Phenol, 2,4-dinitro-, sulfurized, leuco derivatives

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 20 May 2009 and 23 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study has been performed in compliance with the: Swiss Ordinance relating to Good Laboratory Practice adopted May 18th, 2005 [SR 813.112.1]. This Ordinance is based on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted on November 26th, 1997 by decision of the OECD Council [C (97)186/Final]. These principles are compatible with Good Laboratory Practice regulations specified by regulatory authorities throughout the European Community, the United States (EPA and FDA), and Japan (MHLW, MAFF and METI). There were no circumstances that may have affected the quality or integrity of the data.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Leuco Sulfur Black 1

IUPAC Name:

Leuco Sulfur Black 1

Constituent 2

Reference substance name:

Phenol, 2,4-dinitro-, sulfurized, leuco derivatives

EC Number:

266-273-5

EC Name:

Phenol, 2,4-dinitro-, sulfurized, leuco derivatives

Cas Number:

66241-11-0

Molecular formula:

Molecular formula is not available.

IUPAC Name:

Reaction products of 2,4-dinitrophenol with polysulfide, leuco derivatives, sodium salt

Details on test material:

Description: Thixotrophic, black, wet paste
Batch Number: ESA0007595
Composition: Leuco Sulfur Black 1: 52.75% (calculated by amount of by-compounds)
2,4-Dinitrochlorobenzene: < 20 ppm
2,4-Dinitrophenole: < 20 ppm
Sulfate: 970 ppm
Chloride: 750 ppm
Sodium: 1.3% (w/w)
Water: 45.75% (w/w) by Karl Fischer titration
Stability of Test Item: Stable under storage conditions
Stability of Test Item in Vehicle: Seven days
Expiry Date (Retest Date): 11-Jul-2011
Storage Conditions: At room temperature (20 ± 5 °C), light protected
Safety Precautions: Routine hygienic procedures (gloves, goggles, face mask).

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals: Rat, HanRcc: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories Ltd., Laboratory Animal Services, Wölferstrasse 4, 4414 Füllinsdorf, Switzerland
Number of Animals: 40 males (10 per group), 40 females (10 per group)
Age (at Start of Treatment): 11 weeks
Body Weight Range (at Start of Treatment): Males: 290 to 324 g, Females: 284 to 212 g
Identification of Prental Animals: Cage card and individual animal number (ear tattoo).
Identification of Pups: On day 1 post partum, pups were individually tattooed with Indian ink.
Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 37.5 - 80.9%). There was 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
Diet: Pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum (batch no. 15/09).
Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Dose Formulations

The dose formulations were prepared weekly using the test item as supplied by the Sponsor.
Leuco Sulfur Black 1 was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Storage of Dose Formulations

Dose formulations were stored at room temperature (20 ± 5 °C) in brown glass beakers.
Based upon the results of stability analyses performed within the Harlan Laboratories study no. C31076, dose formulations were stable for at least one week.

Treatment

Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
Target Dose Levels:
Group 1: 0 mg/kg bw/day (control group)
Group 2: 100 mg/kg bw/day
Group 3: 300 mg/kg bw/day
Group 4: 1000 mg/kg bw/day
Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range finding toxicity study in Han Wistar Rats, Harlan Laboratories Study C31076, using dose levels of 116, 348 and 1160 mg/kg/day adjusted for 52.75% Leuco Sulfur Black 1 dye stuff in the product and corresponding to 220, 660 and 2200 mg/kg bw/day of product. The NOAEL of this study resulted to be 1160 mg dye stuff /kg bw/day or 2200 mg product/kg bw/day.
Dose Volume: 10 mL/kg body weight

Details on mating procedure:

During the pairing period, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if:
- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.
The day of mating was designated day 0 post coitum.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of Dose Formulations

On the first treatment day duplicate samples from the control group as well as three duplicate samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (4 hrs and 7 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were transferred into tightly closed vessels protected against oxygen. One set of samples was delivered at ambient temperature to Dr. K. Morgenthal (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at room temperature (20 ± 5 °C) until analysis. The remaining set of samples was stored at the facility at room temperature (20 ± 5 °C) in case of repeated analysis.

