Reaction mass of lithium sodium hydrogen 4-amino-6-({5-[(5-chloro-2,6-difluoropyrimidin-4-yl)amino]-2-sulfonatophenyl}diazenyl)-5-hydroxy-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-2,7-disulfonate and lithium sodium hydrogen 4-amino-6-({5-[(5-chloro-2,6-difluoropyrimidin-4-yl)amino]-2-sulfonatophenyl}diazenyl)-5-hydroxy-3-{[4-(vinylsulfonyl)phenyl]diazenyl}naphthalene-2,7-disulfonate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

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

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 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

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:

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

Details on study schedule:

NA

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Remarks:

Corresponding concentration: 12.5 mg/mL

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Corresponding concentration: 50 mg/mL

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 ora 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

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 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

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

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 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.

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

other: broken-off incisors (fluorosis), probably related to 0.3% fluoride impurity

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. This effect could be due to fluorosis of the rats' teeth caused by the 0.3% fluoride impurity.

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

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.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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).

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

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).

Effects on developmental toxicity

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17-07-2001 to 08-08-2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Requirements for Safety Evaluation of Agricultural Chemicals, 59 NohSan No. 4200, Japanese Ministry of Agriculture, Forestry and Fisheries

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

- Age at start of study: approximately 8-10 wk
- Supplier: Fa. Harlan Winkelmann GmbH, Gartenstr. 27, 33178 Borchen
- Housing: Macrolon type III cage, individually on softwood granulate
- Temperature: 20-25°C
- Relative humidity: 30-70%
- Photoperiod: 12 h light/dark cycle
- Acclimatisation before study: atleast 5 d
- Feed: pelleted Ssniff R/Z (V1324), ad libitum
- Water: tap water in plastic bottles, ad libitum

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

- Dose volume: 5 mL/kg

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Virgin female animals in the pre-oestrus or oestrus phase were mated overnight with sexually mature males in the ratio 1 male : 1 female and were caged individually after the detection of sperm in vaginal smears. The day of sperm detection was defined as Day 0 of gestation. Pregnancy was confirmed at necropsy by the detection of implantation sites or normally developed corpora lutea.

Duration of treatment / exposure:

Day 6 to Day 19 of gestation

Frequency of treatment:

once daily

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

23

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a preliminary range-finding study at dose level of 1000 mg/kg/day, the doses for the study was selected.
- Rationale for animal assignment (if not random): randomization based on a computer-generated algorithm (archived with raw data)
- Observations: during the study period, the treated animals were observed for body weight, food consumption, mortality, cinical signs and examinations related to the uetrine contents.

Maternal examinations:

The animals were sacrificed on Day 20 of pregnancy and the fetuses removed by Caesarean section. All animals were examined externally and internally (thoracic and abdominal contents) for macroscopically visible changes, with emphasis on the uterus. Gravid uterus weight was recorded.

Ovaries and uterine content:

The live and dead fetuses in the uterus as well as the conceptuses undergoing resorption and corpora lutea were counted, identified in numerical sequence from cervix to ovary and examined macroscopically. The implantation sites in the uterus were counted after staining with ammonium sulphide.

Fetal examinations:

Approximately 50% of the fetuses of each litter and the dead fetuses were fixed in alcohol, necropsied, sexed and checked for anomalies of the internal organs. After staining with alizarin red S and Alcian blue, the skeletons were examined and checked for stage of development and abnormalities. Visceral and skeletal changes were subdivided into four categories (major defects, minor defects, variations and retardations) based on the severity and/or the spontaneous incidence of the finding.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Slightly lower mean body weight in the highest dose of 1000 mg/kg/day was marked as statistically significant on Day 20. However, body weight gains were regular during the entire study in the high dose group. This was hence considered an incidental finding.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Transient reduction in absolute food consumption in the highest dose during Days 6 to 9 on on Days 3 to 6 prior to treatment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

cf. under Results (fetuses)

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: A minimally lower final body weight and a minimally (not statistically significant) lower number of live fetuses were considered incidental findings and not related to test compound toxicity.

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

Minimal reductions in the number of live fetuses (not statistically significant) were observed in the high dose. These findings were within the biological variation of this strain.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

A minimally higher post-implantation loss (not statistically significant) was observed in the high dose. This finding was within the biological variation of this strain.

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

other: slight increase in post-implantation loss in high dose

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: A minimally higher post-implantation loss and a minimally lower number of live fetuses (both not statistically significant) seen in the high dose group were considered incidental findings and not related to test compound toxicity.

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Under the study conditions, the no observed effect level (NOEL) of the test substance in Sprague-Dawley rats is 250 mg/kg bw/day for maternal toxicity and embryotoxicity. A minimally lower final body weight in high-dose dams and a minimally higher post-implantation loss and lower number of live fetuses (both not statistically significant) seen in the high dose group were considered incidental findings and not related to test compound toxicity. These findings might however be related to dental fluorosis as seen in the one generation study due to relatively high fluoride content in the test substance. This effect is only relevant for rats however and does not pose a hazard for humans.
The test substance was not teratogenic in the rats (Ehling, 2002).

