reaction mass of: tetrasodium 4-amino-6-(5-(2,6-difluoropyrimidin-4-ylamino)-2-sulfonatophenylazo)-5-hydroxy-3-(4-(sulfatoethylsulfonyl)phenylazo)naphthalene-2,7-disulfonate;tetrasodium 4-amino-6-(5-(4,6-difluoropyrimidin-2-ylamino)-2-sulfonatophenylazo)-5-hydroxy-3-(4-(2-sulfatoethylsulfonyl)phenylazo)naphthalene-2,7-disulfonate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Daily oral administration of the test substance to rats during the premating, mating, gestation and lactation period had no adverse effect on parental reproductive performance and F1 offspring development.

The only adverse effects seen in the source substance were secondary effects as a consequence to broken incisors provoked by a 0.3 % impurity of fluoride in the test substance. A 3-fold increase in fluoride concentration within incisors were observed in affected rats. This effects is not relevant for human assessment, as the teeth in humans are not as sensitive to a higher fluoride concentration in food. Hence the NOAEL for all endpoints is considered to be 1000 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-07-31 to 2001-12-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

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 weeks
- Housing: single
- Diet (ad libitum): sniff R/M-Z (V1324)
- Water (ad libitum): tap
- Acclimation period: at least five days


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

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

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

VEHICLE: deionized water
- Concentration in vehicle: 12.5 mg/mL, 50 mg/mL 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: three 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:

not specified

Details on analytical verification of doses or concentrations:

-

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

Remarks:

Doses / Concentrations:
12.5 mg/mL, 50 mg/mL and 200 mg/mL
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
62.5, 250 and 1000 mg/kg body weight per day
Basis:

No. of animals per sex per dose:

28

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose rationale was based on a subacute 28-day oral toxicity study with the test compound 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 body weight per day were selected for the present study.

Positive control:

NA

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No data

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

Oestrous cyclicity (parental animals):

daily during mating period

Sperm parameters (parental animals):

Parameters examined in all P male parental generations: testis weight, epididymis weight, prostate weight, seminal vesicles weight, histopathology of testis, epididymis, prostate, 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: Epididymides, 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 heart, spleen, lung, pancreas and gastro-intestinal tract from those animals with macroscopically visible changes, i.e., blueish colored pigmentation storage of the test compound.
The following organs were weighed: Epidymides, 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 phosphorous content (two high dose and two control animals, data not presented, filed in the raw data). Secondly they were extended to fluoride, calcium and phosphorous 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 days 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:, Nonparametric 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

Clinical signs:

effects observed, treatment-related

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

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no intercurrent deaths in the control-, low- and mid-dose group animals. In the high dose group (1000 mg/kg body weight), 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 killed by mistake 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, 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 body weight 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) was slightly to moderately decreased. 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.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no test item related differences in 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, birth index were comparable in all groups. In addition, supernumerary
implantation sites, percentage of implantations, were not influenced by administration of the test compound.
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.

Dose descriptor:

NOAEL

Remarks:

General health

Effect level:

62.5 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: broken-off incisors (fluorosis) from 0.3% fluor impurity

Dose descriptor:

NOAEL

Remarks:

Reproductive performance

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

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

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 bw.) from day 14, post partum onwards. Mean body weight was not affected in any other group

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Reproductive effects observed:

not specified

Conclusions:

Daily oral administration of the test substance to rats during the premating, mating, gestation and lactation period at dose levels of 62.5 or 250 mg/kg body weight had no adverse effect on parental reproductive performance and F1 offspring development. 1000 mg/kg bw/day of test substance affected reproduction performance and body weight gain of pubs. The observed effects in the high dose group are considered to be secondary as a consequence to broken incisors provoked by a 0.3 % impurity of fluoride in the test substance. A 3 fold increase in fluoride concentration within incisors were observed.

Executive summary:

In a one-generation reproduction toxicity study the test substance was administered to 28 (27 for high dose females) Sprague Dawley rats/sex/dose in water by gavage at dose levels of 0, 62.5, 250 and 1000 mg/kg bw/day.

In the P-generation there were no effects in the low dose group and in the mid dose group no effects regarding clinical chemistry and anatomic pathology apart from pigment storage (dark brownish or bluish discoloration) in the kidney were observed. 4 males in the mid dose group had broken-off incisors during the late treatment period (weeks 6 -12), yet this was without effect.

 

The high dose group 7 males and 5 females were found dead or killed on human grounds due to starvation and bad general health condition as a cause of broken off incisors and subsequent disability of food uptake. 1 female was killed with dead pups at birth, another 1 with live pups was killed on lactation day 6 due to inability to suckle them properly. Mean food consumption and body weight development was decreased during pre-mating (males) and during the lactation period (surviving females). Mean gestation length was not affected. Because of these unscheduled deaths the number of pregnancies was markedly reduced (12 cf./22 of control). The absolute number of females at term with live pups was reduced (11 cf./21 of control), with lower absolute number of implantations. One dam had dead pups only. However, relative numbers of live pups, the mean number of implantations and birth index, was not adversely affected when related to the number of females at term with live pups.

