Reaction products of 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid, coupled twice with diazotized 2-[(4-aminophenyl)sulfonyl]ethyl hydrogen sulfate, sodium salts

Toxicity to reproduction

Some information in this page has been claimed confidential.

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 June 2006 to 10 November 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

Test material information

Constituent 1

Test material form:

solid: particulate/powder

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 maintenance
- 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 stable 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 / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

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 studies 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:

NA

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):

NA

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 (parental animals):

SACRIFICE
- Male animals: All surviving animals were subjected to necropsy following terminal anaesthesia 4 -5 days after the mating period
- Female animals:
- Non-mated female animals were subjected to necropsy following terminal anaesthesia 5 days after the mating period.
Occasional implantations in the uterus were checked. If there was implantation in the uterus, corpora lutea were counted, too
- Animals, which failed to deliver up to gestation day 24 were subjected to necropsy following terminal anaesthesia
- Dams with viable pups were subjected to necropsy following terminal anaesthesia after post-partal day 21

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs of parental animals or representative samples thereof were preserved all in 4 % neutral formaldehyde solution but testis in Bouin's solution for histological examination:
Gross lesions, lymph nodes (submandibular, mesenteric) sternum, skin and female mammary gland, salivary glands (submandibular), larynx, femur + bone marrow, spinal cord (cervical, lumbar, thoracic level), pituitary, thymus, trachea, lungs (with main stem bronchi), heart, thyroid + parathyroid, oesophagus, stomach, caecum, duodenum, ileum, jejunum, colon, rectum, urinary bladder, liver, pancreas, spleen, kidneys, adrenals, prostate, epididymides, ovaries, uterus with vagina, brain (including cerebrum, cerebellum, pons and medulla oblongata), eyes with optic nerve, Harderian glands and lachrymal gland, seminal vesicle, muscle (quadriceps), sciatic nerve, aorta.
Gross lesions, the ovaries, uterus, cervix, vagina, testes, epididymides, seminal vesicles, prostate, coagulating gland and pituitary gland were subjected to histological examinations.
Histological examinations were conducted in all control and high dose treated animals. A full histological examination was performed for animals, which were found dead during the study.
Reproductive organs of animals suspected of fertility were subjected to microscopic examination in low and medium dose groups.

Postmortem examinations (offspring):

SACRIFICE
- All dead pups were dissected to find the cause of the death. It was determined whether the dead newborn was live-born or stillborn by lung flotation test.
- The F1 offspring were sacrificed without necropsy following terminal anaesthesia on postnatal day 21, 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, 21
- 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 heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. 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 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
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

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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 during 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 copulatory index.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No test item related macroscopic findings

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment related alterations were found

Effect levels (P0)

1

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: clinical signs: polyuria

2

Dose descriptor:

NOEL

Remarks:

reproductive performance

Effect level:

750 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: gross pathology; histopathology; mating index; fertility index; duration of pregnancy; birth index; live birth index; pregnancy index; litter size; litter weight; pup weight; sex ratio

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

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)
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)

Sex:

male/female

Basis for effect level:

other: viability index

Overall reproductive toxicity

Reproductive effects observed:

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 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

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