Registration Dossier

Administrative data

Endpoint:
additional toxicological information
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Histopathology start date 24 March 2017 Experimental completion date 12 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
The objective of this study was to perform a histopathological assessment on fixed guinea pig tissues (identified as SMK-PH-16-0617) provided by Phycher Bio Développement, to investigate macroscopic tissue abnormalities seen following a sensitisation study performed under Envigo project number SS90QG.
Cross-reference
Reason / purpose:
other: Supporting histopathological data

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Type of study / information:
The objective of this study was to perform a histopathological assessment on fixed guinea pig tissues (identified as SMK-PH-16-0617) provided by Phycher Bio Développement, to investigate macroscopic tissue abnormalities seen following a sensitisation study performed under Envigo project number SS90QG.
Test guideline
Qualifier:
no guideline available
Version / remarks:
The objective of this study was to perform a histopathological assessment on fixed guinea pig tissues (identified as SMK-PH-16-0617) provided by Phycher Bio Développement, to investigate macroscopic tissue abnormalities seen following a sensitisation study performed under Envigo project number SS90QG.
Principles of method if other than guideline:
Regulatory Testing Guidelines
The study was designed to meet the requirements of the following guidelines:
The UK Good Laboratory Practice Regulations (Statutory Instrument 1999 No. 3106, as amended by Statutory Instrument 2004 No. 994).
OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17.
EC Commission Directive 2004/10/EC of 11 February 2004.
The study was conducted in accordance with the requirements of current, internationally recognised Good Laboratory Practice Standards.
GLP compliance:
yes (incl. certificate)

Test material

Specific details on test material used for the study:
• Sponsor’s identification: Dichlorobis(η-cyclopentadienyl)titanium (CAS# 1271-19-8)
• Batch No.: 0708501022
• Chemical name: bis(cyclopentadienyl) titanium dichloride • CAS No.: 1271-19-8
• Date received: 16 Novembre 2016 • Storage: room temperature, darkness
• Container: smoked glass flask (n=1) • Form: cristalline powder
• Quantity: 186.00 g (container + content) • Colour: Red
• Production date: not specified • Expiry date: 03 April 2019
• Composition: see CoA in appendix

Results and discussion

Any other information on results incl. tables

 Results

Decedents

There were no early decedents during the Main study.

Macroscopic Pathology

Animals Killedat Scheduled Termination

Non-protocol skin

At the macroscopic examination, a hard mass, which was adherent to the overlying skin, was seen under the right leg of 5/10 treated animals (Group 2 animal Nos. C8071, C8072, C8074, C8075 and C8076).

Microscopic Pathology

Animals Killedat Scheduled Termination

Treatment Related Findings

Changes related to treatment with dichlorobis(η-cyclopentadienyl)titanium were seen in the skin of the inter-scapular area, the site of intradermal injection and subsequent topical application.

Dermal injection site

Minimal or slight crystalline foreign body granuloma(ta) with associated mineralization and pigmented macrophages were seen in the dermis and/or subcutis of 3/10 treated animals.

Summary of treatment related findings at the dermal injection site for animals killed at at Scheduled Termination

Group/sex

1F

2F

 

 

 

Granuloma, crystalline foreign material, with associated mineralization and pigmented macrophages, dermal/subcutaneous

 

 

Minimal

0

2

Slight

0

1

Total

0

3

 

 

 

Number of tissues examined

5

10

 

Procedure-related findings

Inter-scapular area

At the inter-scapular area used as the site for intra-dermal injection (1stinduction) and subsequent topical application (2ndinduction), a consistent finding in the skin of all control and treated animals was a slight to marked macrophage infiltrate in the dermis and subcutis. This infiltrate was considered to be an expected response to the injection of Freund’s Complete Adjuvant, a macrophage–attractant, at two of the three pairs of intra-dermal injection sites during the 1stinduction procedure. Combinations of other findings were seen in the epidermis (scabs, ulceration, erosion and hyperplasia), dermis/subcutis (abscessation, inflammatory cell infiltrate and hair shaft/keratin microgranuloma(ta)) and panniculus muscle (myofibre atrophy/degeneration/necrosis), at similar or lower relative incidences in treated animals (Group 2) compared with controls (Group 1). These changes were considered related to the combination of the intra-dermal and topical application procedures, the application of sodium lauryl sulphate (a local irritant) and the vehicles (i.e. olive oil and liquid paraffin) and/or solvent used.

