Disodium hydrogen bis[3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]naphthalene-1-sulphonato(3-)]chromate(3-)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2012 to 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study performed on the analogue substance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

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)

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsichereit, München, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium bis[3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(3-)

EC Number:

260-906-9

EC Name:

Trisodium bis[3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(3-)

Cas Number:

57693-14-8

Molecular formula:

C40H20CrN6O14S2.3Na

Test material form:

other: solid and liquid preparations

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test SystemSpecies/strain:Wistar rats, Crl: WI(Han) (Full Barrier) Source:Charles River, 97633 Sulzfeld, GermanySex:male and female;the female animals were non-pregnant and nulliparous.Age at the start ofthe treatment period:males: 9-10 weeks old, females: 9-10 weeks old.Body weight at the allocation of the animals to the experimental groups:males: 240 - 276 g(mean: 257.33 g, ± 20% = 205.86 – 308.79 g)females: 164 - 195 g(mean: 176.48 g, ± 20% = 141.18 – 211.77 g)The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.Housing and Feeding Conditions-Full barrier in an air-conditioned room-Temperature: 22  3°C-Relative humidity: 55  10%-Artificial light, sequence being 12 hours light, 12 hours dark-Air change: 10 x / hour-Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0939)-Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)-The animals were kept individually in IVC cages (except during the mating period when one female will be paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 300512)-Certificates of food, water and bedding are filed at BSL BIOSERVICE-Adequate acclimatisation period (at least 5 days) under laboratory conditionsNumber of Sex and Animals80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group)Preparation of the AnimalsPrior to the start of the treatment period a detailed clinical observation outside the home cage was made. Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test item was weighed into a tarred plastic vial on a precision balance.The dose formulations were prepared by adding the required volume of sterile water and further vortexing it for 2-3 minutes.The vehicle was selected based on the test item’s characteristics (stability and homogeneity). The test item formulations were prepared freshly on each administration day before the administration procedure. The time of preparation and time of dosing was recorded for all dosing formulations.The homogeneity was guaranteed by intensive stirring during application.According to the results of the dose range finding study and in consultation with the sponsor the following doses (Table 1) are selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C). The animals were treated with the test item formulation or vehicle on 7 days per week for a period of maximum 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.The following doses were administered:GroupDose [mg/kg bw]Control0Low Dose100Mid Dosw300High Dose1000The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same dose volume.The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight.For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Details on mating procedure:

- M/F ratio per cage: 1:1- Length of cohabitation: 14 days maximum- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing concentration was analyzed for nominal concentration. Stability and homogeneity of the test item in the vehicle was analyzed for the LD, MD and HD dosing formulation.Samples for the nominal concentration verification were taken in study week 1 (first week of pre mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation) of control and all treatment groups (16 samples in total).Samples for homogeneity were taken from the top, middle and bottom of HD, MD, and LD preparation in study week 1 and 5 (18 samples in total).All formulation samples were stored at -20 oC until the analysis. The samples were analyzed at BSL BIOSERVICE Scientific Laboratories GmbH. The procedure followed for the sample analysis was mentioned in phase plan.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a period of maximum 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:100 mg/kg bwBasis:actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:300 mg/kg bwBasis:actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:1000 mg/kg bwBasis:actual ingested

No. of animals per sex per dose:

80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes - Time schedule: dailyDETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: dailyGeneral clinical observations were made once a day. Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.BODY WEIGHT: Yes - Time schedule for examinations:The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours off parturition (day 0 post-partum) as well as day 4 post-partum along with pups.FOOD CONSUMPTION:Food consumption was measured weekly on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.WATER CONSUMPTION AND COMPOUND INTAKE: No

Litter observations:

STANDARDISATION OF LITTERS- Performed on day 4 postpartum: noPARAMETERS EXAMINEDEach litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by tattooing.

