reaction mass of: trisodium 3-(5-(2,6-difluoropyrimidin-4-ylamino)-2-sulfonatophenylazo)-5-(4-fluoro-6-morpholin-4-yl-1,3,5-triazin-2-ylamino)-4-hydroxy-2,7-naphthalenedisulfonate; trisodium 3-(5-(4,6-difluoropyrimidin-2-ylamino)-2-sulfonatophenylazo)-5-(4-fluoro-6-morpholin-4-yl-1,3,5-triazin-2-ylamino)-4-hydroxy-2,7-naphthalenedisulfonate

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Jul 2001 to 12 Dec 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.34 (One-Generation Reproduction Toxicity Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Lithium sodium hydrogen 4-amino-6-(5-(5-chloro-2,6-difluoropyrimidin-4-ylamino)-2-sulfonatophenylazo)-5-hydroxy-3-(4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo)naphthalene-2,7-disulfonate

EC Number:

401-560-2

EC Name:

Lithium sodium hydrogen 4-amino-6-(5-(5-chloro-2,6-difluoropyrimidin-4-ylamino)-2-sulfonatophenylazo)-5-hydroxy-3-(4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo)naphthalene-2,7-disulfonate

Cas Number:

108624-00-6

Molecular formula:

Hill formula: C28H(21-x-y)ClF2N8O16S5.xLi.yNa CAS formula: C28H21ClF2N8O16S5.xLi.xNa

IUPAC Name:

Lithium sodium hydrogen-4-amino-6-(5-(5-chloro-2,6-difluoropyrimidine-4-ylamino)- 2-sulfonatophenylazo)-5-hydroxy-3-(4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo)naphthalene-2,7-disulfonate

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Gartenstrasse 27, 33178 Borchen, Germany
- Age at study initiation: approximately 6 wk
- Housing: single
- Diet (ad libitum): sniff R/M-Z (V1324)
- Water (ad libitum): tap
- Acclimation period: at least 5 d


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 16-20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 31 July 2001 To: 12 December 2001

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved daily in deionized water at concentrations of 12.5, 50 and 200 mg/mL.

VEHICLE: deionized water
- Concentration in vehicle: 12.5, 50 and 200 mg/mL
- Amount of vehicle: 5 mL/kg body weight

Details on mating procedure:

- M/F ratio per cage: 1:1 (1:2 mating was performed in three high dose females because of mortality in males)
- Length of cohabitation: 3 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy
- After successful mating each pregnant female was individually caged

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability of the test substance in water was analytically verified. The quantification of the test substance in water was performed by HPLC seperations (reverse phase, UV-detection) of aqueous solutions of the test substance. At 0 and 1 hour after substance preparation 102 to 104% of the nominal test substance concentration were analytically found in both concentrations tested (1 and 250 mg/mL). Hence the test substance concentrations were prepared daily and used within 1 hour after preparation.

Duration of treatment / exposure:

Males: 10 weeks pre-mating, treatment continued during mating (ca. 3 weeks)
Females: 4 weeks pre-mating, treatment continued during mating (ca. 3 weeks) and during lactation until Day 21 post partum

Frequency of treatment:

daily

Details on study schedule:

NA

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Remarks:

Corresponding concentration: 12.5 mg/mL

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Corresponding concentration: 50 mg/mL

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Corresponding concentration: 200 mg/mL

No. of animals per sex per dose:

28

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose selection rationale was based on a subacute 28-day oral toxicity study with the test substance in rats, which did not show any adverse findings up to and including the limit dose of 1000 mg/kg body weight. Accordingly, dose levels of 0, 62.5, 250 and 1000 mg/kg bw/day were selected for the present study.

Positive control:

NA

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly in both sexes during the pre-mating period, in females on Days 0, 7, 14 and 21 during gestation and on Days 0, 4, 7, 14 and 21 of lactation period.


FOOD CONSUMPTION: Food consumption was recorded together with the body weights (except the mating period for both genders, and except on Day 4 of lactation for the females).


