Tetrasodium 7-[[4-[[4,6-bis[(3-sulfonatopropyl)thio]-1,3,5-triazin-2-yl]amino]-3-methoxyphenyl]azo]naphthalene-1,3-disulfonate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 August 2014 to 29 September 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, performed in accordance with valid guidelines and the study was conducted under GLP conditions.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium 7-[[4-[[4,6-bis[(3-sulfonatopropyl)thio]-1,3,5-triazin-2-yl]amino]-3-methoxyphenyl]azo]naphthalene-1,3-disulfonate

EC Number:

428-480-0

EC Name:

Tetrasodium 7-[[4-[[4,6-bis[(3-sulfonatopropyl)thio]-1,3,5-triazin-2-yl]amino]-3-methoxyphenyl]azo]naphthalene-1,3-disulfonate

Cas Number:

214559-61-2

Molecular formula:

C26H24N6O13S6Na4

IUPAC Name:

tetrasodium 7-[[4-[[4,6-bis[(3-sulfonatopropyl)thio]-1,3,5-triazin-2-yl]amino]-3-methoxyphenyl]azo]naphthalene-1,3-disulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Appearance: Orange powder
- Storage condition of test material: Controlled room temperature (15 - 25 °C, <70 RH %) protected from light and humidity

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:WI
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: 360 - 404 g (males); 212 - 256 g (females)
- Housing: Rodents were group-housed as practical, up to 3 animals of the same group per cage (during the mating and gestation/delivery period, they were paired or individually housed, respectively). Animals were housed in Type II and III polypropylene/polycarbonate cages with Lignocel® Hygienic Animal Bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.6 - 25.0 °C
- Humidity: 33 - 66 % (relative)
- Air changes: 15 - 20 air exchanges per hour
- Photoperiod: 12 hours of darkness / 12 hours of light (06:00 to 18:00)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

(distilled)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was formulated in the vehicle as a visibly stable homogenous formulation at the appropriate concentrations according to the dose level and volume selected. Formulations were prepared fresh prior to administration to animals or at the appropriate frequency to allow their use according to stability assessment results.

ADMINISTRATION OF DOSING SOLUTIONS:
The dosing solutions were administered using a bulb-tipped gavage needle attached to a syringe. A volume of 5 mL/kg bw was administered to all animals. The actual volume administered was calculated and adjusted based on each animal’s most recent body weight. Control animals were treated concurrently with the vehicle only.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Triplicate samples were taken from the top, middle and bottom of the test material formulations on 2 occasions, during the first and the last weeks of treatment. Two sets were taken for analysis (collected in replicates, as practical) and one set was taken as a back-up for any confirmatory analyses, as necessary. Similarly, one sample was taken in duplicate from the vehicle (control / Group 1) solution for concentration measurements.

- HPLC conditions for analysis of test material concentration:
Column: ACE 5 AQ 125 × 4.6 mm, 5 μm
Mobil Phase: Methanol/Buffer pH = 6 (5 mM TEA/5 mM NaH₂PO₄) = 4/6
Flow: 1.0 mL/min.
Injection volume: 10 μL
Temperature: 25 °C
Detector: UV at 255 nm
Retention time: 4.5 min ± 10 %

Details on mating procedure:

- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: Females remained with the same male until the copulation occurred
- Proof of pregnancy: vaginal plug or sperm in vaginal smear was referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing, replacement of first male by another male
- After successful mating each pregnant female was caged individually

Duration of treatment / exposure:

Females were dosed for 14 days pre-mating, for up to 16 days mating period, through gestation and up to and including the day before necropsy, 4 days post-partum dosing. The day of birth (viz. when parturition was complete) was defined as Day 0 post-partum.

Frequency of treatment:

Daily during the treatment period (on a 7 days/week basis)

Duration of test:

Females and pups were sacrificed on day 4 post partum.

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (vehicle control), 100, 300, 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

12 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on available data, including the results of a dose range finding study.
- Rationale for animal assignment (if not random): All parental (P) animals were sorted according to body weight by computer and divided into weight ranges. There were an equal number of animals from each weight group in each of the experimental groups assigned by randomisation to ensure that animals of all test groups were as close as practicable to a uniform weight. The grouping was controlled by SPSS/PC+4.0 software according to the actual body weight, thereby verifying the homogeneity/variability between/within the groups and cages. Males and females were randomised separately.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Parental animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). General clinical observations were made once a day, after treatment, at approximately the same time as practical. Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality, were monitored.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: More detailed examinations were made once before the first exposure (to allow for within-subject comparisons), and at least once a week thereafter. These observations were made outside the home cage in a standard arena. Signs evaluated included monitoring for any changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture, and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition, or bizarre behaviour (e.g. self-mutilation, walking backwards). Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed on Day -1, on Day 0 and at least weekly thereafter until termination. Parent females were weighed on gestation Days (GD) 0, 7, 14 and 20 and on post-partal Days (PPD) 0 (within 24 hours after parturition) and on PPD3. The females were additionally weighed on GD 4, 10 and 17 in order to give accurate treatment volumes.

FOOD CONSUMPTION: Yes
- Time schedule: Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then at least weekly thereafter.