Postmortem examinations (parental animals):

All male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29, while female animals were sacrificed on post-natal day 4 using an anaesthesia (ketamine/xylazin, 2:1, medistar Arzeneimittel, lot no: 00212, expiry date: 03/2014, Actavis, lot no: 146066, expiry date: 10/2014, Serumwerk, lot no: 00711, expiry date: 08/2013 and Narcoren®, Merial; lot no.: 221022; expiry date: 28/02/2015) was used. Pups were killed on PND 4 by decapitation. Animals 7, 27, 14, 35, 28, 16, 10, 38, 29 and 19 were killed using high dose of sodium pentabarbitol as no blood sampling was necessary for these animals.Female (animal 73) showing no evidence of copulation up to 14 days of the mating period was sacrificed on day 26 (study day 54) after the last day of the mating period.All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Postmortem examinations (offspring):

Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism 5.01 software (p<0.05 was considered as statistically significant).

Reproductive indices:

The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.

Offspring viability indices:

Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

not adverse

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

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)

MortalityThere was no mortality noted in treated and control groups during the study period.Clinical ObservationsThere were clinical signs namely moving the bedding, salivation and piloerection noted occationally or intermittently on treated groups of male or female animals, which were assumed to be caused by local irritation. The clinical signs reddish nasal discharge, abnormal breathing, slightly reduced spontaneous activity, respiratory sound and diarrhea were observed transiently in few animals of male and female treated groups. These findings were considered not likely to be adverse.Alopecia was noted in few isolated animals of LD or MD groups, which were considered incidental.During the weekly detailed clinical observations, no significant changes or differences between the groups were found. There were no ophthalmoscopic findings in any of the animals of this study.Functional ObservationsNo relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.Body Weight DevelopmentIn both males and females, no treatment related changes were noted for body weight during the study period. The statistical evaluation of data revealed significant decrease in mean body weight of female MD group on lactation day 0 when compared to control. As there was no dose response pattern noted, this finding was not related to treatment. There was slight decrease in body weight change in male HD group when compared to corresponding control during the study period. Slight decrease was also observed transiently in female HD group. However, these changes did not corroborate food intake. Hence, the changes were considered not likely to be related to treatment.Food ConsumptionIn both males and females, no treatment related changes were noted for treated group when compared to control. The statistical evaluation of data revealed no significant changes between the treated and the corresponding control group.Haematology and CoagulationThere was a statistically significant decrease noted for mean red blood cell count value in female MD group. But in the absence of a dose response pattern the above change was not related treatment. There was also increase in mean Neutrophil value noted in female treated groups in a dose response pattern, but without attaining the statistical significance. The values being within the historical range, the changes were considered not likely to be adverse.There were no treatment related changes noted for other haematological parameters and blood coagulation parameters (prothrombin time and activated partial thromboplastin time) in treated groups when compared to corresponding control.Clinical BiochemistrThere was statistically significant increase in mean value of cholesterol in male HD group. There was also increase in mean alkaline phosphatase value in male HD group without attaining statistical significant. The increase in alkaline phosphatase values were due to higher values in one male animal of the group. In females, there was statistically significant decrease in mean creatinine value in MD group which did not follow a dose-dependent pattern and considered to be incidental. There was also increase in mean creatinine and alkaline phosphatase values noted in HD group without attaining the statistical significane. The changes noted in cholesterol value of male HD group and creatinine and alkaline phosphatase value in female HD group could be attributed to histological changes noted in kidney. As the histological changes in kidney were due to test item deposition, the changes in cholesterol, creatinine and alkaline phosphatase in male or female animals were considered not likely to be adverse.UrinalysisThe urinalysis performed in male animals revealed no treatment related effect in any of the treated groups compared to the control