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Diss Factsheets

Administrative data

Endpoint:
one-generation reproductive toxicity
Type of information:
other: Data sharing dispute
Adequacy of study:
key study
Study period:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
tetrasodium 4-amino-5-hydroxy-3,6-bis[(E)-2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl]naphthalene-2,7-disulfonate
Molecular formula:
C26H25N5O19S6.4Na
IUPAC Name:
tetrasodium 4-amino-5-hydroxy-3,6-bis[(E)-2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl]naphthalene-2,7-disulfonate

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: TOXI COOP Ltd. 1032 Budapest, Cserkesz u. 90.
- Age at study initiation: Males: less than 9 weeks old
Females: at least 9 weeks old
- Housing: Before mating: 4 male animals/ cage
5 female animals/ cage
Mating period: male animals: individual caging,
Mating hours: 1 male and 1 female / cage
During pregnancy: in groups of 1 to 4 animals
Delivery and nursing: individual caging
- Diet (ad libitum): sniff SM R/M-Z+H autoclavable complete feed for rats and mice - breeding and m
aintenance
- Water (ad libitum): tap
- Acclimation period: 29 days for male rats
6 days for female rats
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): -
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 29 June 2006 To: 10 November 2006

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in distilled water in concentrations of 10 mg/mL, 30 mg/mL and 75 mg/
mL. The pH of dosing solutions were adjusted to pH=7.0 with 10 M NaOH (solid NaOH: Batch No.:
KBM 5080902, Expiry date: November 2009) during the first three weeks treatment. The results of
the preliminary study (two days treatment) regarding the pH influence on digestive system were not
supported by the observations after two weeks treatment therefore pH was not adjusted after three
weeks treatment. Formulations were prepared daily except weekends. Dosing solutions were stab
le for at least 24 hours at room temperature and 4 days refrigerated. The stock solution was stable
for at least 10 days at 2 to 8°C. Homogeneity of test item in this vehicle was analytically proven.
Analytical control of dosing solutions was performed five times during the treatment period on Days 0,
56, 77, 99, and 131.
VEHICLE: Distilled water
- Concentration in vehicle: 10 mg/mL, 30 mg/mL and 75 mg/mL
- Amount of vehicle: 10 mL/kg body weight
- Lot/batch nos.: 0503-0306; 85 10604; 3490306; 3530306; 3630306; 53 10506; 6650706
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: two weeks; 3 hours/day in the morning
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged in groups of 1 to 4 animals
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing solutions was performed five times during the treatment period on Days 0,
56, 77, 99, and 131 with a validated HPLC method.
Duration of treatment / exposure:
Males: pre-mating: 81 days
pairing: 12 days
post-mating: 4 to 5 days
Females: pre-mating: 27 days
pairing: 12 days
gestation: 22 to 24 days
lactation: 21 to 23 days
Frequency of treatment:
daily
Details on study schedule:
Pre-treatment period
Animal arriving: Male (M): 31 May 2006; Female (F): 16 August 2006
Veterinary control, acclimatisation: M: 31 May - 28 June 2006 (29 days); F: 16 - 21 August 2006 (6
days)
Animal identification/body weight measurement/randomisation: M: 28 June 2006; F: 21 August 2006
Treatment period
Pre-mating period: M: 29 June - 17 September 2006 (81 days); F: 22 August - 17 September 2006
(27 days)
Examination of estrous cycle: 22 August - 29 September 2006
Pairing period: 18 - 29 September 2006 (12 days)
Mating period: 18 - 27 September 2006 (10 days)
Gestation periods: 18 September - 19 October 2006 (32 days)
Lactation periods: 10 October - 10 November 2006 (32 days)
Clinical observation: M: Daily from 29 June 2006 - up to the necropsy; F: Daily from 22 August 2006 -
up to the necropsy
Body weight measurement: M: 29 June 2006, then weekly
F: 22 August 2006, then weekly prior to and during the mating period,
- On gestational days 0, 4, 7, 10, 14, 17 and 21
- On postpartal days 0, 4, 7, 10, 14, 17 and 21
Food consumption measurement: M: 06 July 2006, then weekly prior to the mating
F: 29 August 2006, then weekly prior to the mating
- On gestational days 0, 7, 14 and 21
- On postpartal days 0, 4, 7, 14 and 21
PUPS
Body weight measurement: 10 October - 09 November 2006
- On postnatal days 0, 4, 7, 14 and 21
Surface righting reflex: 10 - 20 October 2006 - At the birthday
Pinna detachment: 12 - 22 October - On postnatal day 2
Eye opening: 24 October - 03 November 2006 - On postnatal day 14
Termination
Necropsy: M: 03, 04 October 2006
Dead animals: No.: 408: 28 September 2006
No.: 418: 05 August 2006
Dams delivered: 31 October - 10 November 2006
Dams not delivered: 25 October 2006
Females not mated: 04 October 2006
Offspring: sacrificed on postnatal day 21, 22 or 23
The end of the in life phase: 10 November 2006
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
analytical conc.
Dose / conc.:
300 mg/kg bw/day
Remarks:
Doses / Concentrations:
300 mg/kg/day
Basis:
analytical conc.
Dose / conc.:
750 mg/kg bw/day
Remarks:
Doses / Concentrations:
750 mg/kg/day
Basis:
analytical conc.
Dose / conc.:
10 other: mg/ml
Remarks:
Doses / Concentrations:
10 mg/ml
Basis:
analytical conc.
Dose / conc.:
30 other: mg/ml
Remarks:
Doses / Concentrations:
30 mg/ml
Basis:
analytical conc.
Dose / conc.:
75 other: mg/ml
Remarks:
Doses / Concentrations:
75 mg/ml
Basis:
analytical conc.
No. of animals per sex per dose:
24 males/group
25 females/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were chosen on the basis of the results of previous rodent st
udies and a preliminary toxicity study with the test item in rats. A group of male animals was treated at
1000 mg/kg bw/day dose for about 2 weeks before mating. During this period, a decision was made
to eliminate this dose level from main part of the study before the female treatment has been started
because of deaths of several male rats (9/24) treated with 1000 mg/kg bw/day.
Positive control:
not reported

