Tetrasodium [μ-[3-[[2-amino-5-hydroxy-6-[(2-hydroxy-5-nitro-3-sulphophenyl)azo]-7-sulpho-1-naphthyl]azo]-2-hydroxy-5-sulphobenzoato(8-)]]dichromate(4-)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to OECD and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium [μ-[3-[[2-amino-5-hydroxy-6-[(2-hydroxy-5-nitro-3-sulphophenyl)azo]-7-sulpho-1-naphthyl]azo]-2-hydroxy-5-sulphobenzoato(8-)]]dichromate(4-)

EC Number:

276-538-7

EC Name:

Tetrasodium [μ-[3-[[2-amino-5-hydroxy-6-[(2-hydroxy-5-nitro-3-sulphophenyl)azo]-7-sulpho-1-naphthyl]azo]-2-hydroxy-5-sulphobenzoato(8-)]]dichromate(4-)

Cas Number:

72252-58-5

Molecular formula:

C23H8Cr2N6O16S3.4Na

IUPAC Name:

tetrasodium [μ-[3-[[2-amino-5-hydroxy-6-[(2-hydroxy-5-nitro-3-sulphophenyl)azo]-7-sulpho-1-naphthyl]azo]-2-hydroxy-5-sulphobenzoato(8-)]]dichromate(4-)

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

The Wistar rat was selected due to the large amount of background data available for this strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Vivo Bio Tech Ltd., Sy. #349/A, Pregnapur-502311, Gajwel Mandal, Medak District, Telangana

- Females (if applicable) nulliparous and non-pregnant: [Yes]
- Age at study initiation: (P) 14-15 weeks;
- Weight at study initiation: (P) Males: 339.40 to 420.20 g; Females: 208.57 to 251.37 g;

- Housing:
Pre-mating: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes.

Mating and post-mating: During mating, two rats (one male and one female) were housed in standard polysulfone cages with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles. After confirming the presence of sperm in the vaginal smear or vaginal plugs (Day ‘0’ pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term.

Enrichment: Polycarbonate rat huts were provided to the animals as environmental enrichment objects during pre-mating period and post-mating period for males and pre-mating period for females. Enrichment was changed along with cage at least once a week.

- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet (Certified) manufactured by Harlan Laboratories (Envigo), P.O. Box 44220, Madison, WI 53744-4220, was provided ad libitum to animals

- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Limted., Mumbai 400 001, India was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.

- Acclimation period: Start: 12 November 2016 End: 16 November 2016

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20−23°C
- Humidity (%): 49−66%
- Air changes (per hr): 12 - 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle
IN-LIFE DATES: From: 17 November 2016 To: 16 February 2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Required quantities of the test item was weighed in to a pre-calibrated beaker* and the vehicle [0.1 % (w/v) of carboxymethyl cellulose in Milli-Q® water] was added up to the pre-mark and mixed using glass rod. The final concentration achieved was 10, 30 and 100 mg/mL for the G2, G3 and G4 groups, respectively. The suspensions were mixed well by stirring using a magnetic stirrer.
VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.1% carboxymethyl cellulose sodium salt (low viscosity) in Milli-Q® water was used as vehicle for dose formulation preparation as the same vehicle was used in the dose range finding toxicity study (study no. N3062).
- Concentration in vehicle: G2: 10, G3: 30, G4: 100 mg/mL
- Amount of vehicle (if gavage): 2.0 g of Sodium carboxy methyl cellulose (low viscosity) was added to about 1800 mL of Milli-Q® water in a 2000 mL pre-marked beaker and stirred on a magnetic stirrer
- Lot no. (if required): 051M0191V

Details on mating procedure:

- M/F ratio per cage: 1 : 1
- Length of cohabitation: 21 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0] of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [No]
- After successful mating each pregnant female was caged (how): One per cage
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during 2nd month of the treatment period and was analysed in-house. For each set, duplicate sample was drawn from top, middle and bottom layers of each preparation and in case of control duplicate samples from the middle layer were drawn.
The analysis was done as per the method validated under Advinus Study No.: G11857. One set of samples were analysed for concentration (a.i) analysis.
Dose formulations were considered acceptable as the overall mean results were within ± 15.0% of the theoretical concentration and the overall relative standard deviation (RSD) was less than 10.0%.

