Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
one-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:
weight of evidence
Study period:
23 Jul 2001 to 12 Dec 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to EU test guidance in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
See below

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 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

Administration / exposure

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:
yes
Details on analytical verification of doses or concentrations:
First and last week of treatment
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 / concentrationsopen allclose all
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

Examinations

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 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 examinated 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:
Copulatory index (%): Number of sperm positive females x 100 / Number of mated females
Fertility index - Males (%): Number of fertile males x 100 / Number of mated males
Fertility index - Females (%): Number of pregnant females x 100 / Number of mated females
Gestation index (%): Number of females with viable pups x 100 / Number of pregnant females
Sex ratio: (Number of pups examined - Number of males (females)) x 100 / Number of pups examined
Offspring viability indices:
Intra uterine mortality: (Number of implantations - Number of newborns) x 100 / Number of implantations
Total mortality: (Number of implantations - Number of viable pups) x 100 / Number of newborns
Viability index (%): Number of viable pups on day 4 (7, 14, 21) x 100 / Number of viable pups on day 0 (4, 7, 14)
Lactation index (%): Number of viable pups on day 21 x 100 / Number of viable pups on day 0 of lactation
Weaning index (%)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

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 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

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.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
General health
Effect level:
62.5 mg/kg bw/day
Sex:
male
Basis for effect level:
other: Broken-off incisors (fluorose) from 0.3% fluor impurity. This is a species specific effects and not relevant for humans.
Dose descriptor:
NOAEL
Remarks:
General health
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
Reproductive performance
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: All effects observed were due to broken-off incisors resulting in lower food consumption and a lower pregnancy index. This effect was due to fluorosis of the rats' teeth caused by the 0.3% fluor impurity and is solely a species specific effect

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

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Daily oral administration of Structural Analogue 01 to rats during the premating, mating, gestation and lactation period at dose levels of 62.5 or 250 mg/kg body weight did not affect food consumption, body weight development, male or female mating/reproductive performance, fertility, gestation length as well as development of their progenity.
Daily oral administrations of 1000 mg/kg body weight (high-dose group) were well tolerated in rats within the first 5 weeks of treatment, but thereafter, from week 6 onwards, 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 body weight for males, and could be related to the fluoride impurity (0.3%) of this batch tested.
Although there was marked pigment storage of the test compound in several organs, there was no clear functional or histopathological correlate that could be related to compound-induced systemic toxicity and/or specific reproductive toxicity. 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 and threshold dose of 250 mg/kg bw for this finding, there was no evidence of selective reproductive toxicity in rats for the Structural Analogue 01, according to the classification criteria of Commission Directive 2001/59/EC.
Executive summary:

The present study was conducted in order to determine the effects of the Structural Analogue 01 on reproduction when administered orally by gavage to male and female Sprague Dawley rats during pre-mating, mating, gestation and lactation.

Groups of 28 male and 28 (27 in the high-dose group) female Spraque Dawley rats received the Structural Analogue 01 orally once daily at dose levels of 0, 62.5, 250 or 1000 mg/kg body weight 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 littering. The dosing volume was 5 mL/kg, corresponding to concentrations of 0, 12.5, 50 and 200 mg/mL. At start of the study, the animals were 5-9 weeks of age with mean body weights of 240 g for males, and 206 g for females.

Behavior 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 behavior of the

progeny were assessed during lactation. The dams as well as surviving pups were killed on day 22-24 post partum. Animals scheduled for necropsy on weekend were killed the next weekday.

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 calculated. Special attention was paid to the organs of the reproductive system. Histopathology of listed organs was performed in case of macroscopic visible changes. Moreover,

dental mineral analyses (fluoride, calcium and phosphorus) were performed externally. In addition, clinical chemistry investigations, in particular for serum electrolytes, were performed in 10 animals per sex and group as amended to the protocol.

Body weights, food consumption, clinical chemistry data, absolute and relative organ weights and litter parameters were analyzed with the aid of a statistical program to show differences compared to the controls.

RESULTS

High-dose group (1000 mg/kg body weight): There were 7 males and five females that were found dead or killed on humane grounds due to starvation and bad general health condition as a cause of broken off incisors and subsequent disability of food uptake. In addition, one female was killed with dead pups at birth, another one with live pups was killed on lactation day 6 due to

inability to suckle them properly. Teeth trimming were carried out to insure food uptake during mating procedures for as many animals concerned as possible. Mean food consumption and body weight development was decreased during pre-mating (males) and during the lactation period (surviving females). Mean gestation length, (ca. 23.0 days), 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 implatations 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 humane grounds, as they were not able to rear their healthy offspring due to starvation. The remaining 7 females reared their healthy offpring 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.

Apart from significantly decreased total bilirubin levels, clinical pathology was unobstrusive, also with regard to serum electrolytes. 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 carcasse 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, probably as a

result of irritating effects of the test compound. 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.

Mid-dose group (250 mg/kg body weight): There were no premature deaths. No compound related clinical findings were recorded for the females. Four males had broken-off incisors during the late treatment period (weeks 6 -12). However, food consumption, body weight development, mating and reproductive performance, fertility, mean gestation length, rearing and development of their

offspring remained unaffected by administration of the test compound. Clinical chemistry, as well as anatomic pathology (necropsy, organ weights, histopathology) in particular of the sexual organs were generally unobstrusive, apart from pigment storage (dark brownish/or bluish discolorations) in the kidneys.

Low-dose group (62.5 mg/kg body weight): There were no premature deaths. No compound-related clinical signs were recorded in the P-generation male and female animals. Food consumption, body weight development, mating and reproductive performance, fertility, mean gestation length, rearing and development of their offspring remained unaffected by administration of the test compound. Clinical Chemistry, as well as anatomic pathology (necropsy, organ weights, histopathology) in particular of the sexual organs were unobstrusive.

By read across to the Structural Analogue 01, Reactive Orange DYPR 1410 is also deemed to have no toxicity to reproduction effects associated with it.