Tetrasodium 4,4'-bis[[4-[bis(2-hydroxyethyl)amino]-6-(4-sulphonatoanilino)-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate]

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 30, 1998 to January 13, 1999.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium 4,4'-bis[[4-[bis(2-hydroxyethyl)amino]-6-(4-sulphonatoanilino)-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate]

EC Number:

240-521-2

EC Name:

Tetrasodium 4,4'-bis[[4-[bis(2-hydroxyethyl)amino]-6-(4-sulphonatoanilino)-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate]

Cas Number:

16470-24-9

Molecular formula:

C40H44N12Na4O16S4

IUPAC Name:

tetrasodium 2,2'-ethene-1,2-diylbis[5-({4-[bis(2-hydroxyethyl)amino]-6-[(4-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)benzenesulfonate]

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Reason for the Species selection: the rat is a universally used model for evaluating toxicity of various classes of chemicals and for which there is a large historical database.
- Number of animals: 134 female.
- Source: Charles River Laboratories, Portage, Michigan.
- Age at study initiation: 8 weeks.
- Weight at study initiation: 170 to 223 g.
- Fasting period before study: None.
- Housing: Animals were individually housed in suspended, stainless steel, wire-mesh cages.
- Acclimation period: 5 days.
- Observation during the acclimation period: daily for any clinical signs of disease, given a detailed clinical examination prior to dosing.
- Identification: unique identification includes cage number, group, applied individually by a metal ear tag .
- Diet: certified Rodent Chow #5002, PMI Feeds, Inc., St. Louis, Missouri, ad libitum (each diet lot were certified by the manufacturer, but no additional analysis was conducted).
- Water: tap water, ad libitum (monitored for specific contaminants at periodic intervals according to SOP by MPI Research, but no additional analysis was conducted)
No contaminants were known to have been in the water or feed which could have altered the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 68 to 72°F.
- Humidity: 48 to 71 %.
- Photoperiod: 12h day/12h night with an automatic timer.
- Parameters monitoring and recording: daily.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 %, low viscosity, white powder, by Sigma Chemical Company.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
Before the test no adjustments for purity were made. The test article was mixed with aqueous 0.5% CMC to achieve the desired concentrations. The required amount of test article was weighed directly into a calibrated beaker and suspended in vehicle using a stainless steel spatula. Additional vehicle was added to the beaker to yield the required amount of prepared test material. The contents of the beaker were stirred using a magnetic stir bar and stir plate and were dispensed into amber glass containers using a syringe and stored refrigerated.

VEHICLE
The required amount of powder CMC was weighted and mixed with deionized water using a Warning blender. The solution was transferred into a volumetric flask and additional deionized water was added. The flask was shaken and transferred into a suitable container. Additional vehicle was added to the container to yield the required amount of prepared vehicle. The contents of the container were mixed thoroughly and stored refrigerated. Prior to handling the test article the required amount of vehicle was dispensed into amber glass containers and stored refrigerated.

ADMINISTRATION
Test and control articles were drawn into an appropriate sized syringe equipped with an 8 French umbilical vessel catheter approximately 2 inches long threaded over a 15 gauge blunt needle.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test article was characterized by MPI Research, using a combination of :
High-performance liquid chromatography (HPLC).
1H and 13C Nuclear magnetic resonance spectroscopy (NMR).

DOSING SUSPENSION ANALYSIS
The homogeneity, stability and concentration verification analyses were conducted by AvTech Laboratories, Inc., Kalamazoo, Michigan.

HOMOGEINICITY CONTROL
Homogenicity of test batches of the test article suspensions at low and high concentrations was assessed.
The dosing suspensions prepared for all dose levels-were homogeneous and accurately prepared.

STABILITY ANALYSIS
Dosing solutions prepared for the 100 and 1000 mg/kg/day groups were found to be stable when stored under refrigeration for 0, 7, and 14 days.

