Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented, according to accepted guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
GLP compliance:
yes (incl. QA statement)
Remarks:
MPI Research, Inc. 54943 North Main Street. Mattawan, MI 49071-9399
Limit test:
yes

Test material

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
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]
Details on test material:
-Physical state: solid
- Analytical purity: 88.3%
- Lot/batch No.: DSR 9710004-006
- Stability under test conditions: September, 2001
- Storage condition of test material: Room temperature, protect from light

Test animals

Species:
rat
Strain:
other: CD (Crl:CD (SD)IGS BR) albino
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan
- Age at study initiation: approx 6 weeks
- Weight at study initiation: 52 to 78 g
- Fasting period before study: None
- Housing: individually in suspended, stainless steel, wire-mesh type cages except during mating where females were individually housed in plastic cages containing wood chip bedding
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): certified meal Rodent chow #5002 from PMI Nutritionals International Inc. St. Louis, Missouri, was available ad libitum
- Water (e.g. ad libitum): Tap water available ad libitum
- Acclimation period: 2-week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 34 to 77
- Photoperiod (hrs dark / hrs light): 12/12

From: Jan. 17, 2000 To: Nov. 3, 2000

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The required amount of test article was weighed and suspended in vehicle and mixed using a Polytron tissue homogenizer. The solutions were stirred with a magnetic stir bar, and then dispensed into amber glass containers using a syringe. Fresh suspensions were prepared for each dose group weekly, and stored refrigerated and protected from light for approximately 1 week.
VEHICLE
- Concentration: 0.5%
- Lot/batch no. (if required): 108H0070
Details on mating procedure:
M/F ratio per cage: 1/1 (sequential, non-random matching; sibling matings avoided for F1 generation)
- Length of cohabitation: when pregnant or 14 days had elapsed
- Proof of pregnancy: copulatory plug or sperm in vaginal smear referred to as day 0 of gestation
- After 14 days of unsuccessful pairing: non-pregnant females moved to plastic cages with woodchip bedding
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually (stainless steel, wire-mesh cages, then moved to plastic cages with woodchip bedding on gestational day 20).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were diluted with methanol and analyzed by HPLC (UV detection) for verification of concentration.
Duration of treatment / exposure:
Treatment of the parental (P) generation began 10 weeks prior to mating and continued until euthanasia. F1 and F2 generation offspring were potentially exposed in utero and during lactation. The F1 offspring selected to comprise the F1 parental group were exposed for ≥70 days prior to mating (beginning at age ≥28 days), until euthanasia.
Frequency of treatment:
Once daily
Details on study schedule:
- F1 parental animals not mated until 11 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 14 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg body weight/day
Basis:
actual ingested
No. of animals per sex per dose:
26 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Further details on study design



- Dose selection rationale: Selected by the sponsor on the basis of a range-finding study (MPI study # 795-005)
- Rationale for animal assignment (if not random): random
Positive control:
Not used

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes / No / No data
- Time schedule for examinations:

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

OTHER:
Oestrous cyclicity (parental animals):
Vaginal smears performed daily beginning 3 weeks prior to mating period in all P-generation females.
Sperm parameters (parental animals):
Sperm production, motility, and morphology performed following dissection
Litter observations:
STANDARDISATION OF LITTERS
- Performed on lactation day 4: maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were euthanized and necropsied.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring: litter size, number of stillborn pups, number of live-born pups, gross pup abnormalities, and pup weight by sex. .

GROSS EXAMINATION OF DEAD PUPS: yes, any intact dead pups were necropsied
Postmortem examinations (parental animals):
SACRIFICE
Performed on:
- P, F1, and F2 animals dying spontaneously or euthanized in extremis
- P and F1 females that showed evidence of mating but failed to deliver
- P and F1 females that showed no evidence of mating and failed to deliver
- P and F1 animals surviving to scheduled termination
- F1 and F2 pups culled on day 4 of lactation
- three F1 pups not chosen to continue on study per sex from each litter at weaning
- all F2 pups at weaning

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs and tissues were prepared for microscopic examination: brain, adrenal glands, epididymis, kidney, liver, pituitary gland, prostate, seminal vesicles with coagulating glands, spleen, testis, and ovaries.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age were subjected to postmortem examinations macroscopic and microscopic examination
- intact pups dying during lactation were also subjected to postmortem examinations macroscopic and microscopic examination

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations
Statistics:
The following parameters were analyzed statistically for significant differences: body weights, change in body weights, food consumption, fertility indices, reproductive organ weights, sperm parameters, follicle counts, number of uterine implantations, litter parameters (size, weight, and viability), and developmental indices.
Reproductive indices:
Calculated
Offspring viability indices:
Calculated

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

P GENERATION:
CLINICAL SIGNS AND MORTALITY
Free of any observable abnormalities. 3 females died or were euthanized in extremis during the P generation: 2 in the 1000 mg/kg group and 1 in the 300 mg/kg group. It was concluded that the observed deaths were unlikely to be related to treatment.

