[[4-[[4-(anilino)phenyl][4-(phenylimino)-2,5-cyclohexadien-1-ylidene]methyl]phenyl]amino]benzenesulphonic acid

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:

from June 11th, 2008 to December 8th, 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline test with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

C.I.Pigment Blue 61

IUPAC Name:

C.I.Pigment Blue 61

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification Structure: C.I.Pigment Blue 61
molecular formula: C37H29N3O3S
Molecular weight: 596
CAS Number:1324-76-1
Description: dark blue powder with granules
Batch: 2084701
Composition: 96.830%
Test substance storage: at room temperature in the dark
Stability under storage conditions:stable
Expiry date: 2018-01-02

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System:Rat: Cri:Wl(Han) (outbred, SPF-Quality). Untreated animals and virgin females were used at initiation of the study
Rationale: This species and strain of rat has been recognized as appropriate for general and reproductive toxicity studies. NOTOX BV has general and reproductive historical data in this species from the same strain and source. This animal mode has been proven to be susceptible to the effects of reproductive toxicants.
Source: Charles River Deutschland, Sulzfeid, Germany
Age at start F0-treatment: Approximately 10 weeks.
Number of F0-animals: 40 females and 40 males.
Acclimatisation F0: At least 5 days prior to start of treatment.
Health check F0: A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.

Animals were housed in room 16. Conditions:
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21± 3°C(actual range: 19.8-22.4°C ), a relative humidity of 30-70% (actual range: 41-87%) and 12 hours artificial light and 12 hours darkness p er day. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of functional observations in the room. Cleaning procdures in the room might have caused the temporary fluctuations above the optimal level of 70% for relative humidity. Based on laboratory historical data these conditions were considered not to have affected the study integrity.
Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiaten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants of each batch were examined and archived.
Water
Free access to tap-water. Certificates of analysis (performed quarterly) were examined and archived.
Analysis of bedding, paper, diet and water did not reveal any findings that were considered to have affected study integrity.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

Oral gavage, using a plastic feeding tube.
Formulations were placed on a magnetic stirrer during closing.
dose volume: 5 ml/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Details on mating procedure:

Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating:Females were caged together with males on a one-to-one-basis in Macrolon cages (Mill type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/sex/cage. Females were individually housed in Macrolon cages (Mill type, height 18 cm).
Following a minimum of 14 days of exposure for the mates and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates). Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post- coitum. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed on a single occasion during the treatment phase (30 June 2008) according to a validated method (NOTOX Project 488235) as specified below:
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions, or 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation was s 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test substance was detected in the Group 1 formulations. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation <=10%). Formulations of Group 2 and Group 4 were stable when stored at room temperature for at least 6 hours.

Duration of treatment / exposure:

Males were exposed for 30 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-45 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.
Female 78 was not dosed for one occasion as this female was littering at the time of dosing.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.

Details on study schedule:

Parturition F0:The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.
Lactation F0: Deficiencies in maternal care, such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding, were recorded.
Number of pups: Approximately 480 pups (40 litters x 12 pups).
Identification offspring: On Day 1 of lactation, all pups were individually identified by means of intracutaneous injection of Indian ink.
Lactation: Offspring was Kept with the dam until termination.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 250, 1000 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Randomisation F0: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Identification F0: Earmark and tattoo

Positive control:

no data

Examinations

Parental animals: Observations and examinations:

Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.
Mortality / Viability: At least twice daily
Clinical signs: At least once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually. The time of onset, degree and duration was recorded. All symptoms were recorded and graded according to fixed scales:
Maximum grade 1: grade 0 = absent, grade 1 = present Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe.
Cage debris of pregnant females were examined to detect abortion or premature birth, if applicable. Signs of difficult or prolonged parturition were recorded, if applicable.

Functional Observations: The following tests were performed in the first 5 mated males and
the first 5 females with live offspring, from each group:
- hearing ability, pupillary reflex, static righting reflex and grip strength (Score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test {recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).
During the motor activity test, males were caged individually and females were caged with their offspring.
The assigned males were tested during week 4 of treatment and the assigned females were tested during lactation (all before blood sampling).
In order to avoid hypothermia of pups, dams were removed from the pups for not more than 30-40 minutes.
Body weights:Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 of gestation, and during lactation on Days 1 and 4.
Food consumption:Weekly, for males and females. Food consumption was not recorded during the breeding period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post- coitum and after delivery on Days 1 and 4 post-partum.
Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Oestrous cyclicity (parental animals):

no applicable

Sperm parameters (parental animals):

Parameters examined in male parental generations:
(testis weight, epididymis weight, sperm morphology)

Litter observations:

Mortality / Viability The numbers of live and dead pups at the First Litter Check (= check at Day 1 of lactation) and daily thereafter were determined. If possible, defects or cause of death were evaluated
Clinical signs: At least once daily, detailed clinical observations were made in all animals.
Body weights :Live pups were weighed during lactation on Days 1 and 4.
Sex: Was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