Duration of treatment / exposure:

Males: The dose formulation was administered orally by gavage to specific group of rats at approximately the same time each day (varying by ± 3 hours), for a minimum period of 28 days (which includes two weeks prior to mating, during the mating period and post mating) after which they were sacrificed after overnight fasting.
Females: The dose formulation was administered orally by gavage to the specific group of rats at approximately the same time each day (varying by ± 3 hours), two weeks prior to the mating period and was continued through mating, pregnancy and up to LD 13. On LD 14, the females were sacrificed after overnight fasting.
The animals in the vehicle control group were handled in an identical manner to the treatment group and were administered vehicle only.
The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control group at an equivolume of 10 mL/kg Bwt.

Frequency of treatment:

Daily

Details on study schedule:

- Age at mating of the mated animals in the study: [14 to 15] weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels of 100 (G2), 300 (G3) and 1000 (G4) mg/kg/day were selected for this study based on the results of 14-Day Repeated Dose Oral Toxicity Study in Wistar Rats (Study No.N3062) and in consultation with the Sponsor.
In addition to the test doses, vehicle control group was included. Animals in the vehicle control were handled in a manner similar to the treatment groups except for test item administration.

- Rationale for animal assignment (if not random): Grouping was done by the method of body weight stratification and distribution. On the day of randomization, based on the given temporary animal identification number, each animal with normal oestrous cyclicity (4-5 day cycle) was weighed and the corresponding body weights was recorded. The data was transferred for data input (temporary identification number and body weight) into an excel spread sheet. The body weights recorded were stratified in ascending order.
Statistical analysis and ensured that the weight variation was minimal and inter group variation did not exceed ± 20% of the mean body weight in each sex. Rats with extreme body weights were discarded. Grouping was done one day prior to initiation of the treatment.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily once
- Cage side observations checked in table [No.2] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

Oestrous cyclicity (parental animals):

Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select for the study females with regular 4-5 days cyclicity. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating to determine treatment-related effects on mating or pre-coital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal. The overall pattern of each female was characterized as regular cycling (having recurring 4–5 day cycles) and irregular cycling (having cycles with a period of diestrus longer than 3 days or a period of estrus more than 2 days). Incomplete cycles (having prolonged periods of either estrus or diestrus) were not included in calculating the mean cycle length.
Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [Yes]
- If yes, maximum of [8] pups/litter ([4]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, other:] Yes

GROSS EXAMINATION OF DEAD PUPS:
[Yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [5 of Appendix 20 Pathology Report] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

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

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [6 of Appendix 20 Pathology Report] were prepared for microscopic examination and weighed, respectively.

Statistics:

Statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package Version 12.0. All data of quantitative variables like body weight, food consumption, oestrous cycle length, hormone levels, ano-genital distance and organ weights and organ weight ratios data were tested for homogeneity of variances (Levene’s test) within the group before performing One-Way Analysis of Variance (ANOVA). When the data found to be non-optimal (non-normal or heteroschedastic), ANOVA was done using suitable transformation. Comparison of means between treatment groups and vehicle control group was done using Dunnett’s test when the overall treatment ‘F’ test was found significant.
Post implantation loss (%), number of nipples/areolae in male pups, no. of implantations, pre-coital interval (days), mean litter size, sex ratio and gestation length (days) were analysed after suitable transformation (√ x + ½) of the data. One-way analysis of variance (ANOVA) was carried out for the transformed data. Dunnett’s pair-wise comparison of the treated means with the control mean was performed for testing the differences in proportions for mating, fertility and survival indices.
Z test was performed for testing the differences in proportions for mating, fertility and survival indices
All analyses and comparisons were evaluated at the 5% (p<0.05) level. Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated throughout the report as stated below:
+/-: Significantly higher (+)/lower (-) than the vehicle control group

