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

 The LD50 oral of substance registered is in the range of >300 - 2000 mg/kg bw.  Based on the results from the available study, an oral LD50 cut-off value of 500 mg/kg bw was determined.
 The LD50 dermal was greater 2000 mg/kg bw. 

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

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

Adequacy of study:

key study

Study period:

2007-09-24 to 2008-01-28

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 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Laboratory Animal Services, Wölferstrasse 4, 4414 Füllinsdorf / Switzerland
- Age at study initiation: 11 weeks
- Weight at study initiation: 184.3 - 203.5 g
- Fasting period before study: treatment applied after being fasted for approximately 17 to 18 hours (access to water was permitted). Food was provided again approximately 3 hours after dosing.
- Housing: In groups of three in Makrolon type-4 cages with wire mesh tops and standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard rat/mouse maintenance diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 7 days

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

IN-LIFE DATES: From: 2007-09-21 To: 2007-10-11

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 200 and 30 mg/l, resp.
- Amount of vehicle (if gavage): 10 ml/kg
- Justification for choice of vehicle: Purified water was found to be a suitable vehicle. The vehicle was chosen after a non-GLP solubility trial which was performed before the study initiation date.
- Lot/batch no. (if required):
- Purity: Purified water prepared at RCC Ltd (deionised water which was processed and treated by the PURELAB Option-R unit. This latter links four purification technologies: reverse osmosis, adsorption, ion-exchange and photo oxidation).

MAXIMUM DOSE VOLUME APPLIED: 2.035 ml

DOSAGE PREPARATION (if unusual):
The dose formulations were made shortly before each dosing occasion using a magnetic stirrer, as homogenizer. The test item was first ground with a pestle and a mortar. The pulverized test item was then weighed into a tared glass beaker on a suitable precision balance and the vehicle was added (weight:volume). Homogeneity of the test item in the vehicle was maintained during administration using a magnetic stirrer.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: no data

Doses:

300 and 2000 mg/kg bw

No. of animals per sex per dose:

6 females in dose group 300 mg/kg bw
3 females in dose group 2000 mg/kg bw

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Mortality / Viability: Daily during the acclimatization period, during the first 30 minutes and at approximately 1, 2, 3 and 5 hours after administration on test day 1 (with the clinical signs) and twice daily during days 2-15.
Body Weights: On test days 1 (prior to administration), 8 and 15.
- Necropsy of survivors performed: yes
- Other examinations performed:
Clinical signs: Daily during the acclimatization period, during the first 30 minutes and at approximately 1, 2, 3 and 5 hours after administration on test day 1. Once daily during days 2-15. All abnormalities were recorded.
Further: body weight,organ weights

Statistics:

No statistical analysis was used.

Sex:

female

Dose descriptor:

LD50

Effect level:

> 300 - < 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: Analogue substance.

Mortality:

MORTALITY:
The following animals were treated and percentage of mortality was observed:
3 females treated at 2000 mg/kg 100 %
1 females treated at 300 mg/kg 17 %
One female treated at 300 mg/kg had to be killed in extremis 3 hours post-dose and the three females treated at 2000 mg/kg died spontaneously within the first hour after treatment.

Clinical signs:

other: The first group of females treated at 300 mg/kg showed a slightly ruffled fur 1 hour after treatment which persisted up to test day 2 in two females. A hunched posture was visible in two females 1-2 hours post-treatment up to the 5- hour observation. A

Gross pathology:

All three females treated at 2000 mg/kg/bw were found with liquid contents in their stomachs. Otherwise, no macroscopic findings were recorded at necropsy.

Other findings:

no data

Interpretation of results:

Category 4 based on GHS criteria

Remarks:

Migrated information: H302, harmful if swallowed

Conclusions:

The median lethal dose of test item after single oral administration to female rats, observed over a period of 14 days is: 300 mg/kg body weight < LD50 (female rat) < 2000 mg/kg body weight. Based on the proposed read across approach, the LD50 of the substance registered can be assumed to be in the same range as for the analogue substance.