Postmortem examinations (parental animals):

Clinical laboratory investigations
Blood samples were collected from the first 5 mated males and the first 5 females with live offspring from each group under iso-flurane (Abbott Laboratories Ltd., Zwolle, The Netherlands) anaesthesia immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m. The animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided. Biood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One, Bad Halier, Austria) prepared with EDTA for haematological parameters (0.5 ml), with citrate for clotting tests (0.9 ml) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 ml).
Termination
All animals were fasted overnight (with a maximum of 20 hours) prior to planned necropsy, but water was provided.
Females Which Deliver
On lactation Day 5, the F0-females were anaesthetised using iso~flurane (Abbott Laboratories Ltd., Zwolle, The Netherlands) and subsequently exsanguinated. Organ weights were collected and tissues were preserved for possible future histopathological examination as described in the following paragraphs. The number of former implantation sites were counted and recorded. Corpora lutea were also counted and recorded. Gross lesions were saved for possible future histopathological examination.
Females Which Failed to Deliver
On post-mating Day 25-27 {females with evidence of mating), the F0- females which had not delivered were anaesthetised using iso-flu「ane (Abbott Laboratories Ltd., Zwolle, The Netherlands) and subsequently exsanguinated. No organ weights were collected. Tissues were preserved for possible future histopathological examination as described in the following paragraphs, with the following exceptions: Nongravid uteri were stained using the Salewski technique (Salewski, 1964) in order to determine any former implantation sites (Salewski staining prepared at NOTOX using Ammoniumsulfide-soSution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)). If evidence of macroscopic implantations was present, the number of implantation sites and corpora lutea was recorded. Gross lesions were saved for possible future histopathological examination.
Termination Procedures for Fn-Males
Following completion of the mating period (29 days of dose administration), the F0-ma!es were anaesthetised using iso-fturane (Abbott Laboratories Ltd., Zwolle, The Netherlands) and subsequently exsanguinated. Organ weights were collected and tissues were preserved for possible future histopathological examination as described in the following paragraphs.
Macroscopic examination
After sacrifice ail parental animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities was recorded. Samples of the tissues and organs were collected and fixed in neutral phosphate buffered 4% formaldehyde solution (Klinipath, Duiven, The Netherlands).
Organ weights
The following organ weights (and terminal body weight) was recorded:
From the first 5 males per group and the first 5 females with live offspring per group: Kidneys, Adrenal glands, Brain, Epididymides, Heart, Liver, Spleen, Testes, Thymus.
From all remaining males: Epididymides, Testes.
Histotechnology
All organ and tissue samples, as defined under Histopathoiogy (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).
Of the selected 5 males/group of the control and high dose group, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes was processed, sectioned at 3-4 microns, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).
Histopathoiogy
The delegated phase was performed by the Principal Investigator for Histopathoiogy.
Histological examination was extended to the mesenteric lymph nodes of the selected males
and females of Group 2 based on possible treatment-related changes noted for mid and high dose animals.
Ali abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Reproductive organs included cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testis, uterus, and vagina.

Postmortem examinations (offspring):

Termination
Pups were killed by decapitation on Day 5 of lactation or shortly thereafter.
Macroscopic examination
All offspring was sexed and externally examined if practically possible. The stomach was examined for the presence of milk.
Descriptions of all macroscopic abnormalities were recorded.
if possible, defects or cause of death was evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin, for possible further examhaUon.

Statistics:

For each dose group reproduction parameters were expressed in two ways:
-As a mean (with standard deviation) of the number observed for each litter
-Relative to a second parameter and calculated on a total group basis
The following statistical methods will be used to analyse the data:
-If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett,
1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
-The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
-The Fisher Exact-test (Fisher, 1950) was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
No statistical analysis was performed on histopathology findings.

Reproductive indices:

Percentage mating, Fertility index, Conception rate, Gestation index, Duration of gestation

Offspring viability indices:

Percentage live mates at First Litter Check, Percentage live females at First Litter Check, Percentage of postnatal loss Days 0-4 post partum, Viability index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