Analysis of homogeneity and concentration was performed gravimetrically by weighing the residue after drying by lyophilization. Stability of dose formulations was confirmed by IR-spectroscopy.

The application formulations investigated during the study were found to comprise Leuco Sulfur Black 1 in the range of 81.7% to 102.5%. The homogeneous distribution of Leuco Sulfur Black 1 in the preparations was approved because individual recoveries found did not deviate more than 14.2% (<15%) from the corresponding mean. In addition, the test item was found to be stable in application formulations when kept for 4 hours and for 7 days under storage conditions due to similar IR spectra compared to the reference spectrum obtained from the test item. Thus, identity of the test item in application formulations was confirmed. In conclusion, the results indicate the accurate use of the test item Leuco Sulfur Black 1 and highly purified water as vehicle during this study.

Duration of treatment / exposure:

Males: 29 days
Females: Approximately 7 weeks

Frequency of treatment:

Daily

Details on study schedule:

Males
Acclimatization: 7 days
First Test Item Administration : Day 1 of pre-pairing
Pre-Pairing: 14 days
Pairing: 14 days maximum
Treatment Ends: On day before sacrifice
Necropsy: After treatment of at least 28 days, when no longer needed for assessment of repro-ductive effects

Females
Acclimatization: 7 days
First Test Item Administration: Day 1 of pre-pairing
Pre-Pairing: 14 days
Pairing: 14 days maximum
Gestation: Approximately 21 days
Treatment Ends: On day 4 post partum
Necropsy:On day 5 post partum (pups on day 4 post partum)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Other:

Positive control:

Not required.

Examinations

Parental animals: Observations and examinations:

VIABILITY / MORTALITY
Twice daily

CLINICAL SIGNS
Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION
Males: Weekly during pre-pairing and after pairing periods
Females: Pre-pairing period days 1 - 8 and 8 - 14; gestation days 0 - 7, 7 - 14 and 14 - 21 post coitum, and days 1 - 4 post partum.
No food consumption was recorded during the pairing period.

BODY WEIGHTS
Recorded daily from treatment start to day of necropsy.

DETAILED CLINICAL OBSERVATIONS
Once prior to the first administration of the test item and weekly thereafter, detailed clinical observations were performed outside the home cage. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

FUNCTIONAL OBSERVATION BATERY
At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum) relevant parameters were performed with five P generation males and five P generation females from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following:
- Cage-side observations: unusual body movements (e.g. tremors, convulsions), abnormal behavior (e.g. circling, stereotypy) and posture as well as resistance to removal
- Hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex and reaction to handling.
- Open field observations: level of ambulatory activity including rearing (one minute evaluation), responsiveness to sharp noise, paw pinch, gait evaluation, quantity of urine and fecal pellets voided.
- Categorical observations (can be made any time during the FOB): hair coat, behavior, respiration, muscle movements, eyes, hearing ability (Preyer’s reflex), urine or feces, soiling, general abnormalities, posture.
- Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.
Any abnormal findings were recorded and, where appropriate, graded in severity.
Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.

CLINICAL LABORATORY INVESTIGATIONS
Blood and urine samples were obtained on the day of the scheduled necropsy from 5 males from each group. Males were kept in metabolism cage and fasted overnight (but allowed access to water ad libitum) for approximately 18 hours.
Blood samples from 5 lactating females from each group were obtained on day 5 post partum. The females were fasted for approximately 18-20 hours before blood sampling but allowed access to water ad libitum.
Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

HEMATOLOGY
The following hematology parameters were determined:

Complete Blood Cell Count:
Erythrocyte count
Reticulocyte Count
Hemoglobin
Hematocrit
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Leukocyte count, total
Differential leukocyte count
Platelet count

Coagulation:
Prothrombin time (= Thromboplastin time)
Activated partial Thromboplastin time

CLINICAL BIOCHEMISTRY
The following clinical biochemistry parameters were determined:
Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Aspartate aminotransferase
Alanine aminotransferase
Alkaline phosphatase
Gamma-glutamyl-transferase
Bile acids
Sodium
Potassium
Chloride
Calcium
Phosphorus
Protein, total
Albumin
Globulin
Albumin/Globulin ratio

URINE ANALYSIS
The following urine parameters were determined:
Relative density
colour
Appearance
pH
Protein
Glucose
Nitrite
Ketones
Urobilinogen
Bilirubin
Erytrocytes
Leukocytes
Volume (18 hours)

Oestrous cyclicity (parental animals):

Not reported.