Reproductive indices:

a. Male mating index (%) = (Number of males with evidence of mating / Number of males cohabited) x 100

b. Male fertility index (%) = (Number of males siring a litter / Number of males cohabited) x 100

c. Female mating index (%) = (Number of females mated / Number of females cohabited) x 100

d. Female fertility index (%) = (Number of pregnant females (confirmed at necropsy) / Number of females used for mating) x 100

e. Mean number of implantations/group = Total number of implantations / Total number of pregnant animals

f. Post implantation loss (%) = ((Number of implantations - Number of live pups) / Number of implantations) x 100

Offspring viability indices:

a. Mean litter size per group = (Total Number of pups / Total Number of littered animals)

b. Mean viable litter size = (No. of viable pups on Day 1 / No. of females littered)

c. Live birth index (%) = (No. of viable pups born (at first observation) / Total no. of pups born (at first observation)) x 100

d. Day 4 survival index (%) = (Number of viable pups on lactation Day 4 / Number of viable pups born) x 100

e. Sex Ratio (%) = (No. of male pups born / Total No. of pups born) x 100

f. Ano-genital Distance Ratio (mm/g1/3 ) = (Ano-genital distance / Cube root of body weight)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs observed both during the treatment period at all the treated groups in both sexes. However, grey coloured faeces at 100 mg/kg Bwt/day and black coloured faeces at 300 and 1000 mg/kg Bwt/day doses in both sexes were observed during the treatment period. The colored feces were attributed to the nature of the test item and not considered an adverse finding.

There were no abnormalities observed in pups.

Mortality:

no mortality observed

Description (incidence):

There were no mortalities observed both during the treatment period at all the treated groups in both sexes.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights and net weight gains were unaffected by the treatment at all the doses tested when compared to vehicle control in both sexes.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption was not altered by the treatment in both the sexes at all the doses tested, when compared to vehicle control.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test item-related changes observed in thyroid stimulating hormone (TSH) and thyroxin hormone (T4) levels in adult males (at termination), adult females (on lactation Day 14) and pups (on Days 4 after birth and 13) across the treatment groups

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related histopathological changes in the reproductive organs in both males and females.
The staging of spermatogenesis did not reveal any stage specific changes. The spermatogenic cycle observed in the different seminiferous tubules of testes were complete and none of the stages of spermatogenesis were arrested in all the animals examined.
There was no test item-related microscopic change observed in thyroid gland of parents and pups at all the tested doses.
Test item related non-adverse histopathological changes were observed only in non-reproductive organs of male and female rats.
Microscopically dark deposits were observed in various organs (jejunum, lungs, mandibular lymph nodes, mesenteric lymph nodes, ileum, liver and kidney) across the treatment groups. Dark deposits was characterized by accumulation of gray to green coloured granules in lamina propria of jejunum, hepatocytes in liver, tubular epithelium in kidneys and in macrophages of lungs, mandibular and mesenteric lymph nodes. These changes were attributed to the physical appearance of test item and considered as test item-related non-adverse change in absence of inflammatory/degenerative changes in any of the organs examined microscopically.
All the other microscopic findings observed across the groups in male and female rats were considered as incidental/spontaneous changes and not related to test item administration as the findings were distributed randomly across the groups and normally observed at this age group animals.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cyclicity was evaluated for its length and normality by examining the vaginal smears daily for two weeks during treatment period (prior to cohabitation).
The calculated mean oestrous cycle length was 3.94, 4.13, 4.13 and 4.05 days in vehicle control, 100, 300 and 1000 mg/kg Bwt/day doses, respectively. The mean oestrous cycle length in the treated groups was not significantly different from the vehicle control group.

Reproductive performance:

no effects observed

Description (incidence and severity):

The duration of gestation (gestation length), pre-coital time, fertility indices were not affected at any of the doses tested, when compared to vehicle control group. However, incidence of significantly lower male and female fertility indices ((80%) was observed at 1000 mg/kg Bwt/day. These significant differences were considered incidental as the difference is within the historical control data (HD range: 80 to 100 %).