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

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

Mortality
No mortality occurred during the study period.
Clinical Signs
There were no clinical signs of toxicity noted during the observation period.
Blue discolouration of the faeces was noted in all animals of both sexes at 50, 250, and 1000 mg/kg. Blue staining of several body parts (tail, back, mouth, snout, abdomen, forelegs, flank) was also observed at these dose levels but at a lower incidence. These findings were considered due to the staining properties of the test substance and not to be toxicologically relevant.
Slight salivation was noted for one low dose male (1 day), five mid dose males (1-3 days), six high dose males (2-18 days) and two high dose females (1 day). Salivation was considered to be a physioiogicaJ response to the taste of the formulation rather than a sign of systemic toxicity, considering the nature, minor severity of the effect and its time of occurrence (i.e. after dosing). Therefore, this was considered not toxicologically relevant.
Incidental findings that were noted included alopecia and scabbing on several body parts, rales, piloerection and a broken tail apex. These findings are commonly noted in rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.
Body Weights
Body weights and body weight gain of treated animals remained in the same range as controls over the study period.
Food Consumption
Food consumption (absolute and relative) of treated animals in all groups remained in the same range as controls over the study period.
Two female rats (numbers 55 and 60) of the low dose group showed very low food consumption on Days 11-14 post-coitum. As body weight gains for these dams were normal, these low values were probably caused by a weighing error rather than being true values and were therefore considered not toxicologically relevant.
Functional observations
No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the animals treated with test article, when compared to control animals.
The variation in motor activity did not indicate a relation with treatment.
Haematology
Haematological parameters of treated rats were not affected by treatment up to 1000 mg/kg.
Females treated with 1000 mg/kg showed statistically significantly lower prothrombin time than controls. This finding is considered to be of no toxicological significance since the value was within normal limits and no corroborative findings were noted. In addition, the statistically significantly increased haematocrit value for low dose females was not considered treatment related as no dose response relationship was noted.
Clinical Biochemistry
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.
Males treated with 1000 mg/kg had statistically significantly higher cholesterol measures than controls. This was considered to be of no toxicological significance as the value was within normal limits and no corroborative findings were noted.

Macroscopic Examination
No toxicologically relevant macroscopic findings were noted up to 1000 mg/kg body weight/day.
Greenish or bluish discolouration of the mesenteric lymph nodes was noted for three low dose animals, seventeen mid dose animals and all high dose animals. Seven high dose females had bluish contents in the gastro intestinal tract. Bluish discolouration of the tail was observed for two males. These findings were considered due to the staining properties of the test substance and not to be toxicologically relevant.
Incidental findings includeci bent tail, hard yellowish nodule on the tail of the epididymidis, alopecia at several body parts, reduced seminal vesicle size, dark red or reddish discolouration of the mandibular iymph nodes, reddish discolouration of the pancreas, and red-brown discolouration of the adrenal glands. These findings are occasionally seen among rats used in these types of studies and in the absence of correlated microscopic findings they were not considered changes of toxicological significance.
Organ Weights
No toxicoiogically relevant organ weight changes were noted for animals treated up to 1000 mg/kg body weight/day.
The statistically significantly increased relative spleen weight noted for high dose males was considered not to be a sign of toxicity in the absence of corroborative (microscopic) findings.
Microscopic Examination
No treatment related histopathological changes were observed in the tissues of any animal receiving test substance.
No abnormalities were noted on evaluation of PAS stained sections of testes of F0 males. All seminiferous tubules were normal with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages.
No pathological findings were present in the reproductive organs of male and female animals that failed to produce offspring.
Small amounts of blue material were observed in the lumen of the duodenum, ileum, jejunum, colon or rectum in a few group 4 animals. Blue material was also noted in the superficial skin (keratin layer) of the tail in one group 4 male. These findings are considered to represent the presence of the test substance and in some cases correlated with the macroscopic observations.
No microscopic findings were observed in the mesenteric lymph nodes to account for the discolouration of these tissues recorded at necropsy. St is considered that the discolouration is due to the presence of the test substance and that the pigment was lost from the tissues in the processing or staining procedures.
All other microscopic findings were considered to be incidental and unrelated to treatment with the test substance.

Reproduction parameters were unaffected by treatment up to 1000 mg/kg body weight/day.
No treatment related findings were observed for mating performance, fertility parameters, duration of gestation, number of dead and living pups at first litter check, number of implantation sites and number of corpora lutea.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related toxicoiogicaHy significant changes were noted in any of the parameters investigated in this study.

Remarks on result:

other: Generation: P and F1 (migrated information)

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:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

Breeding data:
Breeding parameters were unaffected by treatment up to 1000 mg/kg body weight/day.
Postnatal loss and viability index were similar for the control and treated groups.
Pup Development:
Development of pups was unaffected by treatment up to 1000 mg/kg body weight/day.
(Mean) body weights were similar for the control and treated groups.
Incidental clinical symptoms consisted of small size of the pup, red spot on the nose or head, and pale appearance. At macroscopic examination, autoiysis and absence of milk in the stomach was noted for a few dead pups. No relationship with treatment was established for these observations and they were considered to be within the normal biological variation for rats of this age and strain.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the reproduction/developmental NOAEL for test article is greater than 1000 mg/kg/day.

Executive summary:

This study was conducted based on OECD guideline No. 422 with GLP to evaluate the potential of test article to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development when administered to rats for a minimum of 28 days. Four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 50, 250 and 1000 mg/kg/day. Males were exposed for 30 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41 to 45 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. No treatment-related toxicoiogically significant changes were noted in any of the parameters investigated in this study (i.e. mortality, clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, microscopic examination, reproduction, breeding and pup development).

From the results presented in this report, the parental, reproduction, breeding and developmental No Observed Adverse Effect Level (NOAEL) for test substance of at least 1000 mg/kg/day was established.