Pyridinium, 1,1'-[(6,13-dichloro-4,11-disulfo-3,10-triphenodioxazinediyl)bis[imino-2,1-ethanediylimino[6-[(2,5-disulfophenyl)amino]-1,3,5-triazine-4,2-diyl]]]bis[3-carboxy-, dihydroxide, bis(inner salt), hexasodium salt

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 day dosing males, typical 55 days dosing females.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Good 2016 study which includes observation of colour changes in organs and GI tract.

Data source

Reference

Reference Type:

other company data

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Pyridinium, 1,1'-[(6,13-dichloro-4,11-disulfo-3,10-triphenodioxazinediyl)bis[imino-2,1-ethanediylimino[6-[(2,5-disulfophenyl)amino]-1,3,5-triazine-4,2-diyl]]]bis[3-carboxy-, dihydroxide, bis(inner salt), hexasodium salt

EC Number:

279-256-2

EC Name:

Pyridinium, 1,1'-[(6,13-dichloro-4,11-disulfo-3,10-triphenodioxazinediyl)bis[imino-2,1-ethanediylimino[6-[(2,5-disulfophenyl)amino]-1,3,5-triazine-4,2-diyl]]]bis[3-carboxy-, dihydroxide, bis(inner salt), hexasodium salt

Cas Number:

79771-28-1

Molecular formula:

C52H38Cl2N16O24S6.6Na

IUPAC Name:

Pyridinium, 1,1'-[(6,13-dichloro-4,11-disulfo-3,10-triphenodioxazinediyl)bis[imino-2,1-ethanediylimino[6-[(2,5-disulfophenyl)amino]-1,3,5-triazine-4,2-diyl]]]bis[3-carboxy-, dihydroxide, bis(inner salt), hexasodium salt

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Everzol SB26
- Substance type: Powder
- Composition of test material, percentage of components: 78.12%
- Lot/batch No.: 3540
- Storage condition of test material: Ambient

Specific details on test material used for the study:

Purity ca 78%
Results expressed as whole test material and not adjusted for purity.

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: BioLASCO Taiwan Co., Ltd., Taipei, Taiwan
- Age at study initiation: about 9-week old
- Weight at study initiation: Males: 311-379 g; Females: 199-240 g
- Housing: Except for the mating period, animals were housed individually in polycarbonate cages. While mating, animals were pair-housed in stainless steel wire mesh cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 11 days
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 50 ± 20%
- Photoperiod: 12-hrs dark / 12-hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

water for injection (WFI)

Details on exposure:

Animals were dosed for the following periods:
All rats 14 day pre-maitng
All rats14 day mating period, with males terminated after this period
Females doed for duration of gestation (typically 23 days) and until 4 days after birth.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and concentration confirmation using HPLC-UV methods
Samples collected from middle of control, low, mid and high dose samples

Duration of treatment / exposure:

28 day dosing males, typical 55 days dosing females.

Frequency of treatment:

Daily gavage

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Dose group 1

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Dose group 2

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Dose group 3

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Dose group 4

No. of animals per sex per dose:

Male: ten
Female: ten

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

Clinical observations twice daily
Weighings made weekly with food consumption measured

Litter observations:

Litters observed as as possible after delivery for gross abnormalities
Live pups were counted, sexed and weighed.

Postmortem examinations (parental animals):

Males were terminated on Day 29; females typically after 55 days
Number of implantation sites were examined in females
Full necropsy performed.

Postmortem examinations (offspring):

Gross examinations were performed

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Overall, no death was observed in males and females during the study period. The test article-related clinical observations were blue colored/soft feces, blue/blue-green colored urine, blue coloured body skin and blue-purple hair stain on the back in males and/or females.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Blue colour of urine

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

not measured/tested

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Dose Formulation Analysis

On the first and last preparation, the difference between the mean value and the targeted concentration for all dose group formulations were within ± 15.0%. The homogeneity verification revealed that the precision of samples for the low and high dose formulation were ≤ 10.0%.

