Reaction mass of 5,5'-{(phenylmethanediyl)bis[benzene-4,1-diyl-diazene-2,1-diyl]}bis{1-[3-(dimethylamino)propyl]-4-methyl-6-oxo-3-(pyridinium-1-yl)-1,6-dihydropyridin-2-olate} hydrochloride and 5,5’-[3,4’-(phenylmethanediyl)diphenylene]bis(diazene-2,1-diyl)bis{1-[3-(dimethylamino)propyl]-4-methyl-6-oxo-3-(pyridinium-1-yl)-1,6-dihydropyridin-2-olate} hydrochloride

Endpoint summary

Description of key information

While substance registered is considered to be not irritating to skin, it is suspected to cause irreversible effects on eyes, which supports the respective classification.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

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

Adequacy of study:

key study

Study period:

2007-10-02 to 2007-12-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations, conducted with the analogue substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands BV, Kreuzelweg 53, NL-5961 NM Horst / The Netherlands, Postbus 6174, NL-5960 AD Horst / The Netherlands
- Age at study initiation: 11 weeks (male), 13 weeks (females)
- Weight at study initiation: First Day of Acclimatization: 2027, 2547, 2512 g; Last Day of Observation: 2697, 3377, 3346 g
- Housing: Individually in stainless steel cages equipped with feed hoppers and drinking water bowls.
- Diet (e.g. ad libitum): Pelleted standard rabbit maintenance diet ad libitum
- Water (e.g. ad libitum): Community tap water ad libitum.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2007-10-10 To: 2007-10-29

Type of coverage:

semiocclusive

Preparation of test site:

other: clipped with an electric clipper

Vehicle:

water

Controls:

not required

Amount / concentration applied:

500 mg

Duration of treatment / exposure:

4 h

Observation period:

14 days

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: left flank
- % coverage:
- Type of wrap if used: test item was placed on a surgical gauze patch (ca. 2.5 cm x 2.5 cm). This gauze patch was applied to the intact skin of the clipped area. The patch was covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with tape.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): after remocval of dressing the skin was flushed with lukewarm tap water
- Time after start of exposure: 4 hours

SCORING SYSTEM: The skin reaction was assessed according to the numerical scoring system listed in the Commission Directive 2004/73/EC, April 29, 2004.

Irritation parameter:

erythema score

Basis:

mean

Remarks:

average of animals 1, 2, and 3

Time point:

other: overall at 1, 24, and 48 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 7 d

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

other: 72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

erythema score

Basis:

animal: 1 and 2

Time point:

other: 72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

other: mean score for all three animals

Time point:

other: overall at 1, 24, 48 and 72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

Reversibility of any observed effect: Changes fully reversible within 7 days at latest

Other effects:

VIABILITY/MORTALITY/CLINICAL SIGNS: No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.
IRRITATION: The skin assessment of all animals was prevented by a marked yellow staining produced by the test item 1-48 hours after treatment and persisted as slight staining up to day 14. When assessable at the 72-hour observation a very slight erythema was noted in one female, only. No oedema was present in all animals during the whole observation period.
COLORATION: A marked yellow staining produced by the test item was recorded in all animals at the 1-hour observation and persisted as slight staining up to day 14.
CORROSION:
Neither alterations of the treated skin were observed nor were corrosive effects evident on the skin.
BODY WEIGHTS: The body weights of all rabbits were considered to be within the normal range of variability.

Interpretation of results:

not irritating

Remarks:

Migrated information Erythema score could not be investigated in detail, as substance left a yellow staining on the treated skin. However based on the available data the substance still can be considered "not irritating". Criteria used for interpretation of results: OECD GHS

Conclusions:

Based on the classification criteria according to CLP and DSD, the analogue substance is considered to be “not irritating” to rabbit skin regarding the oedema and erythema. Non-classification is likely to apply to the substance registered as well.

Executive summary:

The primary skin irritation potential of the analogue substance was being investigated following the testing protocol as given in OECD guideline 404.

The analogue substance was applied by topical semi-occlusive application of 0.5 g to the intact left flank of each of three young adult New Zealand White rabbits. The duration of treatment was four hours. The scoring of skin reactions was performed 1, 24, 48 and 72 hours, as well as 7, 10 and 14 days after removal of the dressing.

The skin assessment of all animals was prevented by a marked yellow staining produced by the test item 1-48 hours after treatment and persisted as slight staining up to day 14. When assessable at the 72-hour observation a very slight erythema was noted in one female, only. No oedema was present in all animals during the whole observation period.