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
BODY WEIGHT: Yes
- Time schedule for examinations: weekly
FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly
OTHER:
- Observation of the delivery process
- Observation of the nursing instinct
Oestrous cyclicity (parental animals):
Vaginal smear of all the females was prepared daily during the pre-mating period four weeks before
the mating period and during the mating period. The vaginal smears were stained with 1 % aqueous
methylene blue solution and were examined with light microscope
Sperm parameters (parental animals):
not reported
Litter observations:
Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as
possible. All changes were recorded
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
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, physical or behavioural abnormalities, development
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 (offspring):
SACRIFICE
- All dead pups were dissected to find the cause of the death. It was determined whether the dead ne
wborn was live-born or stillborn by lung flotation test.
- The F1 offspring were sacrificed without necropsy following terminal anaesthesia on postnatal day
2 1,22 or 23.
Statistics:
PARENTAL MALES (P)
- Clinical observations
- Body weight (g)
- Body weight gain (g)
- Food consumption (g)
- Number of pairings
- Number of fertile pairings
- Number of infertile males
- Fertility index (%)
- Necropsy findings (%)
PARENTAL FEMALES (P)
- Clinical observations
- Body weight (g)
- Body weight gain (g)
- Food consumption (g)
- Number of not mated females
- Number of pregnant females
- Number of sperm positive, but non-pregnant females
- Copulatory index (%)
- Fertility index (%)
- Gestation index (%)
- Number of oestrous periods (until mating)
- Oestrous length (day, until mating)
- Duration of pregnancy (day)
- Implantations 1 dams
- Intrauterine mortality
- Total mortality (intra and extra uterine mortality)
- Necropsy findings (%)
- Histopathological findings (%)
OFFSPRING (F1)
- Mean body weight per litter on postnatal days 0,4,7, 14 and 21
- Mean body weight gain per litter between postnatal days 0-4,4-7, 7-14, 14-21 and 0-2 1
- Number of live births per litter, and number of viable pups per litter on postnatal days 0,7, 14 and 21
- Extra uterine mortality of pups on postnatal days 0,2 1
- Viability index (%)
- Lactation index (96)
- Sex ratio % (on postnatal days 0 and 21)
- Surface righting reflex (%)
- Pinna detachment (%)
- Eye-opening (%)
- Necropsy findings (%)
The statistical evaluation of appropriate data was done SPSS PC+4.0. The homogeneity of variance
between groups was checked by Bartlett's homogeneity of variance test. Where no significant he
terogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained resu
lt was significant Duncan Multiple Range test was used to access the significance of inter-group dif
ferences. At significant result at Bartlett's test the Kruskal-Wallis analysis of variance was used and
the inter-group comparisons was performed using Mann- Whitney U-test. The Chi² test was performed if feasible.
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 implantati
ons
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
Surface righting reflex, Pinna detachment, Eye-opening: (Number of pups examined - Number of pups
with negative response) x 100 / Number of pups examined

