 889.4

DETAILS ON STIMULATION INDEX CALCULATION
ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes

EC3 CALCULATION
The EC3 value could not be calculated since all SIs were below 3.

CLINICAL OBSERVATIONS
No deaths occurred during the study period.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

SIGNS OF TOXICITY
No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.
The measured ear thickness of all animals treated was recorded prior to the 1stapplication and prior to treatment with 3HTdR. A relevant increase in ear thickness gain was not observed after treatment with the test item.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was concluded to be no skin sensitiser.

Executive summary:

In the study the test item was dissolved in acetone:olive oil (4+1) was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5, and 10 %. The GLP study was performed according to OECD 429. The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.  A relevant increase in ear thickness gain could not be observed after treatment with the test item. In this study Stimulation Indices (S.I.) of 1.60, 2.02, and 1.12 were determined with the test item at concentrations of 2.5, 5, and 10 % in acetone:olive oil (4 +1), respectively. Since the S.I. was not increased above 3 at any test group, and EC3 could not be determined. From the data it can be concluded that the test item is not a skin sensitiser under the conditions of this assay.

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

April 10 - May 17, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed in compliance with the Good Laboratory Practice (GLP) regulations (revised in 1997, ENV/MC/CHEM(98)17). The method followed that described in the OECD Guidelines for Testing of Chemicals, Updated Guideline No 406 Skin Sensitisation (adopted 17 Julyl 1992).

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The data have been generated before teh OECD 429 protocol was mandatory for REACH.

Species:

guinea pig

Strain:

other: HsdPoc:DH

Sex:

male/female

Details on test animals and environmental conditions:

Test System: Guinea pig, HsdPoc:DH, females
Breeder: Charles River Kisslegg
Age: about 7 weeks

The mean initial body weight at the start of the study was 400 g (range from 362 to 446 g).

In a large number of tests, with a variety of test materials, the guinea pig and the specific strain used has proven to be a species well suited for skin sensitization studies.

Identification and adaption
Healthy young animals were allocated to the study groups at least 7 days before dosing to allow them to acclimatize. The guinea pigs were identified by colour mark, whereas the pretest animals were marked with a different colour than the animals of group 1 and 2 (details are documented in the raw data).
Assignment
30 guinea pigs were used in this study.
Group 1: negative control group (5 females + 5 males)
Group 2: test material group (10 females + 10 males)

Housing:
The guinea pigs were housed in a 10 m2 room of the Test Institute. Lighting was controlled by a timer to provide a 12-hour light - 12-hour dark regime.
Five guinea pigs were housed in type GM/5 (EBECO) Makrolon cages with a shelter and placed on mobile racks. The animals were kept on conventional softwood granulate as the bedding. The cages had been machine cleaned before the start of the study. The bedding was changed two times a week.
Temperature and atmospheric humidity were measured by a thermohygrograph. The room temperature within the study period was 23 to 26 °C and the relative atmospheric humidity 45 to 65 %.
Diet and community tap water from Makrolon drinking bottles were available to the guinea pigs ad libitum. The diet, Altromin Standard Diet, was checked according to the specifications of the manufacturer by independent laboratories. Analysis included both qualitative and quantitative evaluation for heavy metals, aflatoxins, pesticides and antibiotics. The tap water was analyzed periodically according to the German regulations for human drinking water. The softwood granulate was analytically checked by independent laboratories.

Route:

other: intradermal and topical

Vehicle:

paraffin oil

Concentration / amount:

see details on study design

Route:

other: topical

Vehicle:

paraffin oil

Concentration / amount:

see details on study design

No. of animals per dose:

Control group: 10 (5/5)
Test group 20 (10/10)

Details on study design:

PRE TEST
To determine the concentrations suitable for the main study, a pretest with single intradermal or topical administrations of the vehicle and of test material preparations was performed. Intradermal (i.d.) injections were given to one animal. Topical (top.) applications were given to another two animals.
In the pretest the following concentrations were used:
1. Liquid paraffin ( vehicle) intradermal: undiluted
2. Test material: intradermal: 50, 25, 10, 5, and 1 g/L paraffin
topical: 250, 100, 50, and 10 g/L paraffin

MAIN STUDY
A1. INDUCTION EXPOSURE (intradermal injection)
After shaving of the shoulder region, six intradermal injections were given (three on each side of the spinal column in a total area of 2 x 4 cm). The guinea pigs received the following injections of 0.1 mL each:

Group 1 (vehicle: Liquid paraffin)
cranial: 0.05 mL Freund's complete adjuvant + 0.05 mL sodium chloride solution
medial: 0.1 mL Liquid paraffin
caudal: 0.05 mL Freund's complete adjuvant + 0.05 mL sodium chloride solution

Group 2 (test material)
cranial: 0.05 mL Freund's complete adjuvant + 0.05 mL sodium chloride solution
medial: 0.1 mL (25 g test material/L ad liquid paraffin)
caudal: 0.1 mL Freund's complete adjuvant + sodium chloride solution (25 g test material/L preparation)

A2. INDUCTION EXPOSURE (topical application)
One week after the intradermal injections, the shoulder area of the guinea pigs was shaven
again and on injection sites covered with a filter paper patch of about 8 cm2, fully soaked with 1 mL of the test material preparation or the vehicle. The patches were attached for 48 hours under occlusive conditions.

