Hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-4,2-diyl]imino]]bis(benzene-1,4-disulphonate)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Principles of method if other than guideline:

The dose-range finding experiment was performed with 3 groups of treated pregnant female rats and a control group to find appropriate dose for the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-4,2-diyl]imino]]bis(benzene-1,4-disulphonate)

EC Number:

273-468-9

EC Name:

Hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-4,2-diyl]imino]]bis(benzene-1,4-disulphonate)

Cas Number:

68971-49-3

Molecular formula:

C40 H38 N12 O22 S6 .6Na (molecular formular in EC inventory is not correct)

IUPAC Name:

hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-4,2-diyl]imino]]bis(benzene-1,4-disulphonate)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Selection of animal species: laboratory rat has been chosen because our testing laboratory has long experience with this species and because rat is recommended according to the test guideline
Strain: Wistar CRL (SPF quality - guaranteed)
Sex: females (males – only for mating)
Total number of animals: 6 females and 6 males per group
Acclimatization: 18 days

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test item concentration at single dose level was adjusted so that the administered volume was constant at all dose levels: 1 ml/100 g body weight.
The animals were without feed two hours before application and two hours after application of the test item.

Details on mating procedure:

After acclimatization, females were mated with males (1 male and 1 female). Control of fertilization was made by the help of the vaginal smears. Vaginal smears were carried out after 24 hours of the first removing to male and then daily at the same time and the presence of sperms were examined. Day 0 of pregnancy was the day on which sperms in vaginal smear were found out.

Duration of treatment / exposure:

The test item was administered in graduated dose levels to pregnant females next day after confirmed mating (on day 1) to 19th day of pregnancy

Frequency of treatment:

daily - 7 days per week at the same time (8.00 – 10.00 am)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

Body weight: 1st, 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy
Health condition check: daily, before the application of test item
Clinical observation: twice a day

Litter observations:

Macroscopic examination of all foetuses

Postmortem examinations (parental animals):

Haematology examination: basic parameters – 20th day of pregnancy
Pathological examination of females: 20th day of pregnancy
Number of implantations, corpora lutea and resorptions

Postmortem examinations (offspring):

Pathological examination of foetuses: 20th day of pregnancy

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No serious changes of animal health status and clinical symptoms of intoxication were observed in treated animals.

Description (incidence):

Female No. 136 (at the dose level 1000 mg/kg/day) died at the 3rd day of application - intubation error, this unrelated with the test item treatment.

Description (incidence and severity):

Decreased body weights of pregnant females at the dose level 1000 mg/kg/day was recorded during the whole study. The body weights of pregnant females at the dose levels 100 and 300 mg/kg/day were similar compared to of pregnant control females.

Haematological findings:

no effects observed

Description (incidence and severity):

Haematological examination did not show significant differences among dose levels and control group

Reproductive function / performance (P0)

Description (incidence and severity):

After the mating probably pregnant females were randomly assigned to the groups. During necropsy on the 20th day of pregnancy the foetuses and implantations were not found out in all females. The number of females without foetuses was following: 2 – 0 – 0 – 1.

Number of implantations, corpora lutea and resorptions
The numbers of implantations site were slightly reduced at the dose level 100 mg/kg/day.
The numbers of resorptions were slightly increased at the dose levels 100 and 300 mg/kg/day.

Effect levels (P0)

Dose descriptor:

LOEL

Remarks:

Used for selection of maximum tested dose in the main study

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Mortality / viability:

no mortality observed

Description (incidence and severity):

No death of foetuses was recorded in any litter. The average number of foetuses per litter were slightly decreased at the dose level 100 mg/kg/day compared to the control group

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic changes of soft tissues and external alteration were found during the pathological examination of the foetuses at all dose levels.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

Dose used for selection of maximum tested dose in the main study

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Results of DRF study

	control	100	300	1000	notes
Number of pregnant females	4 (non-pregnant and no sperm)	6	6	5
Number of non-pregnant females	2	0	0	0
Mortality	0	0	0	1	died due to intubation error
Health condition and clinical observation	no effects	no effects	no effects	no effects
Body weight	no effects	no effects	no effects	effects	Decreased body weights of pregnant females at the dose level 1000 mg/kg/day during the whole study.
Haematological examination	no effects	no effects	no effects	no effects	WBC (10^3/µl), RBC (10^6/µl), HGB (g/dl), HCT (%), MCV (fl), PLT (10^3/µl)
Pathological examination of females	no effects	no effects	no effects	no effects
Implantations ± SD	17.50 ± 3.00	14.50 ± 4.23	17.83 ± 1.17	16.20 ± 2.39	Slightly reduced at the dose level 100 mg/kg/day.
Resorptions ± SD	0.25 ± 0.50	0.50 ± 0.55	0.67 ± 1.21	0.20 ± 0.45	Slightly increased at the dose levels 100 and 300 mg/kg/day.
Corpora lutea ± SD	17.75 ± 2.75	16.50 ± 1.87	18.33 ± 1.21	16.60 ± 1.52
Total number of live foetuses	69	84	103	80
Total number of dead foetuses	0	0	0	0
Average number of live foetuses ± SD	17.25 ± 2.87	14.00 ± 4.20	17.17 ± 1.47	16.00 ± 2.35	Slightly decreased at the dose level 100 mg/kg/day.
Average number of dead foetuses ± SD	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
Patholoical examination of foetuses	no effects	no effects	no effects	no effects	No macroscopic changes of soft tissues and external alteration

Applicant's summary and conclusion

Conclusions:

Dose levels selected for the main Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test: 80, 250 and 750 mg/kg/day

Executive summary:

The oral administration of the test item to pregnant females by gavage from the 1stto the 19thday of pregnancy at the dose levels 100, 300 and 1000 mg/kg/day did not cause mortality of pregnant females.

No adverse changes of health condition and no clinical symptoms of intoxication were found in females at any dose level after administration of the test item.

The slight decreased body weights of pregnant females at the dose level 1000 mg/kg/day was recorded.

Haematological examination did not show significant differences among dose levels.

Pathological examination of females and foetuses revealed no serious macroscopic changes.

Reproduction parameters were very slightly changed, but these changes were not related with the test item treatment: at the dose level 100 mg/kg/day – slightly decreased number of implantations sites, at the dose levels 100 and 300 mg/kg/day – slightly increased number of resorptions and at the dose level 100 mg/kg/day – slightly decreased number of foetuses per litter.

   

On the basis of the results given above the following dose levels – 80, 250 and 750 mg/kg/day will be used for the main Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the rat.