Sperm parameters (parental animals):

testes weight, epididymes weight, stages of spermatogenesis

Litter observations:

The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

Postmortem examinations (parental animals):

TERMINATION OF THE STUDY
Males were sacrificed after treatment of 29 days, when no longer needed for the assessment of reproductive effects. Dams were sacrificed on day 5 post partum. When birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 26 post coitum.

NECROPSY
All animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated. All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred. For the parent animals, special attention was directed at the organs of the reproductive system.

The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

ORGAN WEIGHTS
The testes and epididymides of all parental males were weighed as pairs.
In addition, from 5 males and females killed at the end of the study which were selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken.
Adrenal glands (weighed as pairs)
Brain
Heart
Kidneys (weighed as pairs)
Liver
Thymus
Spleen

TISSUE PRESERVATION
The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:
Prostate
Seminal vesicles with coagulating gland
Testes (in Bouin’s fixative)
Epididymides (in Bouin’s fixative)

The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:
Ovaries

In addition, from the five males and females per group selected for organ weights and from all animals found dead or killed in extremis, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
Gross lesions
Brain
Spinal chord
Small and large intestines (incl. Peyer’s patches)
Stomach
Liver
Kidneys
Adrenals
Spleen
Heart
Thymus
Thyroids, and parathyroids if possible
Trachea and lungs (preserved by inflation with fixative and then immersion)
Uterus (with vagina)
Urinary bladder
Lymph nodes (mesenterial, mandibular)
Peripheral nerve (sciatic)
Bone marrow

HISTOTECHNIQUE
All organ and tissue samples to be examined by the principal investigator were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin.

HISTOPATHOLOGY
Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator. The same applied to all occurring gross lesions.
Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of ovaries was carried out on females that did not give birth.

Postmortem examinations (offspring):

TERMINATION OF THE STUDY
Pups were sacrificed on day 4 post partum.

NECROPSY
All animals were sacrificed by an injection of sodium pentobarbital. Dead pups, except those excessively cannibalized, were examined macroscopically. All pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

Statistics:

The following statistical methods were used to analyze food consumption, body weights, organ weight, locomotor activity and macroscopical findings and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios.

Offspring viability indices:

From the on-line recorded reproduction data, the following parameters were calculated: dead / live pups at first litter check, postnatal loss (up to day 4 post partum).

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:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

PARENT ANIMALS
 
Detailed Clinical Observations (Daily)
All animals survived until the scheduled necropsy.
 
In group 4, dark discoloration of feces was observed in males and females starting on day 3 of the pre-pairing period onwards. This was likely due to the staining property of the test item at higher concentration.
 
In groups 2 and 3, no clinical signs were observed for the entire duration of the study.
 
(See attached Tables on pp. 1 to 3)
 
Detailed Clinical Observations (Weekly)
No findings were observed during the weekly examination.
 
Functional Observational Battery
None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect.
 
The frequency of rearing number and incidence of urine puddles during the open field observation did not give indication of any test item-related effect. Observational as spontaneous vocalization when the rat was removed from the cage was considered to be of incidental nature.
 
Mean values of grip strength (fore- and hind paws) and landing foot splay gave no indication of any test item-related effect.
 
Body temperature in males was statistically significantly lower in group 4 compared to the control group (37.9 °C compared to 38.7 °C in the control group). However, this value was within the range of the historical control data and therefore was not considered to be a test item-related effect.
 
(See attached Tables on pp. 4 to 7 and attached Historical Control Data on pp. 3 to 14)
 
Locomotor Activity
Locomotor activity was assessed quantitatively in terms of low beam counts in activity monitor and did not give any indication of a test item-related effect.
 