Non-protocol skin (under right forelimb)

It should be noted that only subcutaneous tissue from this untreated site was presented for examination for 4/5 treated animals and that full thickness skin (epidermis, dermis and subcutis) was only present for animal No. C8075. For one animal, (No. C8072), a lymph node, probably the right axillary, was present in the tissue examined and was of normal appearance.

Moderate or marked macrophage infiltration was seen in the subcutis of all five treated animals, which accounted for the macroscopic masses under the right forelimb of each of these animals. For animal No. C8071, a minimal amount of crystalline foreign material, which was similar in appearance to the crystalline material seen in the inter-scapular skin of this animal, with associated inflammatory cell infiltrate was seen in the subcutis. Although no masses were reported in negative control animals (Group 1) in the skin under either forelimb, it was considered that the presence of the macrophage infiltrate at this non-protocol site, and thecrystalline foreign material for animal No. C8071,may have represented the effect of residual adjuvant (Freund’s Complete Adjuvant) and test item material respectively, that had possibly migrated ventrally from the scapular area due to a combination of the movement of the animal during the main study and gravity. Combinations of other findings were seen in the subcutis (abscessation, granuloma(ta) and inflammatory cell infiltrate) and panniculus muscle (myofibre atrophy/degeneration/necrosis). Therefore, in general, the findings seen in the skin caudal to the right forelimb were similar in severity and relative incidence to the findings seen at the inter-scapular skin of control and/or treated animals.

Summary of procedure-related findings in the non-protocol skin for animals killed at Scheduled Termination

Group/sex

1F

2F

 

 

 

Infiltrate, macrophage, subcutaneous

 

 

Moderate

 

1

Marked

 

4

Total

 

5

 

 

 

Crystalline foreign material, with associated inflammatory cell infiltrate, subcutaneous

 

 

Minimal

 

1

Total

 

1

 

 

 

Abscess, subcutaneous

 

 

Minimal

 

2

Slight

 

2

Total

 

4

 

 

 

Granuloma(ta), subcutaneous

 

 

Slight

 

1

Total

 

1

 

 

 

Infiltrate, inflammatory cell, subcutaneous

 

 

Minimal

 

4

Total

 

4

 

 

 

Atrophy/degeneration/necrosis, myofibre, panniculus muscle

 

 

Slight

 

1

Total

 

1

 

 

 

Number of tissues examined

0

5

 

Histopathology – Group distribution of findings

TABLE

Histopathology - group distribution of findings

Request ID: 5169436

 

 

Control

Treated

Dose Group

1

2

 

 

 

Number of animals affected

 

Group/Sex

 

 

 

 

 

1F

2F

 

 

 

 

 

Tissue/Organ and Findings

No. of animals

 

 

 

 

 

5

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Dermal IS

No. examined

 

 

 

 

 

5

10

 

 

 

 

 

    Scab(s)

Minimal

 

 

 

 

 

1

3

 

 

 

 

 

 

Slight

 

 

 

 

 

0

2

 

 

 

 

 

 

Moderate

 

 

 

 

 

3

0

 

 

 

 

 

 

Total

 

 

 

 

 

4

5

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

    Ulceration, Epidermal

Minimal

 

 

 

 

 

1

2

 

 

 

 

 

 

Slight

 

 

 

 

 

0

1

 

 

 

 

 

 

Moderate

 

 

 

 

 

3

0

 

 

 

 

 

 

Total

 

 

 

 

 

4

3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

    Hyperplasia, Epidermal

Minimal

 

 

 

 

 

0

1

 

 

 

 

 

 

Slight

 

 

 

 

 

0

4

 

 

 

 

 

 

Moderate

 

 

 

 

 

4

5

 

 

 

 

 

 

Marked

 

 

 

 

 

1

0

 

 

 

 

 

 

Total

 

 

 

 

 

5

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


 

TABLE

Histopathology - group distribution of findings

Request ID: 5169436

 

 

Control

Treated

Dose Group

1

2

 

 

 

Number of animals affected

 

Group/Sex

 

 

 

 

 

1F

2F

 

 

 

 

 

Tissue/Organ and Findings

No. of animals

 

 

 

 

 

5

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Dermal IS

No. examined

 

 

 

 

 

5

10

 

 

 

 

 

    Infiltrate, Inflammatory Cell, Dermal/Subcutaneous

Minimal

 

 

 

 

 

4

3

 

 

 

 

 

 

Slight

 

 

 

 

 

0

1

 

 

 

 

 

 

Total

 

 

 

 

 