Executive summary:

The acute oral toxicity to rats of test item (analogue substance) was being investigated following the testing protocol as given in OECD guideline 423.

Three groups, each of three female rats were treated with the test item by oral gavage administration at a dosage of 300 mg/kg (two groups) or 2000 mg/kg (one group) body weight. The test item was diluted in vehicle (purified water) at a concentration of 0.2 g/mL or 0.03 g/mL and administered at a dosing volume of 10 mL/kg.

The animals were examined daily during the acclimatization period and mortality, viability and clinical signs were recorded. All animals were examined for clinical signs within the first 30 minutes and approximately 1, 2, 3 and 5 hours after treatment on day 1 and once daily during test days 2-15. Mortality/viability was recorded at approximately 30 minutes, 1, 2, 3 and 5 hours after administration on test day 1 (with the clinical signs) and twice daily during days 2-15. Body weights were recorded on day 1 (prior to administration) and on days 8 and 15.All animals were necropsied and examined macroscopically.

The following animals were treated and percentage of mortality was observed:

3 females treated at 2000 mg/kg                  100 %

1 females treated at 300 mg/kg                    17 %

One female treated at 300 mg/kg had to be killed in extremis 3 hours post-dose and the three females treated at 2000 mg/kg died spontaneously within the first hour after treatment.

The median lethal dose of test item after single oral administration to female rats, observed over a period of 14 days is: 300 mg/kg body weight < LD50 (female rat) < 2000 mg/kg body weight.

Given the applicability of the proposed read across approach (see IUCLID chapter 13), the respective LD50 values of the substance registered can safely be assumed to be in the same range as for the analogue substance, as (1) the molecules of the substance registered and the analogue substance show to a large extent structural similarities, and (2) furthermore the molecular weight of the analogue substance and the substance registered only differ by 2.5%.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

discriminating dose

Value:

500 mg/kg bw

Quality of whole database:

Guidline-conform study under GLP without deviations.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

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

Adequacy of study:

key study

Study period:

2007-09-24 to 2008-01-08

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 402 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Laboratory Animal Services, Wölferstrasse 4, 4414 Füllinsdorf / Switzerland
- Age at study initiation: females 11 weeks, males 8 weeks
- Weight at study initiation: females 197.4 - 213.5 g, males 241 - 261.2 g
- Fasting period before study: no
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard rat/mouse maintenance diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 7 days

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

IN-LIFE DATES: From: 2007-09-25 To: 2007-10-16

Type of coverage:

semiocclusive

Vehicle:

water

Details on dermal exposure:

TEST SITE
- Area of exposure: backs of the animals
- % coverage: ca. no data
- Type of wrap if used: The semi-occlusive dressing was wrapped around the abdomen and fixed with an elastic adhesive bandage.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): skin was flushed with lukewarm tap water and dried with disposable paper towels
- Time after start of exposure: 24 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 6 ml/kg bw
- Concentration (if solution): 333.3 mg/l
- Constant volume or concentration used: yes

VEHICLE
- Amount(s) applied (volume or weight with unit): 6 ml/kg bw
- Purity: Purified water prepared at RCC Ltd (deionised water which was processed and treated by the PURELAB Option-R unit. This latter links four purification technologies: reverse osmosis, adsorption, ion-exchange and photo oxidation).

Duration of exposure:

24 h

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

not required

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Mortality / Viability: Daily during the acclimatization period, during the first 30 minutes and at approximately 1, 2, 3 and 5 hours after administration on test day 1 (with the clinical signs) and twice daily during days 2-15.
Clinical signs: Daily during the acclimatization period, during the first 30 minutes and at approximately 1, 2, 3 and 5 hours after administration on test day 1. Once daily during days 2-15. All abnormalities were recorded.
Weighing: On test days 1 (prior to administration), 8 and 15.
Local Signs: Once daily during days 2-15. All abnormalities will be recorded.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Statistics:

No statistical analysis was used.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: Analogue substance.