group.PathologyAt terminal sacrifice, two females of MD group (Nos. 62 and 65) and one female of HD group (No. 73) were found to be non-gravid after successful copulation. This finding was not related to treatment.Two males of MD group showed yellow(ish) spot(s) on the epididymis, corresponding histologically to spermatic granuloma. As this finding was not dose-related and may be seen spontaneously in male rats of this strain and age, it was not considered to be test item-related.Dark or black discoloration of one or several organs was noted in a dose-related manner in 7/10 females of MD group and in 10/10 males and 9/10 females of HD group. Whereas in MD group organs concerned were most often kidney or lymph node (mesenteric or mandibular), in HD group these were some or all of the following: kidney, testis, lymph nodes, female reproductive organs, liver, thymus, adrenal gland, salivary glands. In addition, in HD group, blackish content was noted in the gastrointestinal tract in one single male rat. In one female animal of MD group (No. 68), lung was not collapsed and had black spots. These color changes were considered to be due to the test item.Other macroscopic organ findings noted were few and not dose-related and thereforeconsidered incidental.Organ WeightIn males, there were statistically significant increase noted for absolute left and total adrenal gland weight in HD group; statistically significant increase of relative (to body weight) left adrenal gland weight in HD group; statistically significant decrease of mean absolute prostate+seminal vesicle+coagulating gland and relative (to body weight) spleen weight in HD group. These findings in the absence of histological changes were not related to treatment. In females a statistically significant increase was noted for absolute and relative (to body weight) heart weight in LD group; statistically significant increase in absolute liver weight in MD and HD groups; statistically significant decrease in absolute and relative (to body weight) thymus weight in HD group. The changes noted for heart and liver had no biological relevance and were not considered related to treatment.Lower thymus weights recorded in females were related to an increased incidence and severity of atrophy/regression of the thymus noted in females of HD group, but the difference to controls was small. Hence, the changes were considered not to be adverse.HistopathologyHistopathologically, a minimal amount of brown pigment in interstitial macrophages of the testis was seen in a dose-related manner in MD and HD groups and was considered to represent test item deposition in macrophages. No test item-related changes were noted in the other male or in the female reproductive organs.In the kidney, minimal amounts of brown pigment in tubuloepithelial cells of the cortex were observed in the majority of animals of HD group and in one single male of MD group. In the mesenteric lymph node, minimal numbers of foamy/brown pigmented macrophages were seen in a dose-related manner in MD and HD groups. In addition, the incidence of sinus histiocytosis was higher in HD group than controls. Altogether, these changes were considered to be due to test item deposition.In the lung, multifocal minor numbers of brown pigmented macrophages were observed in single animals MD and HD groups. Under the conditions of this study, it could not be elucidated further whether this change was due to local presence of test item formulation in the lung (via partial gavaging error or regurgitation) or due to systemic circulation of test item-laden macrophages.Corroborating lower thymus weights recorded, an increased incidence and severity of atrophy/ regression of the thymus was noted in females of HD group, but the difference to controls being small the changes were considered not to be adverse.Dose Formulation AnalysisConcentration analysis of formulation samples was determined in study week 1, 3, 5, and 7 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 86.3%, 91.7% and 82.5% of the nominal concentration, respectively.Homogeneity of formulation samples was determined in study week 1 and 5 for LD, MD and HD dose groups. The mean recovery observed for LD dose group was between 98.6 and 99.6% of the nominal value, for MD dose group between 93.7 and 99.5% and for HD dose group between 80.5 and 94.1%. The coefficients of variation of the different sampling locations (top, middle, bottom) were between 0.9 and 3.3% in LD dose group, between 1.8 and 2.1% in MD dose group and between 0.7 and 1.4% in HD dose group.The recovery of test substance in formulation samples of LD and MD groups in week 3 was lower (56.8 and 74.6 %, respectively). It was assumed to be due to improper homogenization of formulation samples before collecting the samples for the analytical purpose.This was not systematical but rather an isolated mistake on a particular day of sample collection, as the formulation samples are prepared freshly each day and administered to the animals. In addition, a review of the raw data of dose preparation did not reveal any mistakes