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
not specified
Dermal irritation (if dermal study):
not specified
Description (incidence and severity):
not specified
Mortality:
not specified
Description (incidence):
not specified
Body weight and weight changes:
no effects observed
Description (incidence and severity):
not specified
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
not specified
Food efficiency:
not specified
Description (incidence and severity):
not specified
Water consumption and compound intake (if drinking water study):
not specified
Description (incidence and severity):
not specified
Ophthalmological findings:
not specified
Description (incidence and severity):
not specified
Haematological findings:
not specified
Description (incidence and severity):
not specified
Clinical biochemistry findings:
not specified
Description (incidence and severity):
not specified
Endocrine findings:
not specified
Description (incidence and severity):
not specified
Urinalysis findings:
not specified
Description (incidence and severity):
not specified
Behaviour (functional findings):
not specified
Description (incidence and severity):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Description (incidence and severity):
not specified
Histopathological findings: non-neoplastic:
not specified
Description (incidence and severity):
No treatment related alterations were found
Histopathological findings: neoplastic:
not specified
Description (incidence and severity):
not specified
Other effects:
not specified
Description (incidence and severity):
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Description (incidence and severity):
not specified
Reproductive function: sperm measures:
not specified
Description (incidence and severity):
not specified
Reproductive performance:
not specified
Description (incidence and severity):
not specified

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Increased water consumption and urine excretion related to the test item was found at 750 mg/kg bw/
day group (male and female animals) from day 2 until termination of treatment. There were no other
test item related clinical signs at 100, 300 or 750 mg/kg bw/day doses.
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No test item influence on the body weight development was found. A slightly higher mean daily food
consumption was noted for male and
female animals at 750 mg/kg bwlday almost the entire pre-mating period and for female animals durin
g the gestation period.
REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no test item related differences in the estrous cycle.
REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The number and percentage of mated and fertile male animals, the copulatory and fertility indices
were not affected by the treatment. There were no differences between the control and test item
treated groups in the number and percentage of sperm positive (mated) female animals and c
opulatory index.
GROSS PATHOLOGY (PARENTAL ANIMALS)
No test item related macroscopic findings
HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment related alterations were found

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
not specified
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: not specified
Dose descriptor:
NOEL
Remarks:
reproductive performance
Effect level:
750 mg/kg bw/day (nominal)
Based on:
not specified
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
not specified
Dermal irritation (if dermal study):
not specified
Description (incidence and severity):
not specified
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
not specified
Body weight and weight changes:
no effects observed
Description (incidence and severity):
not specified
Food consumption and compound intake (if feeding study):
not specified
Description (incidence and severity):
not specified
Food efficiency:
not specified
Description (incidence and severity):
not specified
Water consumption and compound intake (if drinking water study):
not specified
Description (incidence and severity):
not specified
Ophthalmological findings:
not specified
Description (incidence and severity):
not specified
Haematological findings:
not specified
Description (incidence and severity):
not specified
Clinical biochemistry findings:
not specified
Description (incidence and severity):
not specified
Urinalysis findings:
not specified
Description (incidence and severity):
not specified
Sexual maturation:
not specified
Description (incidence and severity):
not specified
Anogenital distance (AGD):
not specified
Description (incidence and severity):
not specified
Nipple retention in male pups:
not specified
Description (incidence and severity):
not specified
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
not specified
Histopathological findings:
not examined
Description (incidence and severity):
not specified
Other effects:
not specified
Description (incidence and severity):
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified
Description (incidence and severity):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified
Description (incidence and severity):
not specified

Details on results (F1)

VIABILITY (OFFSPRING)
A slightly higher mortality rate of male pups was found at 750 mgkg bwlday between postnatal day
0 and 4, consequently the viability indices for this period was less. This difference was ascribed to
treatment, but was probably related to maternal toxicity (observed as polyuria).
CLINICAL SIGNS (OFFSPRING)
no effects
BODY WEIGHT (OFFSPRING)
no effects
DEVELOPMENTAL TESTS (OFFSPRING)
There was no test item influence on the development of pups (surface-righting reflex, suckling ability,
pinna detachment, eye opening)
GROSS PATHOLOGY (OFFSPRING)
Test item related macroscopic alterations were not found in offspring subjected to gross pathology.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
300 mg/kg bw/day (nominal)
Based on:
not specified
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified
Treatment related:
not specified