The mean score is usually calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar grades and for oedema grades, separately.

The skin assessment of all animals was prevented 1-48 hours after treatment by a yellow staining produced by the test item. Therefore, the mean score was not calculated.

No corrosive effects were noted on the treated skin of any animal at any of the measuring intervals and no clinical signs were observed.

Thus, the test item did not induce significant or irreversible damage to the skin.

Based upon the referred classification criteria (Commission Directive 2001/59/EC of August 2001), the analogue substance is considered to be “not irritating” to rabbit skin regarding the oedema. Although the erythema assessment was not possible from the 1- to 48-hour observations due to a yellow staining, the test item is assumed to be “not irritating” for the erythema, as well.

Given the applicability of the proposed read across approach (see IUCLID chapter 13) non-classification is likely to apply to the substance registered as well.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

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

Adequacy of study:

key study

Study period:

2007-10-22 to 2008-01-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations, conducted with the analogue substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands BV, Kreuzelweg 53, NL-5961 NM Horst / The Netherlands, Postbus 6174, NL-5960 AD Horst / The Netherlands
- Age at study initiation: 13 weeks (male), 13 - 14 weeks (females)
- Weight at study initiation: First Day of Acclimatization: 2187, 2523, 2418 g; Last Day of Observation: 3021, 3411, 3444 g
- Housing: Individually in stainless steel cages equipped with feed hoppers and drinking water bowls.
- Diet (e.g. ad libitum): Pelleted standard rabbit maintenance diet ad libitum
- Water (e.g. ad libitum): Community tap water ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2007-10-24 To: 2007-11-20

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

100 mg

Duration of treatment / exposure:

21 days

Observation period (in vivo):

21 days

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

SCORING SYSTEM: The eye reactions were assessed according to the numerical scoring system listed in the Commission Directive 2004/73/EC, April 29, 2004 (study report page 30). Scleral reddening and ocular discharge were also assessed.

TOOL USED TO ASSESS SCORE: hand-slit lamp

Irritation parameter:

cornea opacity score

Remarks:

degree of corneal opacity

Basis:

mean

Remarks:

animals no. 1, 2, and 3

Time point:

other: overall at 24, 48, and 72 h

Score:

1

Max. score:

4

Reversibility:

other: staining not fully reversible within 21 days in one female,

Remarks on result:

other: One female with a very slight opacity of the cornea on day 21. Another test animal showed fully reversible reaction within 21 days, whereas the third test animal recovered fully within 7 days.

Irritation parameter:

iris score

Basis:

mean

Remarks:

animals no. 1, 2, and 3

Time point:

other: overall at 24, 48, and 72 h

Score:

0.44

Max. score:

2

Reversibility:

fully reversible within: 2 days or 7 days, respectively

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

mean

Remarks:

animals no. 1, 2, and 3

Time point:

other: overall at 24, 48 and 72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 10 days or 14 days, respectively

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animals no. 1, 2, and 3

Time point:

other: overall at 24, 48 and 72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

other: sclera

Basis:

mean

Remarks:

animals no. 1, 2, and 3

Time point:

other: overall at 24, 48, and 72 h

Score:

1.66

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The individual mean scores for corneal opacity were 1.00 for each of the three animals and for iris were 0.00, 1.00 and 0.33, respectively. The individual mean scores for the conjunctivae were 2.00 for reddening and 1.00 for chemosis, for each of the three animals.
One hour after instillation, the opacity of the cornea and the reddening of the conjunctivae as well as the sclerae of the three animals were not assessable due to a marked yellow staining produced by the test item. When assessable at the 24-hour reading, a very slight opacity of the cornea affecting the whole area was observed in all animals. The opacity persisted up to day 10 and 21, respectively affecting more than one quarter or more than half of the cornea and was due to the staining caused by the test item. The iris of both females showed a delayed / reduced light reflex at the 24-hour observation up to the 72-hour reading in one female. Additionally, a moderate reddening of the conjunctivae was noted in all animals 24 hours after treatment and persisted as slight up to day 7 or 10, respectively. An obvious swelling with partial eversion of the lids was recorded in the three animals at the 1-hour observation and persisted as slight swelling up to the 72-hour evaluation. Furthermore, a slight to moderate reddening of the sclerae was present in the treated animals 24-72 hours after treatment.
Moderate ocular discharge was noted in all animals 1 hour after instillation and persisted as slight discharge up to the 72-hour observation.
No abnormal findings were observed in the treated eye of two animals 10 and 21 days after treatment, respectively. One female was still observed with a very slight opacity affecting more than half of the cornea on day 21, i.e. one of three animals showed persistent corneal effect.