During early lactation, 4/11 females had to be killed on human grounds, as they were not able to rear their healthy offspring due to starvation. The remaining 7 females reared their healthy offspring up to the end of the lactation period, however, mean pup body weight gains were significantly decreased from day 14 post partum up to the end of the study.

Mean viability index, weaning index, survival rate at day 21 was not affected. There was 1 unreared litter recorded for this group. The pups did not show any macroscopically visible abnormalities.

Clinical pathology was unobtrusive, a part from significantly decreased total bilirubin levels. Anatomic pathology revealed severe dental lesions (broken off, deformed and white discolored incisors), which were confirmed to contain a 3-fold concentration of fluoride. Fluoride (0.3%) was identified as an impurity of the test compound batch, tested in this study.

Massive bluish discolorations of the whole carcasses and in several inner organs were also detected at necropsy. Microscopy confirmed intratubular pigment storage in the kidneys, increased number of necrotic/apoptotic cells in the liver as a histopathological correlate of clinical starvation, and mixed cellular infiltrations in the submucosal area of the stomach. There were no selective changes in sexual organs that could be related to selective reproductive toxicity in these dose group animals, nor were there any correlates of target organ toxicity.

The NOAEL for parental toxicity is considered to be 62.5 mg/kg bw/day in males/females, based on broken incisors starting time-dependent at higher doses. The NOAEL for parental reproductive and F1 offspring effects is 1000 mg/kg bw/day in males and females.

 

This study is acceptable and satisfies the guideline requirement for a one-generation reproduction toxicity study OECD 415 in rats.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a one-generation reproduction toxicity study the test substance was administered to 28 (27 for high dose females) Sprague Dawley rats/sex/dose in water by gavage at dose levels of 0, 62.5, 250 and 1000 mg/kg bw/day.

In the P-generation there were no effects in the low dose group and in the mid dose group no effects regarding clinical chemistry and anatomic pathology apart from pigment storage (dark brownish or bluish discoloration) in the kidney were observed. 4 males in the mid dose group had broken-off incisors during the late treatment period (weeks 6 -12), yet this was without effect.

 

The high dose group 7 males and 5 females were found dead or killed on human grounds due to starvation and bad general health condition as a cause of broken off incisors and subsequent disability of food uptake. 1 female was killed with dead pups at birth, another 1 with live pups was killed on lactation day 6 due to inability to suckle them properly. Mean food consumption and body weight development was decreased during pre-mating (males) and during the lactation period (surviving females). Mean gestation length was not affected. Because of these unscheduled deaths the number of pregnancies was markedly reduced (12 cf./22 of control). The absolute number of females at term with live pups was reduced (11 cf./21 of control), with lower absolute number of implantations. One dam had dead pups only. However, relative numbers of live pups, the mean number of implantations and birth index, was not adversely affected when related to the number of females at term with live pups.

During early lactation, 4/11 females had to be killed on human grounds, as they were not able to rear their healthy offspring due to starvation. The remaining 7 females reared their healthy offspring up to the end of the lactation period, however, mean pup body weight gains were significantly decreased from day 14 post partum up to the end of the study.

Mean viability index, weaning index, survival rate at day 21 was not affected. There was 1 unreared litter recorded for this group. The pups did not show any macroscopically visible abnormalities.

Clinical pathology was unobtrusive, a part from significantly decreased total bilirubin levels. Anatomic pathology revealed severe dental lesions (broken off, deformed and white discolored incisors), which were confirmed to contain a 3-fold concentration of fluoride. Fluoride (0.3%) was identified as an impurity of the test compound batch, tested in this study.

Massive bluish discolorations of the whole carcasses and in several inner organs were also detected at necropsy. Microscopy confirmed intratubular pigment storage in the kidneys, increased number of necrotic/apoptotic cells in the liver as a histopathological correlate of clinical starvation, and mixed cellular infiltrations in the submucosal area of the stomach. There were no selective changes in sexual organs that could be related to selective reproductive toxicity in these dose group animals, nor were there any correlates of target organ toxicity.

As all effects seen, did not represent test substance related toxicity but were secondary effects resulting from a 0.3 % impurity of fluoride in the test material, these effects are not relevant for the target substance.

Consequently, the NOAEL for parental toxicity, parental reproductive and F1 offspring effects is 1000 mg/kg bw/day in males and females.

Effects on developmental toxicity

Description of key information

In a modified one-generation study with a structural analogue of Reactive Navy FC63805, no adverse effects regarding maternal and embryofoetal toxicity and teratogenicity were observed up to the limit dose of 1000 mg/kg bw/d.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

No adverse effect relevant for classification and labelling have been reported in one OECD 415 study conducted with a structural analogues substance.