CONCENTRATION ANALYSIS
Samples of test article suspension at each concentration were collected from the first study preparation. The 400 mg/kg/day dose group was slightly outside of concentration specifications for the first week of the study. This dose was administered for Week 1 and was reanalysed during the second week of the study to confirm that the concentration was within specifications.

Details on mating procedure:

Females were time mated upon delivery, and the day on which evidence of copulation was observed was designated gestational day 0.

Duration of treatment / exposure:

From gestational Day 6 to through Day 19.

Frequency of treatment:

Once per day.

Duration of test:

From gestational Day 6 until Day 20.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 400, and 1000 mg/kg bw
Basis:
nominal conc.
Constant volume of 10 ml/kg/day in 0.5 % of CMC.

No. of animals per sex per dose:

30 females/group.

Control animals:

yes, concurrent vehicle

Details on study design:

Rats considered suitable for study were weighted following arrival and prior to exposure and randomized into treatment groups using a stratified, by weight, block randomization procedure.
See the following details of the assignment procedure:
Group 1
Dose Level (mg/kg): 0
Initial females: 30
Uterine Exam/Necropsy: 30
Group 2
Dose Level (mg/kg): 100 TS
Initial females: 30
Uterine Exam/Necropsy: 30
Group 3
Dose Level (mg/kg): 400 TS
Initial females: 30
Uterine Exam/Necropsy: 30
Group 4
Dose Level (mg/kg): 1000 TS
Initial females: 30
Uterine Exam/Necropsy: 30

Examinations

Maternal examinations:

CLINICAL FINDINGS
- Observation: at least twice a day, 7 days each week.
- Clinical findings: morbidity, mortality, signs of injury.
- Registration: all findings on the day they were observed.
From Days 6 through 20 of gestation each animal was removed from the cage and given a detailed clinical examination. The detailed examination included observations of the general appearance and condition activity and behaviour, excretory matter, respiration body surface abnormalities (scabbing, hair loss), and oral, nasal, and ocular regions. Observations included location size, and colour when applicable.

BODY WEIGHT
- Time schedule for examinations: on gestational days 0, 6, 9, 12, 15, 18, and 20.
Body weight change was recorded for gestational days 0 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 20, 6 to 20, and 0 to 20.

FOOD CONSUMPTION
- Food consumption reported on gestational days 0 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 20, 6 to 20, and 0 to 20.

POST-MORTEM EXAMINATIONS
On Day 20 of gestation each female was euthanized via carbon dioxide inhalation and immediately subjected to a laparohysterectomy. The carcasses were discarded.
- Organs examined: uterus.

Ovaries and uterine content:

UTERINE EXAMINATION
The uterus was excised, the gravid uterine weight was recorded, and the foetuses were removed. Beginning at the distal end of the left uterine horn, the location of viable and non-viable foetuses, early and late resorptions for each uterine horn position of the cervix and the number of total implantations and corpora lutea were recorded.
The embryonic membrane of each foetus was gently removed, and each foetus was pulled away from the placenta fully extending the umbilical cord. The placentae were grossly examined. Uteri from females that appeared non-gravid were opened and placed in 10 % ammonium sulphide solution for detection of implantation sites (Kopf, 1964).
Maternal gross lesions were saved in 10% neutral buffered formalin for possible microscopic evaluation.

Fetal examinations:

TERATOLOGIC EXAMINATION
Fetuses were individually weighed, sexed, tagged (using a string tag labelled with the darn and foetus number) and examined for external malformations and variations. Each litter, without knowledge of treatment group, was designated "A" (for alcohol) or "B" (for Bouin's).
Mark A: first foetus processed for skeletal examination the second for visceral, and so forth until the entire litter was complete.
Mark B: first foetus processed for visceral examination the second for skeletal and so forth until the entire litter was complete.
Approximately one-half of the foetuses were placed in Bouin's solution for subsequent soft tissue examination using the Wilson razor blade sectioning technique (Wilson 1965).
The remaining foetuses were fixed in alcohol, macerated with potassium hydroxide, stained with Alizarm Red S and Alcian blue, and cleared with glycerine for subsequent skeletal examination of bone and cartilage (Kimmel and Trammel, 1981).
Foetal findings were classified as malformations or developmental variations under the supervision of a developmental toxicologist.