Other effects:

MORTALITY/CLINICAL SIGNS: No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.

COLORATION:
A marked yellow staining produced by the test item was observed in each animal one hour after instillation and persisted as slight up to day 21.

CORROSION:
No corrosion of the cornea was observed at any of the reading times. BODY WEIGHTS: The body weights of all rabbits were considered to be within the normal range of variability.

TEST ITEM REMNANTS:
Yellow test item remnants were evident in the eye or conjunctival sac of each animal 1 hour after treatment and persisted up to the 24- or 72-hour reading.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Remarks:

Migrated information

Conclusions:

One of three test animals showed a persistent corneal effect (i.e. staining) which triggers classification for eye damage. Given the applicability of the proposed read across approach (see IUCLID chapter 13) the same classification also apply to the substance registered.

Executive summary:

The primary eye irritation potential of the analogue substance was being investigated following the testing protocol as given in OECD guideline 405.

The analogue substance was applied by instillation of 0.1 g into the left eye of each of three young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours, as well as 7, 10, 14, 17 and 21 days after test item instillation.

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The individual mean scores for corneal opacity were 1.00 for all three animals and for iris were 0.00, 1.00 and 0.33, respectively. The individual mean scores for the conjunctivae were 2.00 for reddening and 1.00 for chemosis, for each of the three animals.

 

The instillation of the analogue substance into the eye resulted in mild to moderate, early-onset and transient ocular changes, such as very slight opacity of the cornea, delayed/reduced light reflex, reddening of the conjunctivae and sclerae, discharge and chemosis. These effects were reversible in two animals and were no longer evident at the latest 10 and 21 days after treatment, respectively. One female was still observed with a very slight opacity affecting more than half of the cornea on day 21, the end of the observation period. No corrosion was observed at any of the measuring intervals. A marked yellow staining of the treated eyes by the test item was observed which persisted as slight up to day 21, the end of the observation period. Yellow test item remnants were evident in the eye or conjunctival sac of each animal 1 hour after treatment and persisted up to the 24- or 72 hours. No clinical signs were observed.

One of three test animals showed a persistent corneal effect (i.e. staining) which triggers classification for eye damage. Given the applicability of the proposed read across approach (see IUCLID chapter 13) the same classification also apply to the substance registered.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Two valid in-vivo studies on skin irritation and eye irritation have been conducted with the analogue substance. The applicability of the read across approach had been confirmed by the registrant via an expert statement (see IUCLID chapter 13).

While the result of the skin irritation study was "not irritating", the result of the eye irritation study was "irreversible effects on the eye".

No clinical signs of toxicity were observed in the animals during the OECD 404 study and no mortality occurred. The skin assessment of all animals was prevented by a marked yellow staining produced by the test substance 1 to 48 hours after treatment and persisted as slight staining up to day 14. When assessable at the 72 hour observation a very slight erythema was noted in one female only. No edema was present in all animals during the 14 day observation period.

The instillation of the analogue substance into the eye resulted in mild to moderate, early-onset and transient ocular changes, such as very slight opacity of the cornea, delayed/reduced light reflex, reddening of the conjunctivae and sclerae, discharge and chemosis. These effects were reversible in two animals and were no longer evident at the latest 10 and 21 days after treatment, respectively. One female was still observed with a very slight opacity affecting more than half of the cornea on day 21, the end of the observation period. No corrosion was observed at any of the measuring intervals. A marked yellow staining of the treated eyes by the test item was observed which persisted as slight up to day 21, the end of the observation period.

Justification for selection of skin irritation / corrosion endpoint:
The selected study was performed under GLP and in accordance with OECD TG 404. No other studies are available.

Justification for selection of eye irritation endpoint:
The selected study was performed under GLP and in accordance with OECD TG 405. No other studies are available.

Effects on eye irritation: highly irritating

Justification for classification or non-classification

Based on the new CLP regulation, the analogue substance is considered to be classified as "Eye damage 1", H318 "causes serious eye damage", which requires the labelling "corrosive". Based on the former DSD classification, the analogue substance is considered to be "irritant" Xi;R41 "Risk of serious damage to eyes” (labelling St. Andrew's Cross). Given the applicability of the proposed read across approach, the same classification applies to the substance registered as well.