Food Consumption - Males - Pre-pairing and After Pairing Periods
Mean food consumption was not considered to be affected by the treatment with the test item.
 
During the pre-pairing period, the lower food consumption observed in groups 2 and 3 (-6.2 and -4.6% compared to the control group) was considered to be incidental since this reduction did not follow a dose-dependent pattern and in group 4 mean food consumption was similar to the control (-0.4% compared to the control).
 
During the after pairing period, mean food consumption compared to the control was: +2.2, -1.1 and +1.5%, in groups 2, 3 and 4, respectively
 
(See attached Tables on pp. 8 to 11)
 
Body Weights - Males - Pre-pairing, Pairing and After Pairing Periods
Mean body weight and mean body weight gain were not affected by the treatment with the test item for the whole duration of the study. The statistically significantly lower body weight gain observed in group 2 between day 4 and 7 was considered to be incidental since there was no dose-dependency.
 
In order of ascending dose level, body weight gain was: during the pre-pairing period +13.0%, +10.9%, +11.5 and +11.9%, during the pairing period +4.9%, +5.7%, +4.4% and +4.7%, and during the after pairing period +4.1%, +5.6%, +4.8% and +4.5%, respectively.
 
(See attached Tables on pp. 12 to 20)
 
Food Consumption - Females - Pre-pairing, Gestation and Lactation Periods
No test item-related effects were observed in mean food consumption for the entire duration of the study.
 
Mean food consumption in groups 2, 3 and 4, compared to the control group, was: during the pre-pairing period +5.0%, +2.2% and +2.2%, during the gestation period +0.4%, ‑2.1% and -0.4%, and during the lactation period +11.3%, -3.1% and -3.7%, respectively.
 
(See attached Tables on pp. 21 to 26)
 
Body Weights - Females -Pre-pairing, Pairing, Gestation and Lactation Periods
No test item-related effects were noted in mean body weight and mean body weight gain of females during the whole study at any dose level.
 
In order of ascending dose level, body weight gain was: during the pre-pairing period +7.5%, +8.3%, +9.3% and +8.5, during the gestation period +63.5%, +63.3%, +60.2% and +60.5%, and during the lactation +3.1%, +6.2%, +4.6 and +2.7%, respectively.
 
(See attached tables on pp. 27 to 39)
 
CLINICAL LABORATORY INVESTIGATION
 
Hematology
The assessment of the hematology data did not reveal any test item-related effects in males and females.
 
Clinical Biochemistry
Males:
In group 4, the concentration of potassium was statistically significantly higher (+17.8% compared to the control). Since the mean value was within the range of the historical control data, this was not considered to be a test item-related effect.
 
In groups 2 and 3 no changes were noted.
 
Females:
The assessment of the clinical biochenistry data did not reveal any test item-related effects in females.
 
(See attached Tables on pp. 40 to 41 and attached Historical Control Data on pp. 1 to 2)
 
Urine analysis
The assessment of the urine analysis data did not reveal any test item-related effects in males and females.
 
REPRODUCTIONANDBREEDINGDATA- PARENT ANIMALS
 
Please see section "Any otherinformation on results including tables" for the table "Summary of Performance - P Animals Breeding for F1 Litters"
 

 
Mating Performance and Fertility
All females mated within the first pairing period. The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 2.6, 3.1, 2.8 and 3.7 days in groups 1, 2, 3 and 4, respectively. The median precoital time was 3, 4, 3 and 4 days in order of ascending dose level.
 
Due to one female (no. 58) in group 2 which was not pregnant, the fertility index and conception rate were 100% in groups 1, 3 and 4, and 90% in group 2.
 
In group 1, one female (no. 43) did not deliver any pup and had only two implantation sites. Therefore, the gestation index was 90% in group 1, and 100% in groups 2, 3 and 4. 
 
Duration of Gestation
The mean duration of gestation was unaffected by the exposure to the test item. Mean duration of gestation was 21.4, 21.8, 21.5 and 21.4 days, in order of ascending dose level.
 