Executive summary:

A study was conducted to determine the effects of the test substance on embryonic and fetal development in mated Sprague-Dawley rats according to OECD Guideline 414, EU method B.31, OPPTS Guideline 870.3700 and Japanese Ministry Guideline 59 NohSan No. 4200, in compliance with GLP. The test substance was administered orally by gavage once daily to mated female Sprague Dawley rats from Day 6 - 19 of pregnancy at 0, 62.5, 250 or 1000 mg/kg bw/day and the treated rats were sacrificed on Day 20 of pregnancy. Animals were observed daily for mortality and clinical signs of toxicity. Body weight and food consumption were determined regularly throughout the study. At necropsy the dams were examined for macroscopically visible changes. The uterus was opened and the number of live and dead fetuses and the number of conceptuses undergoing resorption were determined. Body weights, crown-rump lengths, sex ratios of the fetuses and placental weights were determined and external, visceral and skeletal examinations of the fetuses performed. Neither any mortality nor substance-related clinical signs of toxicity occurred throughout the study. Body weight gain of all pregnant females was slightly lower in the high dose group (1000 mg/kg bw/day) during the treatment period. This led to a statistically significant (p<0.05) lower body weight at the end of treatment only. This is considered an incidental finding, as body weight gains were within normal ranges and the curve of body weight development is similar to that of the control group. Statistical evaluation showed an initial reduction (p<0.05) of feed consumption in this group (Days 3 -6) with subsequent recovery thereafter, which is considered to be an incidental occurrence. No substance-related adverse effects were observed at necropsy. There was a slight increase in the number of early or late conceptuses undergoing resorption (post-implantation loss), as well as a slight and not statistically significant decrease in the number of live fetuses at birth in the high dose dams. However, these numbers were within the normal ranges for this strain and, taking into consideration the normal intrauterine development of the conceptuses, considered to be of no toxicological significance. No such findings were observed for mid or low dose group females. Fetal crown-rump lengths, litter size, sex ratios, fetal body weight and placental weights remained unaffected by the administration of the test substance in any group. External, visceral and skeletal examinations of the fetuses did not reveal any substance-related alterations in any group. Under the study conditions, the NOEL of the test substance in Sprague-Dawley rats is 250 mg/kg bw/day for maternal toxicity and embryotoxicity. A minimally lower final body weight in high dose dams and a minimally higher post-implantation loss and lower number of live fetuses (both not statistically significant) seen in the high dose were considered incidental findings and not related to treatment-related. Hence the no observed adverse effect level (NOAEL) of the test substance was considered to be 1000 mg/kg bw/day. The test substance was not teratogenic in the rat (Ehling, 2002).

Effect on developmental toxicity: 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 developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A study was conducted to determine the effects of the test substance on embryonic and fetal development in mated Sprague-Dawley rats according to OECD Guideline 414, EU method B.31, OPPTS Guideline 870.3700 and Japanese Ministry Guideline 59 NohSan No. 4200, in compliance with GLP. The test substance was administered orally by gavage once daily to mated female Sprague Dawley rats from Day 6 - 19 of pregnancy at 0, 62.5, 250 or 1000 mg/kg bw/day and the treated rats were sacrificed on Day 20 of pregnancy. Animals were observed daily for mortality and clinical signs of toxicity. Body weight and food consumption were determined regularly throughout the study. At necropsy the dams were examined for macroscopically visible changes. The uterus was opened and the number of live and dead fetuses and the number of conceptuses undergoing resorption were determined. Body weights, crown-rump lengths, sex ratios of the fetuses and placental weights were determined and external, visceral and skeletal examinations of the fetuses performed. Neither any mortality nor substance-related clinical signs of toxicity occurred throughout the study. Body weight gain of all pregnant females was slightly lower in the high dose group (1000 mg/kg bw/day) during the treatment period. This led to a statistically significant (p<0.05) lower body weight at the end of treatment only. This is considered an incidental finding, as body weight gains were within normal ranges and the curve of body weight development is similar to that of the control group. Statistical evaluation showed an initial reduction (p<0.05) of feed consumption in this group (Days 3 -6) with subsequent recovery thereafter, which is considered to be an incidental occurrence. No substance-related adverse effects were observed at necropsy. There was a slight increase in the number of early or late conceptuses undergoing resorption (post-implantation loss), as well as a slight and not statistically significant decrease in the number of live fetuses at birth in the high dose dams. However, these numbers were within the normal ranges for this strain and, taking into consideration the normal intrauterine development of the conceptuses, considered to be of no toxicological significance. No such findings were observed for mid or low dose group females. Fetal crown-rump lengths, litter size, sex ratios, fetal body weight and placental weights remained unaffected by the administration of the test substance in any group. External, visceral and skeletal examinations of the fetuses did not reveal any substance-related alterations in any group. Under the study conditions, the NOEL of the test substance in Sprague-Dawley rats is 250 mg/kg bw/day for maternal toxicity and embryotoxicity. A minimally lower final body weight in high dose dams and a minimally higher post-implantation loss and lower number of live fetuses (both not statistically significant) seen in the high dose were considered incidental findings and not related to treatment-related. Hence the no observed adverse effect level (NOAEL) of the test substance was considered to be 1000 mg/kg bw/day. The test substance was not teratogenic in the rat (Ehling, 2002).

Mode of Action Analysis / Human Relevance Framework

A slight increase in post-implantation loss and a slight decrease in the number of live fetuses at birth in the high dose have been observed in the pre-natal developmental toxicity study, in the presence of a minimally lower final body weight in high-dose dams. Although it is questionable that these are substance-related effects, these findings might however be related to dental fluorosis as seen in the one generation study due to relatively high fluoride content in the test substance. This effect is only relevant for rats however and does not pose a hazard for humans. No such findings were observed for mid or low doses. There were no other adverse effects on the development of fetuses.

Justification for classification or non-classification

Based on the results of a one-generation reproductive toxicity study and a prenatal developmental toxicity study in rats, no classification for reproductive/developmental toxicity is warranted for the test substance according to CLP (EC/1272/2008) criteria.