ORGAN WEIGHTS
Treatment related and significantly increased absolute and relative kidney weight (to brain and body weight) were observed in the 1000 mg/kg-group females when compared to control values. Similar changes were noted in F1 parents in the 1000 mg/kg group. Significantly decreases in absolute and relative (to brain weight) epididymides weights were observed in the 300 and 1000 mg/kg groups compared to control values. As these decreases were not related to any microscopic epididymal changes or epididymal sperm counts, they were not considered to be treatment-related. Significant increases in absolute and relative (to brain weight) pituitary weight was observed in females in the 1000 mg/kg group compared to controls. As these increases were not related to any microscopic changes in the pituitary glands, they were not considered to be treatment-related.

F1 GENERATION:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Free of any observable abnormalities. 8 animals died or were euthanized in extremis during the study (2, 1, and 5 animals in the 100, 300, and 1000 mg/kg groups, respectively). As there were no abnormal clinical observations, and the number of deaths was comparable between treatment groups, these deaths were not considered to be treatment-related. Sporadic significant changes in body weight were observed in the treatment groups compared to the control. As no dose-related pattern was observed, these changes were not considered to be treatment-related.

FOOD CONSUMPTION:
A significant decrease in food consumption was noted in males in the 300 mg/kg group during the first 2 weeks of the premating period. As no dose-related pattern was observed, this decrease was not considered to be treatment-related.

ORGAN WEIGHTS
Significant increases in absolute and relative (to body weight) kidney weights were observed in males and females in the 1000 mg/kg group. Significantly increased relative kidney weight was observed in females in the 300 mg/kg group. Although these changes were not related to microscopic kidney changes, a clear dose-related pattern was observed; thus, the changes observed in the 1000 mg/kg group were considered to be treatment-related. The changes observed in the 300 mg/kg group were considered to be spurious due to lack of change in total body weight, kidney weight relative to brain weight, and similar changes in male animals of the same dose group.
Significantly decreased absolute liver weights were observed in males in the 300 and 1000 mg/kg groups (compared to control values). Relative (to brain and body weights) liver weight was significantly decreased in males in the 300 mg/kg group. As there were no corresponding microscopic liver changes, these differences were not considered to be treatment-related.
Significant increases in absolute and relative adrenal gland weights were observed in females in the 1000 mg/kg group (compared to control values). As there were no corresponding microscopic adrenal gland changes, these differences were not considered to be treatment-related.

HISTOPATHOLOGY
1 control male had epididymal aspermia and testicular atrophy, and 1 male in the 1000 mg/kg group had trace degeneration of the seminiferous tubules. These observations were not considered to be treatment-related.

REPRODUCTIVE FUNCTION (ESTROUS CYCLES)
A significant increase in the number of estrous cycles was observed in the 300 mg/kg group. As no effect on estrous cycles was observed in the 1000 mg/kg group, this increase was not considered to be treatment-related.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
Toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on observation of increased kidney weight in 1000 mg/kg body weight group
Dose descriptor:
NOAEL
Remarks:
Tocicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No effects observed at highest dose
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed at highest dose

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

F1 GENERATION:

BODY WEIGHT
Significantly increased body weight (males and total pups) was observed for pups in the 1000 mg/kg group (compared to controls). This increase was not considered to be biologically meaningful as it was less than 10% difference from control values.

F2 GENERATION:
PARTURITION
A significant increase in the number of implant scars was observed in the 100 mg/kg group. As this increase was not observed in either of the higher dose groups, it was not considered to be treatment-related. A significantly higher sex ratio (% males) was noted in the 300 mg/kg group; however, this change was likely due to a lower % of male pups in the control group. Thus, this difference was not considered to be treatment-related.

BODY WEIGHT
Sporadic but significant increases in pup body weight were noted in all treatment group (versus control). Although these increases were considered to be treatment-related, they were not considered to be adverse.

ORGAN WEIGHTS
Significant increases in brain weight were observed in the 300 and 1000 mg/kg groups (compared to control). As these increases were considered to be the result of increased body weight, and were less than 10% different from control values, they were not considered to be biologically relevant of treatment-related.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
Toxicity
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed at highest dose
Dose descriptor:
NOAEL
Remarks:
Reproduction
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed at highest dose
Dose descriptor:
NOAEL
Remarks:
Growth and Development
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed at highest dose

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEL
Remarks:
Growth and Development
Generation:
F2
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed at highest dose

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion