1,4-Benzenedisulfonic acid, 2,2'-[1,2-ethenediylbis[(3-sulfo-4,1-phenylene)imino[6-[bis(2-hydroxypropyl)amino]-1,3,5-triazine-4,2-diyl]imino]]bis-, sodium salt (1:6)

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

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

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

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

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

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

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

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: oestrous cycle:

effects observed, treatment-related

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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.

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

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

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.

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

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

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

chronic

Species:

rat

Additional information

In a 2-generation rat study with the analogue substance EC 240-521-2 according to EPA Guideline OPPTS 870.3800 / OECD 416 and performed under GLP, 26 rats per sex per group were administered 100, 300 or 1000 mg/kg bw/day by oral gavage. In parental animals, the only test substance-related effect noted was an increased kidney weight. In F0 animals, an increased kidney weight (absolute and relative to body and brain weight) was observed in females at 1000 mg/kg bw/day. In F1 parental animals, there was an increase in kidney weight in males (absolute and relative to body weight) and females (absolute and relative to body and brain weight) at 1000 mg/kg bw/day as well as an increase in kidney weight (relative to body weight) in females at 300 mg/kg bw/day. The statistical change in 300 mg/kg bw/day was considered to be spurious since no changes in absolute weight or kidney weight relative to brain weight were seen, and similar increases were not observed in 300 mg/kg bw/day males. There were no test substance-related effects on reproductive performance noted for either parental generation. No adverse, test substance-related changes in growth or development of offspring were observed in either the F1 or the F2 generations. Based on the results of this study, the NOAEL for parental toxicity was 300 mg/kg bw/day. For parental reproductive performance, the NOAEL was 1000 mg/kg bw/day. For offspring growth and development, the NOAEL was also 1000 mg/kg bw/day (ETAD 2001).

The substance belonging to the category of derivatives of 4,4’-bis(1,3,5-triazinyl-2-yl)amino)stilbene-2,2’-disulfonic acid, each with one anilino and one alkyl amino moiety has with high probability no potential for reproductive toxicity. It is concluded that this negative finding can also be transferred to the substance defined in section 1.

Short description of key information:
No effects concerning reproduction toxicity observed at highest dose tested in a two-generation study with the analogue substance EC 240-521-2in rats performed according to OECD testing guideline 416 and under GLP. The NOAEL was 300 mg/kg bw/day for parental toxicity and 1000 mg/kg bw/day for reproductive performance and offspring toxicity (ETAD 2001)

Effects on developmental toxicity

Description of key information

Two studies with the analogue substance EC 240-521-2 revealed no evidence of teratogenicity in rats and rabbits: rat: NOAEL = 1000 mg/kg bw/day (maternal and fetal toxicity); rabbit: NOAEL = 100 mg/kg bw/day (maternal and fetal toxicity). GLP-compliant OECD 414 studies (ETAD 1999 and 2000)

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

chronic

Species:

rat

Additional information

Two studies on developmental toxicity and teratogenicity according to EPA Guideline OPPTS 870.3700 and under GLP conditions have been performed in rats and rabbits with the analgue substance EC 240 -521 -2. In the rat study, 30 pregnant Sprague-Dawley rats per group were dosed with 100, 400 or1000 mg/kg bw/day by oral gavage on gestation days 6-19. The only substance-related effect observed was discolored feces at 400 and 1000 mg/kg bw/day. At skeletal examination of fetuses, the incidence of misaligned sternebra was slightly increased in all dose groups but was well within historical control range and not dose-related and therefore not considered to be test substance related. The incidence of rudimentary ribs was slightly above the historical control range at 100 and 1000 mg/kg bw/day. As the difference from the concurrent control group was not statistically significant and the increase was not dose-related, these findings were not considered biologically significant or test substance-related. The number of vertebral malformations at 1000 mg/kg bw/day (litter incidence 7.1 %) was very slightly above the historical control range (0 - 7 %) and not statistically different from the vehicle controls. Therefore, this border finding too was considered to be within normal variation and unrelated to test substance administration. As there were no adverse maternal or developmental effects seen at any dose level, the NOAEL for both maternal and fetaltoxicity is the highest dose tested (1000 mg/kg bw/day) (ETAD 1999).

In the definitive rabbit study, 7 pregnant New Zealand White rabbits per group were dosed with 100, 400 or 800 mg/kg bw/day by oral gavage on gestation days 7 - 28. The application of 800 mg/kg bw/day resulted in excessive maternal toxicity as exhibited by death, abortion, increased incidence of clinical and gross pathological findings, and a marked decrease in food consumption and body weight gain. As a consequence this group was terminated prior to study. Abortion or early delivery and soft stool and discolored feces also occurred in some dams at 400 mg/kg bw/day. The fetal body weights were lower in the 400 mg/kg bw/day group than compared to controls, an effect which is considered to be secondary to maternal toxicity. At visceral examination of fetuses, the litterincidence of hemorrhagic iris at 400 mg/kg bw/day was slightly above the historical control range while the slightly increased incidences of gallbladder agenesis, hypoplasia of the gallbladder and azygous lobe of lung absent were within historical control range. Since all the above findings were within or slightly above historical control range, the findings were considered to be spontaneous innature and unrelated to test substance. Also, no substance-related effects were noted at external and skeletal examinations. At a dose level of 100 mg/kg bw/day no substance-related effects were seen in dams or at fetal examinations. The NOAEL for both maternal and fetal toxicity therefore was established as 100 mg/kg bw/day (ETAD 2000).

Both tests consistently yielded negative results indicating that the substance belonging to the category of derivatives of 4,4’-bis(1,3,5-triazinyl-2-yl)amino)stilbene-2,2’-disulfonic acid, each with one anilino and one alkyl amino moiety has with high probability no potential for teratogenicity. It is concluded that this negative finding can also be transferred to the substance defined in section 1.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the category members are not considered to be classified for toxicity to reproduction, effects via lactation, developmental toxicity, and teratogenicity under Directive 67/548/EEC, as amended for the 28th time in Directive 2001/59/EC.

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the category members are not considered to be classified for toxicity to reproduction, effects via lactation, developmental toxicity, and teratogenicity under Regulation (EC) No. 1272/2008.