ANATOMIC PATHOLOGY
Dams euthanized at termination of the study were subjected to a necropsy by trained personnel under the supervision of a veterinary pathologist.
Special emphasis was placed on structural abnormalities or pathologic changes which may have influenced the pregnancy.
Gross lesions and/or target organs from the dams were saved in 10% neutral buffered formalin.

Statistics:

Differences between groups were assessed using:
- Group pair-wise Comparison: Levene’s test, Dunnett’s test, Welch’s test with a Bonferroni correction (homogeneity).
- Arcsin-Square-Root Transformation.
- Fisher's Exact Test with a Bonferroni correction.
- Chi-square test (homogeneity).
- Kruskal-Wallis test, Mann-Whitney U-test (malformations).
- Parson Chi-Square (malformations and developmental variations).
- Descriptive statistics (means, SD).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITY
No mortality at any dose tested.

CLINICAL OBSERVATIONS
Discoloured faeces in 100 % of animals at 400 and 1000 mg/kg/day (sign related to test article administration, with unknown toxicological significance.

BODY WEIGHT
No test article-related changes in body weight or body weight gain were noted in treated groups when compared with the vehicle control group.
Body weights and body weight gain were comparable among all groups, including the vehicle control group.
For further details see Table n.1.

FOOD CONSUMPTION
No test article-related changes in food consumption were noted.
Food consumption was comparable between the vehicle control and treatment groups.
For further details see Table n.2.

NECROPSY OBSERVATION
No test article-related findings were noted at necropsy of the dams.
One dam from the 1000 mg/kg/day group had a liver adhesion and a nodule on the right kidney.
One dam from the 100 mg/kg/day group had granular material on the surface of the right kidney.
All other animals were normal at necropsy. Since only single incidences of all findings were noted and no dose response was evident, these findings were considered to be spontaneous in nature and unrelated to test article administration.

UTERINE EXAMINATION
No test article-related findings in uterine parameters were noted.
Numbers of corpora lutea, implantations, live foetuses and resorptions, foetal weight and sex ratio were similar in the vehicle control and treatment groups.
Pre-and post-implantation loss were comparable among all groups, including the vehicle controls.
Gravid uterine weight was comparable between the vehicle control and treatment groups.
No changes in adjusted body weight or adjusted body weight gain were noted in treatment groups when compared with the vehicle control group.
For further details see Table n.3.

Effect levels (maternal animals)

1

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw (total dose)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw (total dose)

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:
FETAL EVALUATIONS
1) EXTERNAL EXAMINATION
No test article related effects were noted following external examination of foetuses.
In the 1000- mg/kg/day group, 1 foetus from 1 litter had omphalocele and a second foetus from a different litter had an absent tail and anal atresia.
Since only 1 foetus was affected in each case, the findings were considered to be spontaneous in nature and unrelated to treatment.
No other external abnormalities were seen.

2) VISCERAL EXAMINATION
No test article-related effects were noted. Sporadic findings were observed in all groups including the vehicle control, but the incidences were low and no dose relationship was evident.

3) SKELETAL EXAMINATION
No findings were considered to be related to test article administration. Two foetuses in 2 litters from the 1000 mg/kg/day group (7.1 %) had vertebral malformations compared with a 0 % incidence in the vehicle control group.
In addition slight increases in the incidence of rudimentary ribs and misaligned stemebra were noted at 100 and 1000 mg/kg/day when compared with the vehicle control group (78.6 % and 10.7 % respectively).
For further details see Table n.4.