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:

clinical signs

mortality

body weight and weight gain

haematology

clinical biochemistry

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

not adverse

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 DataNo treatment-related changes were noted for the number of still births, number of runts, total number of pups born on PND 0 and number of male and female pups, sex ratio, live pups on PND 0 and PND 4. The statistical evaluation of data revealed no significant differences between the values of treated and control groups.Litter Weight DataNo treatment related changes were noted for the mean litter weight on PND 0 and total litter weight, male and female litter weight on PND 0 and 4 in treated groups when compared to corresponding control. However, there was statistically significant difference noted for group mean litter weight in HD group. There was no marked difference noted biologically. Hence, this change was considered not likely to be an adverse effect.There was also a statistically significant difference noted for female litter weight in LD group on PND 4. There was no dose response pattern noted for this finding. Therefore, the finding was considered incidental.Pup Survival DataNo treatment related changes were noted for survival of the pups from PND 0 to PND 4 in any treated group when compared to controls. However, there was 1 pup each missing for animals 41 (pup no.5) and 56 (pup no. 8). The missing pup was considered to be cannibalized by the dam. There was also 1 pup found dead for animal 48 (pup no.1). The above findings were noted in C and LD groups and were considered to be incidental. Pup External FindingsNo treatment related gross external findings were observed in any of the groups.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In conclusion, the repeated dose administration of the analogue substance to the male (maximum 29 days) and female (maximum 54 days) Wistar rats at dosages of 100, 300 and 1000 mg/kg body weight revealed neither mortalities nor major findings of toxicological relevance in male and female animals. Based on the data generated from this “Combined Repeated Dose Oral Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test with analogue substance, the no observed adverse effect level (NOAEL) for general and reproduction/developmental toxicity is considered to be 1000 mg/kg body weight.

Executive summary:

The aim of this study was to assess the possible effects of analogue substance on male and female fertility and embryofetal development after repeated dose administration in Wistar rats.

The test item was administered daily in graduated doses to3 groups of test animals, one dose level per group for a treatment period of maximally 54 days for females and minimum of 28 days for males.Animals of control group were handledidentically as the dose groups but received same dose volume of the vehicle used in this study. Each of the 4 groups comprised 10 male and 10 female Wistar rats.

During the period of administration, the animals were observed each day for signs of toxicity. At the conclusion of the test, surviving animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

Haematological and clinical biochemistry evaluations were performedon blood samples collected at terminal sacrifice from five males and five randomly selected females from each group. Urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males from each group.

Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behaviour observations were performed in the week before the treatment and at the end of the study on five randomly selected males and females.

After 14 days of treatment to both male and female animals were mated (1:1) for a maximum of 14 days. From subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours off parturition and on day 4 post-partum.

The males were sacrificed after completion of the mating period on days 29/30 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females were sacrificed on day 26 from the day of confirmed mating.

Pups sacrificed on postnatalday 4 and those found dead (1 pup from animal 48), were carefully examined for gross external abnormalities.

 

A full histopathological evaluation of the tissues was performed on high dose and control animals. Organs showing gross alterations were also examined histopathologically. The examinations of kidney, lung, thymus and mesenteric lymph node were also evaluated from five randomly selected males and females of the low and medium dose due to changes in high dose group.

The following doses were evaluated:

Control:                       0        mg/kg body weight

Low Dose:                   100     mg/kg body weight

Medium Dose:             300     mg/kg body weight

High Dose:                  1000   mg/kg body weight

The test item formulation was prepared freshly on each day of administration. The test item was suspended in sterile water and administered daily during 14 days of pre-mating and during mating period in both male and female animals, during the gestation period and up to post-natal day 3 in females. Males were dosed for 28days. Non pregnant females and females with no evidence of mating were treated until day 25 after confirmed mating or from the end of mating period. Dose volumes were adjusted individually based on weekly body weight measurements. Theadministration volume was 5 mL/kg body weight.

No mortality occurred in the control or any of the dose groups during the treatment period of this study.

The clinical signs reddish nasal discharge, abnormal breathing, slightly reduced spontaneous activity and diarrhea were observed transiently in few animals of HD group, which were considered not likely to be adverse. During the weekly detailed clinical observation, no significant changes or differences between the groups were found. There were no ophthalmoscopic findings in any of the animals of this study.

No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.

In both males and females, no treatment related changes were considered for body weight, body weight change and food consumption in treated groups when compared to control.

No treatment-related changes were noted for number of still births, number of runts, total number of pups born on PND 0 and number of male and female pups, sex ratio, live pups on PND 0 and PND 4.

There was statistically significant difference noted for group mean litter weight in HD group. As there was no marked difference noted biologically, this change was considered not likely to be adverse.

No treatment related changes were noted for precoital interval and duration of gestation in treated groups when compared to control. All pregnancies resulted in normal births.