OTHER:
- Clinical Chemistry: 10 male and 10 female animals per group at scheduled sacrifice
- Anatomic Pathology: Macroscopic observation: all animals
Organ weights: all parental animals
Microscopic observation: all control and high-dose animals; all macroscopical changes
- Dental examinations: 5 incisors of control and high dose animals

Oestrous cyclicity (parental animals):

daily during mating period

Sperm parameters (parental animals):

Parameters examined in all males of the parental generation: organ weights - testis, epididymis, prostate and seminal vesicles weight; histopathology of testis, epididymis, prostate and seminal vesicles

Litter observations:

STANDARDISATION OF LITTERS
- Performed on Day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, viability, physical or behavioural abnormalities


GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were killed in the third week of the mating period
- Maternal animals: All surviving animals were killed on Day 22 (or until Day 24, after weekends), after birth. Animals with necropsy date on weekend were killed the next weekday.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations. All abnormal findings with special attention paid to the organs of the reproductive system were recorded


HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues or organs (or pieces of them) were preserved in Bouin's solution (testes) and formaldehyde solution and processed for histopathological investigations: Epididymes, Kidneys, Liver, Ovaries with oviducts, Pituitary, Prostate, Seminal vesicle, Testes, Uterus, Vagina, all other gross lesions.
Histopathological examinations were carried out of the control and high dose animals on these organs, as well as on on heart, spleen, lung, pancreas and gastro-intestinal tract from those animals with macroscopically visible changes, i.e., blueish colored pigmentation storage of the test substance.
The following organs were weighed: Epididymes, Kidneys, Liver, Ovaries, Pituitary, Prostate, Seminal vesicle, Testes, Uterus

OTHER: In order to investigate the cause of the dental findings in the late treatment period of the high dose animals, in total five affected incisors of the high dose males and five incisors of the control animals were analyzed for calcium and phosphorus content (two high dose and two control animals, data not presented, filed in the raw data). Secondly they were extended to fluoride, calcium and phosphorus content on the remaining 3 high dose incisors and control incisors.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 4 d of age.
- Dead or moribund pups and pups killed at Day 4 were examined for defects.

- All surviving F1-animals were killed on Day 22 (or until Day 24, after weekends), after birth. Animals with necropsy date on weekend were killed the next weekday.

Statistics:

All Parameters: The assumption of a monotonic dose-response relationship for all parameters justifies the restriction of the significance level to 5 percent (per parameter and sex), using the method of: HOTHORN L, LEHMACHER W.: A Simple Testing Procedure "Control versus k Treatments" for One-sided Ordered Alternatives, with Application in Toxicology, Biom. J. 33, 179-189, Akademie Verlag
Bodyweights: The changes of parameter values compared to the treatment-free baseline values are analyzed with the t-Test:
HARTUNG J., ELPERT B., KLÖSENER K. H., Lehr- und Handbuch der angewandten
Statistik (1989), R. Oldenbourg Verlag, München
Clinical Pathology Data: Wilcoxon's Test: HOLLANDER M., WOLFE, D. A:, Non-parametric statistical methods
Organ weights (absolute): t-Test
Organ weights (relative to bodyweight): Wilcoxon's Test

Reproductive indices:

Mating Index (%): (Number of mated or inseminated animals/Number of paired animals)*100
Pregnancy Index (%): (Number of pregnant animals/Number of mated or inseminated animals)*100
Fertility Index (%): (Number of pregnant animals/Number of paired animals)*100
Gestation Index (%): (Number pregnancies with live pups/Number of pregnancies)*100

Offspring viability indices:

Live Birth index (%): (Number of pups born alive/Number of pups born)*100
Survival index (%): (Number of pups alive on Day X/Number of pups born)*100
Weaning index (%): (Number of pups alive on Day 21/Number of pups alive on Day 4)*100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Behavior and health status was not affected in control-, low- and mid-dose group animals with the exception of males Nos. 66, 79 and 81 (mid dose group 3; 250 mg/kg body weight), exhibiting broken off incisors from week 7 onwards.
In the high dose group (1000 mg/kg), all males and females exhibited bluish-discolored feces, a few of them later on also bluish-discolored urine. As a major clinical finding, the lower and/or upper incisors were white discolored from week 5 onwards, and generally broke off within a few days. In particular some of those animals developed general clinical signs (stilted gait, hypoactivity, coat bristling, irregular respiration, respiratory sounds diarrhea, snout encrusted blood colored or swollen etc.) and some of those ended up in a general poor condition. This was mainly due to the fact that they could not take up food properly.
This effect was caused by the fluoride impurity and is rat specific.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were no intercurrent deaths in the control-, low- and mid-dose group animals.
Animal No. 128 was killed by mistake on day 51.
In the high dose group (1000 mg/kg body weight), 1 male and 1 female animal was found dead early (days 19 and 5, respectively) with unknown pathogenesis. In addition, further 6/28 males and 4/27 females were found dead or had to be killed on human grounds from study week 6-7 onwards, due to severely broken off- and white-discolored incisors, generally starting to occur from study week 6 onwards.
This effect was caused by the fluoride impurity and is rat specific.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gains were not significantly influenced by the administration of the test compound in the low- and mid-dose group (62.5 and 250 mg/kg). Body weight gain was significantly decreased for high dose males (10% within the first 6 weeks), with subsequent marked loss of body weight up to the end of treatment. In the high dose females, body weight gain was slightly increased during the first 4 weeks (pre-mating period), which, however, had turned back to a slight decrease during the gestation period (based on pregnant females only), and subsequent marked loss of body weight during lactation in surviving females. There was marked loss of body weights in high dose animals that had dental problems.
This effect was caused by the fluoride impurity and is rat specific.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Those high dose animals that were found dead from week 6 onwards or were killed on human grounds did not take up food a few days before death. Mean absolute food consumption in all remaining animals of the high dose group (1000 mg/kg) was slightly to moderately decrerased. This was in line with the lower body weight gains recorded for this group. Hence, relative food consumption was generally comparable in all groups throughout the study, except for high dose females, who exhibited a significant decrease of relative food consumption during the lactation period.
This effect was caused by the fluoride impurity and is rat specific.

Food efficiency:

no effects observed

Clinical biochemistry findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

In the high dose group, only 23/27 females were detected sperm positive and recorded as being successfully mated. Mating for female Nos. 207, 210, 213 was still unsuccessful after 21 days. These three females were killed on day 49 of the study. The mean pre coital interval of the remaining high-dose group females was slightly prolonged, i.e., to 7.9 days, (10.7 days, when taking both intervals together for 2 different males, each mating with female Nos. 199, 201, 204).
This effects was caused by the effects of the fluoride impurity on rat teeth and is rat specific

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no intercurrent deaths in the control, low and mid dose groups. In the high dose (1000 mg/kg/day), 1 male and 1 female animal was found dead early with unknown pathogenesis. In addition, further 6/28 males and 4/27 females were found dead or had to be killed on human grounds from study Week 6-7 onwards. Animal No. 128 was inadvertently killed on Day 51.

Behavior and health status was not affected in low- and mid-dose group animals with the exception of 4 males exhibiting broken off incisors from Week 6 onwards. Several high-dose animals had broken off- and white-discolored incisors, generally starting to occur from study Week 6 onwards. Some of those animals developped general clinical signs (stilted gait, hypoactivity, coat bristling, irregular respiration, abnormal respiratory sounds, diarrhea, snout encrusted blood colored or swollen etc.) and some of those ended up in a general poor condition.

Blue discolored feces were observed in all P-generation male and female animals of the 250 and 1000 mg/kg/day groups.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight gain was significantly decreased for high dose animals that had dental problems.
Those high dose animals that were found dead from Week 6 onwards or were killed on human grounds did not take up food a few days before death. Mean absolute food consumption in all remaining animals of the high dose group (1000 mg/kg/day) was slightly to moderately decrerased. This was in line with the lower body weight gain recorded for this group. Hence, relative food consumption was generally comparable in all groups throughout the study, except for high dose females, who exhibited a significant decrease of relative food consumption during the lactation period.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no test substance-related adverse effects on the estrous cycle.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Due to the lower food consumption resulting from broken-off incisors the pregnancy index was lower in high-dose females.
The mean number of implantations counted, mean live pups/litter and birth index were comparable in all groups. In addition, supernumerary implantation sites, percentage of implantations, were not influenced by administration of the test substance.
Mean gestation length was comparable in all groups.