Vertebral malformations are relatively rare, however, the incidence observed in this study (7.1 %) was only slightly above the historical control range (0 - 7%) and it was not statistically different from the vehicle controls. Therefore, this finding was considered to be within normal variation and unrelated to test article administration.
The incidence of misaligned sternebrae (10.7 %) was considered to be within historical control ranges and it was therefore not considered biologically relevant or treatment-related.
The incidence of rudimentary ribs was slightly above the historical control range (0 - 70 %) a dose-related pattern was not observed, and there was no significant difference in incidence compared to the control group. Thus, the incidence of rudimentary ribs was not considered to be biologically relevant or treatment-related.
Other findings occurred sporadically among all groups and were comparable with concurrent vehicle control incidences and/or within historical control incidences for this laboratory.

Effect levels (fetuses)

Remarks on result:

other: See Details on results

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

TABLE 1: Maternal Body Weight Gain (during gestation)

Study interval	Dose level (mg/kg/day)
(Gestational Day)	0	100	400	1000
0-6	44	42	41	44
6-20	142	141	149	145
0-20	186	184	190	189

TABLE 2: Maternal Food Consumption (during gestation)

Study interval	Dose level (mg/kg/day)
(Gestational Day)	0	100	400	1000
0-6	19.7	19.5	19.9	20.1
6-20	27.3	27	27.7	27.8
0-20	25	24.8	25.4	25.5

TABLE 3: Data on Selected Uterine Parameters

Study interval	Dose level (mg/kg/day)
(Gestational Day)	0	100	400	1000
N. Pregnant	30	29	19.9	28
% Pre-implantation Loss	14.17	27	12.69	11.76
% Post-implantation Loss	4.02	24.8	3.96	4.09
N. Live foetuses	12.77	24.8	12.31	12.43
Fetal weight (g)	4.19	24.8	4.2	4.16
Adjusted maternal Body Weight gain	107.3	24.8	107.5	112.9

TABLE 4: Skeletal Abnormalities (% litter incidence)

Study interval	Dose level (mg/kg/day)
(Gestational Day)	0	100	400	1000
N. Litters examined	30	29	28	28
Vertebral malformation	0	0	0	7.1
Sternbra misaligned	3.3	10.3	7.1	10.7
Rudimentary rib	63.3	75.9	57.1	78.6

Applicant's summary and conclusion

Conclusions:

The test item, was not teratogenic in rats following oral administration of doses up to and including 1000 mg/kg/day.

Executive summary:

Method

The developmental toxicity, including the teratogenic potential, of the test article was assessed in rats in accordance with OPPTS Guideline 870.3700, EPA Guidelines, March 1997. The study consisted of 3 groups of 30 time-mated females/group treated and 1 vehicle control group.

The dose levels of 0, 100, 400 and 1000 mg/kg/day were administered via oral gavage daily from Days 6 through 19 of gestation at a volume of 10 ml/kg. The females were time mated upon delivery. The day on which evidence of copulation was observed was considered to be Day 0 of gestation. 

Observations of dams included clinical signs, gestational body weights and food consumption. Litters were delivered by caesarean section on Day 20 of gestation. Gravid uterine weights were recorded

Total number of implantations, early and late resorptions, live and dead foetuses, and sex and individual body weights of foetuses were recorded. 

External abnormalities of foetuses were recorded.

Approximately one-half of the foetuses were examined for visceral abnormalities, and the remaining foetuses were examined for skeletal abnormalities (bone and cartilage).

Results

No mortalities were observed during the study, and the only test article-related clinical observation noted was discoloured faeces. 

No changes in maternal body weight, body weight gain or food consumption were noted in the treatment groups when compared with the vehicle control group. No test article-related necropsy findings were seen. Uterine parameters, including numbers of corpora lutea, implantations, live foetuses, and resorptions, gravid uterine weight, and adjusted body weight and body weight gain were comparable between vehicle controls and treatment groups. Pre-and post-implantation loss were similar among all groups, and no test article effects were noted.

Foetal external, visceral, and skeletal evaluations did not reveal any test article related effects. All findings were either comparable with the concurrent vehicle control and/or historical control incidences.

Based on the results of this study, the No Observed Effect Level (NOEL) for both maternal and developmental toxicity was 1000 mg/kg/day, the highest dose tested.