Corpora Lutea Count
Mean number of corpora lutea per dam (determined at necropsy) was similar in all groups (16.9, 18.3, 17.2 and 16.4 in order of ascending dose level) and gave no indication of a test item-related effect.
 
Implantation Rate and Post-implantation Loss
The mean number of implantations per dam was not affected by the treatment with the test item.
 
In groups 3 and 4, total post-implantation loss was statistically significantly higher but within the range of the historical control data, therefore was not considered to be test item-related.
 
The mean numbers of implantations per litter were 15.0, 16.1, 14.3 and 14.6 in order of ascending dose level. The mean incidence of post-implantation loss as a percentage of total implantations was 1.5, 4.8, 8.4 and 6.2%, in order of ascending dose level.
 
(See attached Tables on pp. 42 to 43 and attached Historical Control Data on pp. 15 to 38)
 
Litter Size at First Litter Check
At first litter check, mean litter size was not affected by the treatment with the test item.
 
Due to the higher number post-implantation loss which occurred in groups 3 and 4, the birth index was statistically significantly lower but within the range of the historical control data. Birth index was 98.5, 95.2, 91.6 and 93.8% and mean litter size at first litter check was 14.8, 15.3, 13.1 and 13.7, in order of ascending dose level.
 
Postnatal Loss Days 0 - 4 Post Partum
Postnatal loss up to day 4 p.p. was not affected by the treatment with the test item at any dose level.
 
Mean postnatal loss was 0.3, 0.0, 0.0 and 0.1, corresponding to a viability index of 97.7, 100.0, 100.0 and 99.3% in groups 1, 2, 3 and 4, respectively.
 
TERMINAL FINDINGS
 
Organ Weights
No test item-related effects were noted in absolute and relative weight of organs in males and females. All the statistically significant changes were within the range of the historical control data or did not follow a dose-dependent pattern.
 
(See attached Tables on pp. 44 to 49 and attached Historical Control Data on pp. 39 to 62)
 
Macroscopical Findings
The findings noted during the macroscopical examination did not give indication of any test item-related effect.
 
Males:
In group 4, spleen with a constriction was noted in male no. 34. No other findings were noted.
 
Females:
In group 4, female no. 72 was noted to have the liver caudal lobe adherent to the lateral lobe. 
 
In groups 2 and 3 no findings were noted.
 
In group 1, female no. 43 was noted to have tan or dark red or red brown discolored nodules on the left uterine horn. Same female had only two implantation sites and did not deliver any pups.
 
Histopathology Findings
In group 4, in the lungs of animal no. 75 some alveolar macrophages (moderate severity degree) were found to contain dark pigment. This finding was possibly related to accidental aspiration of dose solution. All further lesions recorded during the microscopic investigation were within the range of background alterations that may be recorded in this type of study, and in rats of this strain and age. 
 
In group 1, in one infertile female (number 43) a deciduoma was noted. This lesion was deemed to have contributed to infertility.
 
Qualitative staging of thePAS-stained testes was conducted and there were no abnormal lesions encountered during sperm staging regarding completeness of stages and maturation of cell populations. Individual lesions recorded were within the range of background alterations that may be recorded in this type of study, in rats of this strain and age.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

LITTER DATA- F1 PUPS
 
External Examination at First Litter Check and during Lactation
No abnormal findings were noted at first litter check or during the first 4 days post partum.
 
In group 1, two male pups from same litter and one male pup from another litter were noted to have no milk in the stomach at first litter check.
 
Sex Ratios
Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item.
 
The proportion of males on day 4 post partum was 48, 49, 44 and 50%, in order of ascending dose level.
 
Body Weights to Day 4 Post Partum
Mean pup weights on day 1 post partum were unaffected by treatment with the test item. On day 1 post partum mean pup weights were6.0,6.1, 6.4and6.0 g in order of ascending dose level.
 
During the lactation period (to day 4 post partum), mean pup weight gain was not affected by the treatment with the test item (+41.7, +39.3, +39.1 and 43.3%, in order of ascending dose level). Mean pup weights on day 4 post partum were 8.5, 8.5, 8.9 and 8.6 g in order of ascending dose level.
 