4

4

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

    Atrophy/Degeneration/Necrosis, Myofiber, Panniculus Muscle

Minimal

 

 

 

 

 

0

4

 

 

 

 

 

 

Slight

 

 

 

 

 

1

4

 

 

 

 

 

 

Moderate

 

 

 

 

 

3

1

 

 

 

 

 

 

Total

 

 

 

 

 

4

9

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


 

TABLE

Histopathology - group distribution of findings

Request ID: 5169436

 

 

Control

Treated

Dose Group

1

2

 

 

 

Number of animals affected

 

Group/Sex

 

 

 

 

 

1F

2F

 

 

 

 

 

Tissue/Organ and Findings

No. of animals

 

 

 

 

 

5

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Dermal IS

No. examined

 

 

 

 

 

5

10

 

 

 

 

 

    Infiltrate, Macrophage, Dermal/Subcutaneous

Slight

 

 

 

 

 

0

1

 

 

 

 

 

 

Moderate

 

 

 

 

 

2

8

 

 

 

 

 

 

Marked

 

 

 

 

 

3

1

 

 

 

 

 

 

Total

 

 

 

 

 

5

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

    Microgranuloma(ta), Hair Shaft/Keratin

Minimal

 

 

 

 

 

1

0

 

 

 

 

 

 

Total

 

 

 

 

 

1

0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

    Abscess, Dermal/Subcutaneous

Minimal

 

 

 

 

 

2

1

 

 

 

 

 

 

Slight

 

 

 

 

 

1

0

 

 

 

 

 

 

Total

 

 

 

 

 

3

1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

    Granuloma(ta), Crystalline Foreign Material, with Associated Mineralization and Pigmented Macrophages, Dermal/Subcutaneous

Minimal

 

 

 

 

 

0

2

 

 

 

 

 

 

Slight

 

 

 

 

 

0

1

 

 

 

 

 

 

Total

 

 

 

 

 

0

3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


 

TABLE

Histopathology - group distribution of findings

Request ID: 5169436

 

 

Control

Treated

Dose Group

1

2

 

 

 

Number of animals affected

 

Group/Sex

 

 

 

 

 

1F

2F

 

 

 

 

 

Tissue/Organ and Findings

No. of animals

 

 

 

 

 

5

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Dermal IS

No. examined

 

 

 

 

 

5

10

 

 

 

 

 

    Erosion, Epidermal

Minimal

 

 

 

 

 

0

1

 

 

 

 

 

 

Total

 

 

 

 

 

0

1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Skin, Non-Protocol

No. examined

 

 

 

 

 

0

5

 

 

 

 

 

    Abscess, Subcutaneous

Minimal

 

 

 

 

 

0

2

 

 

 

 

 

 

Slight

 

 

 

 

 

0

2

 

 

 

 

 

 

Total

 

 

 

 

 

0

4

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

    Granuloma(ta), Subcutaneous

Slight

 

 

 

 

 

0

1

 

 

 

 

 

 

Total

 

 

 

 

 

0

1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

    Crystalline Foreign Material, with Associated Inflammatory Cell Infiltrate, Subcutaneous

Minimal

 

 

 

 

 

0

1

 

 

 

 

 

 

Total

 

 

 

 

 

0

1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


 

TABLE

Histopathology - group distribution of findings

Request ID: 5169436

 

 

Control

Treated

Dose Group

1

2

 

 

 

Number of animals affected

 

Group/Sex

 

 

 

 

 

1F

2F

 

 

 

 

 

Tissue/Organ and Findings

No. of animals

 

 

 

 

 

5

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Skin, Non-Protocol

No. examined

 

 

 

 

 

0

5

 

 

 

 

 

    Infiltrate, Macrophage, Subcutaneous

Moderate

 

 

 

 

 

0

1

 

 

 

 

 

 

Marked

 

 

 

 

 

0

4

 

 

 

 

 

 

Total

 

 

 

 

 

0

5

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

    Infiltrate, Inflammatory Cell, Subcutaneous

Minimal

 

 

 

 

 

0

4

 

 

 

 

 

 

Total

 

 

 

 

 

0

4

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

    Atrophy/Degeneration/Necrosis, Myofiber, Panniculus Muscle

Slight

 

 

 

 

 

0

1

 

 

 

 

 

 

Total

 

 

 

 

 

0

1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
Following the main study period, a test item (dichlorobis(η-cyclopentadienyl)titanium) related finding was seen in the dermis and subcutis of the skin of the inter-scapular area (crystalline foreign body granuloma(ta) with associated mineralization and pigmented macrophages).