WATER CONSUMPTION: No

OTHER:
On gestation Day (GD) 13 or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).
In addition, the duration of gestation was recorded and was calculated from Day 0 of pregnancy. Dams were observed for nesting behaviour (whether they made a nest from the bedding material, and covered their new-borns, or not). The efficiency of the suckling was observed by the presence of milk in the stomach. All observations were recorded.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on post partum day 4
Terminally, after completion of treatment, animals were euthanised under pentobarbital anaesthesia, followed by exsanguination. Gross necropsy was performed on all animals. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues, and organs, were observed macroscopically.
Detailed histological examination was performed on the selected list of retained organs in the control and high dose groups and any macroscopic findings observed in all animals.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes

Fetal examinations:

PARAMETERS EXAMINED
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities. Live pups were counted, sexed, weighed individually within 24 hours of parturition (on the first day after parturition was complete, i.e. Day 0 or 1 post-partum) and on Day 4 post-partum with an accuracy of 0.01 g.
All the litters were checked and recorded daily for the number of viable and dead pups. The dead pups found were subjected to necropsy with macroscopic examination. All observed abnormalities were recorded and reported. Some of the pups that were found dead were cannibalised, thus, they were counted and sex determined (when it was possible), but were not further examined macroscopically.

Pups euthanised at PND 4 were carefully examined at least externally for gross abnormalities.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
None of the animals died during the study and no clinical signs were noted which could be attributed to treatment with the test material. One female from the mid dose group was euthanised prematurely since it was diagnosed with severe prolapse of the uterus. Furthermore, there were no effects on body weight or food consumption; any changes were considered incidental and not related to treatment with the test material.
The number of corpora lutea and implantation sites in the treated groups was comparable with the values recorded for the control group. In comparison with the control, a statistically significant decrease was noted in mean pre-implantation loss in the low dose group, which was considered incidental without toxicological relevance.
No test material-related findings were observed at necropsy.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The number of viable pups on PND4 as well as pups survival indices on PND0 and PND4 were comparable to control values at up to, and including, 1000 mg/kg bw/day. Overall there were no treatment-related effects on pup mortality, and there were no treatment related effect on the viability of pups on PND0 and PND 4.
Test material administered to the parental generation at up to 1000 mg/kg bw/day did not lead to mortality or any adverse effects considered related to treatment or any toxicologically significant effects in the F1 generation.
When evaluated on a per-litter basis, the mean litter body weights (PND 0 and 4) and/or weight gains of pups (PND 4) showed no statistically or toxicologically significant differences compared to controls.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Dose Formulation Analysis

Test material content and homogeneity of the dosing formulations was determined two times during the treatment period (during the first and sixth weeks of the treatment). No test material was detected in the control solution samples. All formulations were shown to be homogeneous. All formulations were found to be in the range of 101 to 104 % of nominal concentrations. Based on these results, the formulations were considered acceptable.

Table 1: Ovaries and Uterine Content

Females	Dose (mg/kg bw/day)
	Control	100	300	1000
Mean number of corpora lutea	12.58	13.67	14.08	14.33
Mean number of implantations	12.17	10.83	13.08	13.67
Mean pre-implantation loss (%)	0.42	2.83*	1.00	0.67

*p ≤ 0.05

Table 2: Body Weights (Offspring)

	Dose (mg/kg bw/day)
	Control	100	300	1000
Mean for all pups
Mean body weight (g), PND0	124 pups	127 pups	119 pups	154 pups
	6.521	6.450	6.346*	6.610
Mean body weight (g), PND4	118 pups	127 pups	114 pups	149 pups
	9.892	9.634	9.738	10.356**
Body weight gain (g), PND0-4	3.363	3.184	3.379	3.737**

*p ≤ 0.05

**p ≤ 0.01

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study the no observed adverse effect level for both maternal toxicity, and developmental toxicity, was determined to be 1000 mg/kg bw/day, the highest dose level tested.

Executive summary:

The developmental toxicity of the test material was investigated as part of a reproduction/developmental toxicity screening test. The study was conducted under GLP conditions and in accordance with the standardised guideline OECD 421.

During the study groups of 12 male and 12 female Wistar rats were administered with test material in distilled water at doses of 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day. Males were dosed for up to 34 days (14 days pre-mating and up to 20 days mating/post-mating), then they were euthanised and subjected to necropsy examination. Females were dosed for 14 days pre-mating, for up to 16 days for the mating period, through gestation and up to and including the day before necropsy, 4 days post-partum dosing. The day of birth was defined as Day 0 post-partum.

Adult animals were inspected for signs of morbidity and mortality twice daily. Clinical observations were performed once daily, with detailed examination performed weekly. Special attention was paid to evaluation of the mating, pregnancy, parturition and postpartum

periods, and relevant parameters and/or indices were measured and/or calculated. Body weight and food consumption were measured at least weekly.

After delivery, all pups from each litter were counted, sex established, weighed on post-natal days (PND) 0 and 4, offspring evaluated for presence of stillbirths, live births, runts (pups that are significantly smaller than normal pups) or any gross

abnormalities, then examined clinically at least daily.

Gross necropsy of the adult parental animals was conducted at the end of the treatment period and the weights of a standard list of organs were measured. Pups euthanized at PND 4 were carefully examined at least externally for gross abnormalities. Detailed histological examination was performed on the selected list of retained organs (ovaries, testes, epididymides,) in the control and High dose and all macroscopic findings (abnormalities) from all animals. Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Under the conditions of the study, daily administration of Y-1189 by oral gavage to Wistar rats at up to 1000 mg/kg bw/day did not result in test item related mortality, clinical adverse effects, or changes in body weight or food consumption.

No test item related, or adverse effects were noted at evaluation of the reproductive parameters, either during the mating, gestation, delivery or post-partum/lactation period up to post-partum Day 4.

There were no adverse effects ascribable to test item administration on the F1 offspring viability, clinical signs, development or at observation following euthanasia.

There were no test item-related changes in organ weights, gross findings or histopathology of the adult animals.

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for Y-1189 for parental/adult and F1 effects is considered to be 1000 mg/kg bw/day.