Macroscopical Findings
No abnormal findings were noted during the macroscopical examination of pups.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Summary of Performance - P Animals Breeding for F1 Litters

 

Group (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (1000)
Female numbers	41-50	51-60	61-70	71-80
Number of females paired	10	10	10	10
Number of females mated	10	10	10	10
Number of non pregnant females (A)	0	1	0	0
Numbers of females, which did not deliver any pups (B)	1	0	0	0
Number of females which reared their pups until day 4 post partum	9	9	10	10

(A)  Female No. 58 was not pregnant.

(B)   Female No. 43 had only two implantation sites.

Applicant's summary and conclusion

Conclusions:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item Leuco Sulfur Black 1 (CAS#66241-11-0) to rats over approximately 28 days. Leuco Sulfur Black 1 was administered in highly purified water as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. Leuco Sulfur Black 1 was administered to male rats for 29 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

Administrations at 1000 mg/kg body weight/day caused a dark discoloration of feces in all animals after the onset of the treatment until the end of study. This was likely due to the staining property of the test item and not considered to be adverse.

Food consumption and body weight were not affected for the entire duration of the study. The evaluation of the functional battery observations and the assessment of clinical laboratory investigations parameters did not reveal any test item-related effect.

Relevant reproduction parameters (number of corpora lutea, implantation sites, pre- and post implantation losses, number of pups) were not affected by the treatment with the test item. The litter size at first litter check and on day 4 post partum was also not affected.

All the changes noted in the organ weights were within the background of the biological variation.

No adverse findings were noted during macroscopical and histopathology examinations.

Based on these results a general NOAEL (No Observed Adverse Effect Level) was established at 1000 mg/kg body weight/day.

The NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 1000 mg/kg/day.

Executive summary:

The purpose of this study was to generate preliminary information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provided information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

 

This study should provide information to assess the need to conduct further investigations and may provide guidance in the design of subsequent studies.

 

Dose levels were selected in agreement with the Sponsor, based on the results of a dose range-finding study (Harlan Laboratories Study C31076).

 

Leuco Sulfur Black 1 was administered to male rats for 29 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

The following dose levels were applied:

 

Group 1:                        0 mg/kg body weight/day (control group)

Group 2:                    100 mg/kg body weight/day

Group 3:                    300 mg/kg body weight/day

Group 4:                   1000 mg/kg body weight/day

 

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (highly purified water).

 

The following results were obtained:

 

PARENT ANIMALS

General Tolerability 

All the animals survived until the scheduled necropsy. At 1000 mg/kg body weight/day, a dark discoloration of feces was observed in all animals starting on day 3 of the pre-pairing period onwards.

 

Functional Observational Battery 

No test item-related effects were noted.

 

Food Consumption 

Food consumption was not affected by the treatment with the test item in males and females.

 

Body Weights 

No test item-related effects were noted in body weight and body weight gain in males and females for the entire duration of the study.

 

Clinical Laboratory Investigations 

The assessment of the clinical laboratory investigations parameters did not give indication of any test item-related effect.

 

Reproduction and Breeding Data 

Mean precoital time, fertility and gestation index and conception rate were not affected by the treatment with the test item. 

Implantation rate and post implatation loss were also not affected by the treatment with the test item.

 

Organ Weights 

Weight of organs was not affected by the treatment with the test item. All the alterations noted were within the range of historical control data.

 

Macroscopical Findings and Histopathological Examinations 

No adverse effects of the test item were noted during the histopathological examination.

 

LITTER DATA - F1 PUPS

Findings at First Litter Check and during Lactation 

The mean number of pups at first litter check and on day 4 post partum were not affected by the treatment with the test item. The sex ratio was also not affected. No abnormal pup was noted at any dose level.

 

Pup Weights to Day 4 Post Partum 

Mean pup weight gain to day 4 post partum was not affectd by the treatment with the test item. 

 

Macroscopical Findings 

At necropsy of pups, there were no abnormal findings. 

 

Conclusion

Based on these results a general NOAEL (No Observed Adverse Effect Level) was established at 1000 mg/kg body weight/day. 

The NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 1000 mg/kg body weight/day.