The macroscopic masses in the untreated skin caudal to the right forelimb of 5/10 treated (Group 2) animals were accounted for by macrophage infiltration in the subcutis, a change that was also seen in the treated inter-scapular skin of all control and treated animals.
Executive summary:

Summary

The objective of this study was to perform a histopathological assessment on fixed guinea pig tissues (identified as SMK-PH-16-0617) provided byPhycher Bio Développement,to investigate macroscopic tissue abnormalities seen following a sensitisation study performed under Envigo project number SS90QG.

All dose administration and animal identification performed atPhycher Bio Développementwas as follows:

Female Dunkin-Hartley guinea pigs received two induction administrations followed by a challenge administration of the test material (treated) or sham inductions followed by a test material challenge administration (control)  

Group1 (Control): C8056, C8066, C8067, C8068 and C8069

Group 2 (Treated): C8070, C8071, C8072, C8073, C8074, C8075, C8076, C8077, C8078 and C8079

Histological processing of the tissues provided was conducted at the Eye Test Facility with pathological evaluation performed at the Huntingdon Test Facility of Envigo CRS Limited respectively.

Results

Treatment Related Findings

Changes related to treatment with dichlorobis(η-cyclopentadienyl)titanium were seen in the skin of the inter-scapular area, the site of intradermal injection and subsequent topical application.

Minimal or slight crystalline foreign body granuloma(ta) with associated mineralization and pigmented macrophages were seen at the dermal injection site in the dermis and/or subcutis of 3/10 treated animals.

Procedure-related findings

Inter-scapular area

At the inter-scapular area used as the site for intra-dermal injection (1stinduction) and subsequent topical application (2ndinduction), a consistent finding in the skin of all control and treated animals was a slight to marked macrophage infiltrate in the dermis and subcutis. This infiltrate was considered to be an expected response to the injection of Freund’s Complete Adjuvant, a macrophage–attractant, at two of the three pairs of intra-dermal injection sites during the 1stinduction procedure. Combinations of other findings were seen in the epidermis (scabs, ulceration, erosion and hyperplasia), dermis/subcutis (abscessation, inflammatory cell infiltrate and hair shaft/keratin microgranuloma(ta)) and panniculus muscle (myofibre atrophy/degeneration/necrosis), at similar or lower relative incidences in treated animals (Group 2) compared with controls (Group 1). These changes were considered related to the combination of the intra-dermal and topical application procedures, the application of sodium lauryl sulphate (a local irritant) and the vehicles (i.e. olive oil and liquid paraffin) and/or solvent used.

Non-protocol skin (under right forelimb)

It should be noted that only subcutaneous tissue from this untreated site was presented for examination for 4/5 treated animals and that full thickness skin (epidermis, dermis and subcutis) was only present for animal No. C8075. For one animal, (No. C8072), a lymph node, probably the right axillary, was present in the tissue examined and was of normal appearance.

Moderate or marked macrophage infiltration was seen in the subcutis of all five treated animals, which accounted for the macroscopic masses under the right forelimb of each of these animals. For animal No. C8071, a minimal amount of crystalline foreign material, which was similar in appearance to the crystalline material seen in the inter-scapular skin of this animal, with associated inflammatory cell infiltrate was seen in the subcutis. Although no masses were reported in negative control animals (Group 1) in the skin under either forelimb, it was considered that the presence of the macrophage infiltrate at this non-protocol site, and thecrystalline foreign material for animal No. C8071,may have represented the effect of residual adjuvant (Freund’s Complete Adjuvant) and test item material respectively, that had possibly migrated ventrally from the scapular area due to a combination of the movement of the animal during the main study and gravity. Combinations of other findings were seen in the subcutis (abscessation, granuloma(ta) and inflammatory cell infiltrate) and panniculus muscle (myofibre atrophy/degeneration/necrosis). Therefore, in general, the findings seen in the skin caudal to the right forelimb were similar in severity and relative incidence to the findings seen at the inter-scapular skin of control and/or treated animals.

Conclusion

Following the main study period, a test item (dichlorobis(η-cyclopentadienyl)titanium) related finding was seen in the dermis and subcutis of the skin of the inter-scapular area (crystalline foreign body granuloma(ta) with associated mineralization and pigmented macrophages).

The macroscopic masses in the untreated skin caudal to the right forelimb of 5/10 treated (Group 2) animals were accounted for bymacrophage infiltration in the subcutis, a change that was also seen in the treated inter-scapular skin of all control and treated animals.