Group 1: Liquid paraffin
Group 2: Test material (100 g/L ad Liquid paraffin)

B. CHALLENGE EXPOSURE
Two weeks after the topical induction the challenge was performed by fixing filter papers of about 4 cm2 fully loaded with 0.5 mL of the test material preparations (10 g/l liquid paraffin) to the left and soaked with 0.5 mL liquid paraffin (undiluted) to the right shaven flank ofthe animals. The patches were fixed for 24 hours with occlusive plastic tape.

Observation schedule
Clinical investigations
During the induction phase the guinea-pigs were examined daily for local skin reactions, and the results were documented. The challenge sites were investigated for reactions 48 hours after start of the challenge. Further inspections followed 72 hours after start of the challenge, to detect weak or slowly developing reactions.

Following the grading according to the EEC Directive 2001/59/EEC, a result in an adjuvant method is considered positive if 30 % or more ofthe test animals showed positive reactions.

Body Weight
The body weight of the guinea-pigs was determined prior to the start of the study, then on day 8, 15, 22, and at the end of the study.

Positive control substance(s):

yes

Remarks:

DNCB

Positive control results:

50 % positive reactions with control substance

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25 g/L (intradermal induction I), 100 g/L (topical induction II), 10 g/L (topical challenge)

No. with + reactions:

5

Total no. in group:

20

Clinical observations:

no signs of toxicity

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

25 g/L (intradermal induction I), 100 g/L (topical induction II), 10 g/L (topical challenge)

No. with + reactions:

3

Total no. in group:

20

Clinical observations:

no signs of toxicity

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

0 g/L induction, 0 g/L challenge

No. with + reactions:

1

Total no. in group:

10

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

0 g/L induction, 0 g/L challenge

No. with + reactions:

1

Total no. in group:

10

Reading:

1st reading

Hours after challenge:

48

Group:

other: challenge control

Dose level:

Induction included intradermal injection of the test material formulation (25 g/L with and without FCA) on day 1, and topical application of the test material formulation (100 g/L) on day 8, challenge with liquid paraffin

No. with + reactions:

4

Total no. in group:

20

Reading:

2nd reading

Hours after challenge:

72

Group:

other: challenge control

Dose level:

Induction included intradermal injection of the test material formulation (25 g/L with and without FCA) on day 1, and topical application of the test material formulation (100 g/L) on day 8, challenge with liquid paraffin

No. with + reactions:

3

Total no. in group:

20

Findings in the induction phase

After intradermal injection, the common signs of irritation after injection of Freund's complete adjuvant were observed. The injection sites were swollen and red, later on scabs developed.

 

Findings after challenge

Group 1: negative control group

The challenge was performed on the right flank with the vehicle liquid paraffin. On the left flank, test material preparation (10 g/L paraffin) was applied. In one animal positive reactions in both treated areas were observed. All the other animals showed no positive reactions in the exposed areas.

 

Group 2: test material group

The challenge was performed on the right flank with liquid paraffin and the left flank with test material (10 g/L paraffin) preparation.

With liquid paraffin alone, 4 of 20 animals showed positive reactions after challenge.

After challenge with the test material (10 g/L paraffin), positive reactions in the treated areas were observed in 5 of 20 animals (25 %).

 

Under the given experimental conditions, animal reacted to the test material and not to the vehicle. All the other animals showed positive reactions on both sites. Overall, the course of the reactions was not typical for sensitization and it is concluded that there is some irritating potential. Nevertheless, it was calculated with a sensitization rate of 25 %.

 

Clinical findings and mortality

The clinical behavior of the guinea pigs was normal during the experimental part. All animals survived the experimental part.

 

Body weight

The body weight development corresponded to that of the animals of the vehicle group.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the given experimental conditions, the test material induced no reactions.

Executive summary:

The purpose of this GPMT assay, according to OECD 406 was to identify the contact allergenic potential of the test material. This study should provide a rational basis for risk assessment to the sensitising potential of the test item in man.

Findings in the induction phase

After intradermal injection, the common signs of irritation after injection of Freund's complete adjuvant were observed. The injection sites were swollen and red, later on scabs developed.

Findings after challenge

Group 1: negative control group

The challenge was performed on the right flank with the vehicle liquid paraffin. On the left flank, test material preparation (10 g/L paraffin) was applied. In one animal positive reactions in both treated areas were observed. All the other animals showed no positive reactions in the exposed areas.

Group 2: test material group

The challenge was performed on the right flank with liquid paraffin and the left flank with test material (10 g/L paraffin) preparation.