 

Mortality and Clinical Observations

All animals survived the study period. Bluepurple colored body skin was observed in high dose females since the day of the 36th dosing to the end of study. Blue-purple stained hair on the back was observed in one high dose male (ID# 1037) and in one high dose female (ID# 0039) animals.

 

Body Weights

In males, statistically significantly increased body weight gain compared to the controls was observed in males of Group 3 on D15. There were no statistically significant differences in body weight and body weight gain changes in treated groups compared to control animals in females.

 

Food Consumption

There was no statistically significant difference between treated and control groups in both genders.

 

Cohabitation and Pregnancy

In males, a positive indication of mating was obtained for 9, 10, 10 and 9 of 10 in Groups 1 to 4 animals, resulting in a mating index of 90%, 100%, 100% and 90%, respectively. In females, a positive indication of mating was obtained for 10 of 10 in Group 2 to 4 animals and for 9 of 10 in Group 1 animals. The mating index was 90%, 100%, 100% and 100% for Group 1 to 4, respectively. There was only one animal in Group 4 that showed a pre-coital interval over 5 days. In this female, the mating with the first male failed but the copulation with another male was successful. In Group 2, 3 animals with positive indications of mating were later found not to be pregnant. In Group 4, one animal with positive indications of mating was later found not to be pregnant. All Groups 1 and 3 animals were found to be pregnant. Thus, the fertility index was 100%, 70%, 100% and 90% in the control, low, mid and high dose group, respectively. The average length of gestation was 22.1 or 22.2 days without dosedependency.

 

Litter Observation

All pregnant rats delivered live pups. The gestation index was 100% in all groups. One dead pup was observed at birth in Groups 2 and 4. The incidence of the dams with dead pups at birth was 0%, 14%, 0% and 11% in Group 1, 2, 3 and 4, respectively. On P4, all pups survived so that the 4-day survival index was 100.0% in all groups. No external abnormal pups were observed at birth in the control and treated groups. At birth, 48.5% to 51.4% of pups were male and on P4, 49.2% to 54.7% pups were male. The gender difference at birth and on P4 was a consequence of a difficult sex determination at birth. The litter weights in all groups were increased from P0 to P4.

 

Male Gross Examination, Organ Weight and Histopathology

There were no statistically significant differences in testis and epididymis weightsbetween control and treated groups. Decreased size of testes was observed in one Group 2 (ID# 1011).

 

Females Gross, Uterus Examination and Histopathology

There were no statistically significant differences on number of corpora lutea and implantation between control and treated groups. Treatment-related gross findings included discoloration of the intestine, kidney, lung, mesenteric lymph node, and uterus. Discoloration of the lung was correlated with foamy alveolar macrophages with intracytoplasmic blue pigment in two Group 4 females ((IDs# 0036 and 0040) microscopically.

Applicant's summary and conclusion

Conclusions:

According to OECD 421 test method, the no observed adverse effect level of Reactive Blue 187 in rat’s reproductive performance was 1000 mg/kg/day.

Executive summary:

This test using the procedures outlined in the QPS Study Plan for T65315035-RP and OECD 421. Reactive Blue 187 (100, 300 or 1000 mg/kg/day) was given orally by gavage once daily to male rats for 28 days and female rats for 40 to 53 days, depending on the time of copulation and gestation. Dose-related clinical observations in the treated groups included blue colored/soft feces, blue/blue-green colored urine, blue colored body skin and blue-purple hair stain on the back. Test article-related macro- or microscopic changes were discoloration in intestine, kidney, lung, mesenteric lymph node and uterus, and foamy alveolar macrophages, with intracytoplasmic blue pigment in the lung. However, there were no test article-related effects on male and female rat's reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

The no observed adverse effect level in rat's reproductive performance was 1000 mg/kg/day.