Applicant's summary and conclusion

Conclusions:
The read across substance B induced an increase in the urine excretion (polyuria) at 750 mg/kg/day d
ose level on parental generation of CRL:(WI)BR rats during the course of a one generation reproducti
on toxicity study.
Reproductive performance of males and females were unaffected by the treatment.
The mortality of pups was a slightly higher at 750 mg/kg bw/day, probably as a consequence of
maternal toxicity (polyuria). There was no effect on postnatal development of viable pups.
NOEL for males: 300 mg/kg bw/day
NOEL for females: 300 mg/kg bw/day
NOEL for reproductive performance of the males: 750 mg/kg bw/day
NOEL for reproductive performance of the females: 750 mg/kg bw/day
NOEL for offspring: 300 mg/kg bw/day
Executive summary:

The aim of the one-generation reproduction toxicity study was to provide general information concerning
the effect of the test item read across B on the male and female reproductive performance, such as
gonadal function, estrous cycle, mating behavior, conception, parturition, gestation and lactation (P
generation) and on the neonatal morbidity, mortality, growth and development of the offspring (F1
generation) following oral (by gavage) administration.
CRL:(WI)BR rats (n=24 males/group and n=25 females/group) were involved in the study in a control
and at three dose levels: 100 mg/kg/day, 300 mg/kg/day and 750 mg/kg/day. Treatment was carried
out orally once a day in concentrations of 10 mg/ml, 30 mg/ml and 75 mg/ml corresponding to 10 ml/
kg body weight volume. All animals of the P generation were treated prior to mating (male animals for
81 days, females for 27 days) and throughout mating. For females, treatment was continued though the
gestation and lactation periods up to the necropsy. Observations included mortality, clinical symptoms,
body weight, food consumption, estrous cycle, mating, and delivery process.
The dams of P generation were allowed to litter, and rear their young up to termination on day 21-23
postpartum. Developmental tests were evaluated on litters (surface righting reflex, suckling, pinna
detachment and eye opening). All animals were subjected to gross pathology one day after the last
treatment and offspring were sacrificed. Histopathology examination was performed on reproductive
organs in the control and high dose groups and on gross lesions in the low and middle dose groups.
There were no test item related effect on the general state and behavior of animals, but an increased
urine excretion and water consumption related to the test item was found at 750 mg/kg bw/day group
(male and female animals) from day 2 until the termination of the treatment.
The body weight, body weight gain of males and females were unaffected at the examined dose levels
during the pre-mating period. There was no effect on body weight, body weight gain of dams during


gestation and lactation period at the examined dose levels. The mean daily food consumption was
higher than the control at 750 mg/kg bw/day dose in male animals and in female animals during the
pre-mating and gestation periods.
No test item influence on the estrous was found.
There were no test item related alterations in the delivery data of dams when compared with the control
value.
Reproductive performance of males and females were unaffected by treatment with read across
substance B.
Gross pathology revealed no alterations due to the effect of test item in P generation. No histological
alterations related to the test item effect were found. In the male animals the investigated organs
of reproductive system (testes, epididymides, seminal vesicles, prostate, coagulating gland) were
histologically normal. In dams, the ovaries had a normal structure characteristic of the species, age and
phase of the active sexual cycle. The uterus, cervix, and vagina had a normal structure in accordance
with the phase of sexual cycle in the investigated animals.
Mortality of pups was higher at 750 mg/kg bw/day group (9%vs.3%of control value).
Body weight, body weight gain and sex ratio of pups were not influenced by treatment with Read across
substance B
There was no effect on postnatal development of pups.
Read across substance B induced an increase in the urine excretion (polyuria) at 750 mg/kg/day dose
level on parental generation of CRL:(WI)BR rats during the course of a one generation reproduction
toxicity study.
Reproductive performance of males and females were unaffected by the treatment.
The mortality of pups was a slightly higher at 750 mg/kg bw/day, probably as a consequence of maternal
toxicity (polyuria). There was no effect on postnatal development of viable pups.
NOEL for males: 300 mg/kg bw/day
NOEL for females: 300 mg/kg bw/day


NOEL for reproductive performance of the males: 750 mg/kg bw/day
NOEL for reproductive performance of the females: 750 mg/kg bw/day
NOEL for offspring: 300 mg/kg bw/day