ORGAN WEIGHTS
In high dose males, liver, kidney, testes, epididymides, prostate and seminal vesicles weight were slightly lower, with statistical significance, which was due to the reduction of terminal body weight and hence, not related to target organ toxicity.
The same applied for high dose females, where liver, kidney and uterus weight was slightly lower, with statistical significance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Males and females from the mid-dose group exhibited kidneys with dark brown discolorations. In addition, the kidneys of one male in this groups was bluish discolored.
The main relevant findings were discolorations in several organs animals of the high dose group. Further major alterations were white discolored or broken incisors in nearly all animals of this group.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histopathological findings in parental animals of the high-dose group at terminal killing revealed intratubular pigment in kidneys in 10 male and 5 female animals. Single animals exhibited degenerations or necrosis of tubular cells. Increased number of necrotic/apoptotic cells were found in the liver. Mixed cellular infiltrations in the submucosal area of the stomach were found particular in males.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

General health

Effect level:

62.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Remarks on result:

not determinable because of methodological limitations

Remarks:

As the effects seen are related to an impurity, which causes species-specific effects in the rat teeth that are not relevant for humans, this NOAEL is only related to rats. The NOAEL for effects relevant for humans is most probably 1000 mg/kg bw/day.

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive performance

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects observed were due to broken-off incisors resulting in lower food consumption and a lower pregnancy index.

Target system / organ toxicity (P0)

Critical effects observed:

no

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:

not specified

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
no effects

CLINICAL SIGNS (OFFSPRING)
no effects

BODY WEIGHT (OFFSPRING)
Mean body weight of live pups during lactation was significantly decreased in the high dose offspring (1000 mg/kg/day) from Day 14, post partum onwards. Mean body weight was not affected in any other group.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

other: effects observed on body weight of high-dose pups were due to broken-off incisors in dams resulting in lower food consumption. This effect could be due to fluorosis of the teeth caused by the 0.3% fluoride impurity.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Dental examinations

Analysis of the incisors from remaining 3 high dose males and three respective control males revealed burning concentrations of 140 µg/L fluoride in high dose males and 50 µg/L in the controls, indicating a significantly higher amount of fluoride in particluar on the dental surface.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the reproductive and developmental NOAELs of the test substance were both considered to be 1000 mg/kg/day.

Executive summary:

A study was conducted in order to determine the effects of the test substance on reproduction according to OECD Guideline 415 and EU Method B.34, in compliance with GLP. Groups of 28 male and 28 (27 in the high-dose group) female Sprague Dawley rats received the test substance orally once daily at dose levels of 0, 62.5, 250 or 1000 mg/kg bw/day for a period of 10 weeks (males) and 4 weeks (females), prior to mating. Dosing of males was continued during the whole mating period until sacrifice (approx. Week 11 - 13 of the study). Treatment of mated females was continued until Day 21 after birth. Behaviour and state of health were observed daily in all groups. Body weight development and food consumption were recorded throughout the study in females, and during pre-mating period in males. After the mating period the males were killed and necropsied. The dams were allowed to litter and rear their progeny to the stage of weaning. Growth, development and behaviour of the progeny were assessed during lactation. The dams as well as surviving pups were killed on Day 22-24 post-partum. At the time of sacrifice or death during the study the animals of the P generation were examined for macroscopically visible abnormalities. The main organs were weighed and the organ to body weight ratios were calculated. Special attention was paid to the organs of the reproductive system. No treatment-related effects were observed during the premating, mating, gestation and lactation period at dose levels of 62.5 or 250 mg/kg bw/day. Daily oral administrations of 1000 mg/kg bw/day was well tolerated in rats within the first 5 weeks of treatment, but thereafter, from Week 6, caused mortality due to dental lesions with subsequent disability of food uptake and starvation (clinical picture of dental fluorosis). This finding was time-dependent, with a threshold dose of 250 mg/kg bw/day for males, and could be related to the fluoride impurity (0.3%) of this batch tested as shown by the analysis of broken teeth. Although there was marked pigment storage of the test substance in several organs, there was no clear functional or histopathological correlation. Impairment of reproduction and fertility at high dose parental animals was primarily the result of severe dental problems. In the presence of severe dental problems at 1000 mg/kg bw/day and threshold dose of 250 mg/kg bw/day for this finding, there was no evidence of selective reproductive toxicity in rats for the test substance. Under the study conditions, the reproductive and developmental NOAELs of the test substance were both considered to be 1000 mg/kg bw/day (Ehling, 2001).