With liquid paraffin alone, 4 of 20 animals showed positive reactions after challenge.

After challenge with the test material (10 g/L paraffin), positive reactions in the treated areas were observed in 5 of 20 animals (25 %).

Under the given experimental conditions, animal reacted to the test material and not to the vehicle. All the other animals showed positive reactions on both sites. Overall, the course of the reactions was not typical for sensitization and it is concluded that there is some irritating potential. Nevertheless, it was calculated with a sensitization rate of 25 %.

Clinical findings and mortality

The clinical behavior of the guinea pigs was normal during the experimental part. All animals survived the experimental part.

Body weight

The body weight development corresponded to that of the animals of the vehicle group.

Conclusion

Under the given experimental conditions, the test material induced no reactions. According to EU Regulation No. 1272/2008 (CLP), the test material is not classified as a skin sensitizer.

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

For this endpoint one-to-one read across was performed to chemical similar compounds of the same chemical class with a comparable phys. chem. profile and similar responses in biological assays. The relevant studies were performed according to GLP and the methods applied are fully compliant with OECD TG. Please see chapter 13 for a detailed justificaton report.

Reason / purpose for cross-reference:

read-across source

Reading:

2nd reading

Hours after challenge:

72

Group:

other: challenge control

Dose level:

Induction included intradermal injection of the test material formulation (25 g/L with and without FCA) on day 1, and topical application of the test material formulation (100 g/L) on day 8, challenge with liquid paraffin

No. with + reactions:

3

Total no. in group:

20

Reading:

1st reading

Hours after challenge:

48

Group:

other: challenge control

Dose level:

Induction included intradermal injection of the test material formulation (25 g/L with and without FCA) on day 1, and topical application of the test material formulation (100 g/L) on day 8, challenge with liquid paraffin

No. with + reactions:

4

Total no. in group:

20

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25 g/L (intradermal induction I), 100 g/L (topical induction II), 10 g/L (topical challenge)

No. with + reactions:

5

Total no. in group:

20

Clinical observations:

no clinical signs

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

25 g/L (intradermal induction I), 100 g/L (topical induction II), 10 g/L (topical challenge)

No. with + reactions:

3

Total no. in group:

20

Clinical observations:

no signs of toxicity

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

0 g/L

No. with + reactions:

1

Total no. in group:

10

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

0 g/L

No. with + reactions:

1

Total no. in group:

10

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

No study data with the test item is available for this endpoint. Therefore, a read-across to two read-across source substances with a very similar chemical structure and comparable physico-chemical parameters is used to evaluate the endpoint for the test item.

Key study - OECD 429

In the study the test item was dissolved in acetone:olive oil (4+1) was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5, and 10 %. The GLP study was performed according to OECD 429. The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.  A relevant increase in ear thickness gain could not be observed after treatment with the test item. In this study Stimulation Indices (S.I.) of 1.60, 2.02, and 1.12 were determined with the test item at concentrations of 2.5, 5, and 10 % in acetone:olive oil (4 +1), respectively. Since the S.I. was not increased above 3 at any test group, and EC3 could not be determined. From the data it can be concluded that the test item is not a skin sensitiser under the conditions of this assay.

 

Supporting study - OECD 406 

The purpose of this GPMT assay, according to OECD 406 was to identify the contact allergenic potential of the test material. This study should provide a rational basis for risk assessment to the sensitising potential of the test item in man.

Findings in the induction phase

After intradermal injection, the common signs of irritation after injection of Freund's complete adjuvant were observed. The injection sites were swollen and red, later on scabs developed.

Findings after challenge

Group 1: negative control group

The challenge was performed on the right flank with the vehicle liquid paraffin. On the left flank, test material preparation (10 g/L paraffin) was applied. In one animal positive reactions in both treated areas were observed. All the other animals showed no positive reactions in the exposed areas.

Group 2: test material group

The challenge was performed on the right flank with liquid paraffin and the left flank with test material (10 g/L paraffin) preparation.

With liquid paraffin alone, 4 of 20 animals showed positive reactions after challenge.

After challenge with the test material (10 g/L paraffin), positive reactions in the treated areas were observed in 5 of 20 animals (25 %).

Under the given experimental conditions, animal reacted to the test material and not to the vehicle. All the other animals showed positive reactions on both sites. Overall, the course of the reactions was not typical for sensitization and it is concluded that there is some irritating potential. Nevertheless, it was calculated with a sensitization rate of 25 %.

Clinical findings and mortality

The clinical behavior of the guinea pigs was normal during the experimental part. All animals survived the experimental part.

Body weight

The body weight development corresponded to that of the animals of the vehicle group.

Conclusion

Under the given experimental conditions, the test material induced no reactions. According to EU Regulation No. 1272/2008 (CLP), the test material is not classified as a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin sensitisation, the test item does not require classification for causing skin sensitisation according to Regulation (EC) No 1272/2008 (CLP).