Mortality:

Male: 2000 mg/kg bw; Number of animals: 5; Number of deaths: 0
Female: 2000 mg/kg bw; Number of animals: 5; Number of deaths: 0

Clinical signs:

other: Signs of toxicity related to dose levels: No systemic signs of toxicity were observed. Local toxicity: At removal of the application patch the local skin reactions were not assessable in all animals due to a yellow staining produced by the test item. The

Gross pathology:

Effects on organs: No macroscopic findings were observed at necropsy.

Other findings:

- Organ weights: no data
- Histopathology: no data
- Potential target organs: not applicable
- Other observations:

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The median lethal dose of the test item after single dermal administration to rats of both sexes, observed over a period of 14 days is: LD50 (rat) > 2000 mg/kg body weight. Based on the proposed read across approach, the LD50 of the substance registered can be assumed to be in the same range as for the analogue substance.

Executive summary:

The acute dermal toxicity to rats of the analogue substance was being investigated following the testing protocol as given in OECD guideline 402.

Five male and five female rats were treated with the test item at 2000 mg/kg by dermal application. The test item was diluted in vehicle (purified water) at a concentration of 0.33 g/mL and administered at a volume dosage of 6 mL/kg. The application period was 24 hours.

The animals were examined daily during the acclimatization period and mortality, viability and clinical signs were recorded. Body weights were recorded. All animals were necropsied and examined macroscopically.

No deaths occurred during the study.

At removal of the application patch the local skin reactions were not assessable in all animals due to a yellow staining produced by the test item. The assessment was prevented up to day 6 in the females and day 7 in the males. The staining was present from day 7 in the females and on day 8 in the males.When assessable, a slight local erythema was noted in one male from days 8 to 9 and in all females from day 7 to 9 or 10. Slight scaling was present from day males and from days 7 or 8 up to day 10 in all females. Slight crusts were noted in one female from day 7 to 13.

The body weight of the animals was within the range commonly recorded for this strain and age.

No macroscopic findings were observed at necropsy.

The median lethal dose of the test item after single dermal administration to rats of both sexes, observed over a period of 14 days is: LD50 (rat) > 2000 mg/kg body weight.

Given the applicability of the proposed read across approach (see IUCLID chapter 13), the respective LD50 values of the substance registered can safely be assumed to be in the same range as for the analogue substance, as (1) the molecules of the substance registered and the analogue substance show to a large extent structural similarities, and (2) furthermore the molecular weights of the analogue substance and the substance registered only differ by 2.5%.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

Guidline-conform study under GLP without deviations.

Additional information

Two valid acute toxicity studies on the most relevant exposure routes, oral and dermal, have been conducted with the analogue chemical.

While in the oral acute toxicity study a LD50 in the range of 300 - 2000 mg/kg bw was determined, in the dermal study the LD50 was greater than 2000 mg/kg bw. Given the applicability of the proposed read across approach (see IUCLID chapter 13), the respective LD50 values of the substance registered can safely be assumed to be in the same range, as (1) the close structural comparability of source and target molecule, and (2) the close physic-chemical similarities of source and target molecules, as demonstrated in the read across justification, IUCLID chapter 13.

Justification for selection of acute toxicity – oral endpoint
The selected study was performed under GLP and in accordance with OECD TG 423. No other studies are available.

Justification for selection of acute toxicity – dermal endpoint
The selected study was performed under GLP and in accordance with OECD TG 402. No other studies are available.

Justification for classification or non-classification

Acute oral toxicity:

Based on the former DSD regulation, the test item (analogue substance) is to be classified as harmful Xn; R22 "harmful if swallowed" concerning its acute oral toxicity properties. Referring to the new CLP regulation, the test item is to be classified as "Acute Tox 4 oral", H302 "harmful id swallowed".

Based on the applicability of the proposed read across approach, the same classification (harmful if swallowed) is likely to apply to target chemical (substance registered) as well.

Acute dermal toxicity:

No classification within the EU according to the new CLP and the former DSD regulation. Note that in certain countries outside the EU the OECD-GHS acute toxicity category 5 may be applied, i.e. 2000 - 5000 mg/kg bw. This dose level however has not been covered by this study, due to animal welfare concerns.