No treatment related changes were noted for number of corpora lutea, number of implantation sites, number of live pups born on PND 0 and percentage of pre- and post implantation loss. 

There were no treatment related changes noted for copulation index (%), fertility index (%) and delivery index (%) in treated groups when compared to the corresponding control group.

No treatment related changes were noted on survival of the pups from PND 0 to PND 4 in any treated group when compared with controls.

No treatment-related gross external findings were observed in any of the treated groups.

There were no treatment related changes noted for haematological parameters. However, there was increase noted for mean Neutrophil values of female treated groups. But the values being within the historical control range, the changes were considered not likely to be adverse.

There were no changes noted for coagulation parameters (prothrombin timeand activated partial thromboplastin time) in treated groups when compared to control.

There were changes noted in cholesterol value of male HD group andcreatinineandalkaline phosphatasevalue in female HD group which was attributed to histological changes noted in kidney and the histological changes in kidney were due to test item deposition. The changes in cholesterol,creatinineandalkaline phosphatasein male or female animals were considered not likely to be adverse.

The urinalysis performed in male animals revealed no test- item related effect in any of the treatment groups compared to the control group.

At terminal sacrifice, two females of MD group (Nos. 62 and 65) and one female of HD group (No. 73) were found to be non-gravid after successful copulation.

 

Two males of MD group showed yellow(ish) spot(s) on the epididymis, corresponding histologically to spermatic granuloma. As this finding was not dose-related and may be seen spontaneously in male rats of this strain and age, it was not considered to be test item-related.

 

Dark or black discoloration of one or several organs was noted in a dose-related manner in 7/10 females of MD group and in 10/10 males and 9/10 females of HD group. Whereas in MD group organs concerned were most often kidney or lymph node (mesenteric or mandibular), in HD group these were some or all of the following: kidney, testis, lymph nodes, female reproductive organs, liver, thymus, adrenal gland, salivary glands. In addition, in HD group, blackish content was noted in the gastrointestinal tract in one single male rat. In one female animal of MD group (No. 68), lung was not collapsed and had black spots. These color changes were considered to be due to the test item. Other macroscopic organ findings noted were few and not dose-related and therefore considered incidental.

 

Lower thymus weights recorded in females were related to an increased incidence and severity of atrophy/regression of the thymus was noted in females of HD group, but the difference to controls was small. Hence, the changes were considered not to be adverse.

 

Histopathologically, a minimal amount of brown pigment in interstitial macrophages of the testis was seen in a dose-related manner in MD and HD groups and was considered to represent test item deposition in macrophages. No test item-related changes were noted in the other male or in the female reproductive organs.

In the kidney, minimal amounts of brown pigment in tubuloepithelial cells of the cortex were observed in the majority of animals of HD group and in one single male of MD group.

 

In the mesenteric lymph node, minimal numbers of foamy/brown pigmented macrophages were seen in a dose-related manner in MD and HD groups. In addition, the incidence of sinus histiocytosis was higher in HD group than controls. Altogether, these changes were considered to be due to test item deposition.

 

In the lung, multifocal minor numbers of brown pigmented macrophages were observed in single animals of the MD and HD groups. Under the conditions of this study, it could not be elucidated further whether this change was due to local presence of test item formulation in the lung (via partial gavaging error or regurgitation) or due to systemic circulation of test item-laden macrophages.

Corroborating lower thymus weights recorded, an increased incidence and severity of atrophy/ regression of the thymus was noted in females of HD group, but the difference to controls being small the changes were considered not to be adverse.

Concentration analysis of formulation samples was determined in study week 1, 3, 5, and 7 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 86.3%, 91.7% and 82.5% of the nominal concentration, respectively.

Homogeneity of formulation samples was determined in study week 1 and 5 for LD, MD and HD dose groups. The mean recovery observed for LD dose group was between 98.6 and 99.6% of the nominal value, for MD dose group between 93.7 and 99.5% and for HD dose group between 80.5 and 94.1%. The coefficients of variation of the different sampling locations (top, middle, bottom) were between 0.9 and 3.3% in LD dose group, between 1.8 and 2.1% in MD dose group and between 0.7 and 1.4% in HD dose group.