Details on results (P0)

The purpose of this study in Wistar Rats was to generate limited information concerning the effects of Sanodal-Schwarz 2LW on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.
The test item Sanodal-Schwarz 2LW was suspended in vehicle i.e., 0.1% Carboxymethylcellulose Sodium salt (low viscosity) in Milli-Q® water and administered by oral gavage at the dose levels of 100 (G2), 300 (G3) and 1000 (G4) mg/kg Bwt/day to male and female Wistar rats, at the dose volume of 10 mL/kg Bwt/day. Similarly, vehicle was administered to rats in the vehicle control group. Each group consisted of 10 male and 10 female rats. The prepared dose formulations were administered once daily to specific group of rats prior to mating, during mating and post-mating periods (for males), prior to mating, during mating and pregnancy and after delivery up to Lactation Day (LD) 13 (for females).
The identity of Sanodal-Schwarz 2LW was provided by the study sponsor by a Certificate of Analysis (CoA). The stability and homogeneity of test item in the vehicle was carried out under Advinus Study No.G11857. Based on the results, the test item was found to be stable for up to 48 hours at 1 and 100 mg/mL concentrations, when stored at room temperature. The dose formulations were analysed for active ingredient (a.i.) concentration on Day 1 and during 2nd month of the treatment period. The results indicated that the analysed concentrations were within ± 15 % of variations from the theoretical concentrations.
All rats were observed for clinical signs once daily. Body weight was recorded at the beginning of the treatment, weekly thereafter and at termination. Food consumption was recorded weekly except during the cohabitation period. Female rats were also weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4, 7 and 13. The number, weight, survival and mortality of pups were observed during the lactation period. The ano-genital distance of each pup was measured on LD 4. All the survived male pups were examined for the appearance of nipples/areolae on post-natal day (PND 13). The blood samples were collected for thyroid hormone analysis prior to necropsy from males, on LD 4 and on LD 13 from available pups. The animals were subjected to detailed necropsy at sacrifice and study plan specified tissues were collected. From female rats (dams), the blood was collected on LD 13 prior to necropsy. Gross necropsy was performed on all dams on LD 14 and study plan specified tissues were collected. All the surviving pups were sacrificed on LD 13 and thyroid gland from available one male and one female pup from each litter was collected for histopathological examination.
Tissues collected from all animals in the control and high dose groups were examined microscopically for histopathological changes. Histopathological examination of the testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. All gross lesions were examined in all the groups. The reproductive organs of animals failing to mate were examined in all the dose groups.
Under the experimental conditions employed, the following results were obtained:
• There were no treatment-related clinical signs or mortality observed at any of the doses tested.
• The body weights and food consumption were unaffected by the treatment at all the doses tested.
• The maternal body weight and food consumption during gestation and lactation periods were unaffected by the treatment at all the doses tested.
• Treatment had no effect on pre-coital time or gestation length, oestrous cycle length at all the tested doses. No treatment-related changes were observed in the fertility indices of sires and dams at all the doses tested. The survival indices were not altered by the treatment at all the doses tested.
• There were no treatment-related effects on the uterine/implantation data, mean litter size and mean viable litter size. There were no external abnormalities in live or dead pups in any of the groups.
• No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses tested when compared to the vehicle control group.
• The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.
• There were no test item related changes in the terminal body weights, organ weights and organs weight ratios in both males and females. Gross examination of pups on LD 13 did not reveal any gross changes.
• The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item-administration.
• Gross and histopathology: Grossly, green discoloration of mandibular lymph nodes and mesenteric lymph node in both sexes, green intestinal content in both sexes was observed at 100 mg/kg/day. At 300 mg/kg/day, grossly gray/green discoloration of kidneys and liver in both sexes, green discoloration mandibular lymph nodes and mesenteric lymph node in both sexes, green intestinal content in both sexes; gray discoloration of testes in males, green discoloration of lungs in females; green content/green discoloration of non-glandular mucosa in stomach in females were observed. Microscopically pigment deposits in tubular epithelial cells in kidneys and macrophages in lungs in females, pigment deposits in macrophages in mandibular and mesenteric lymph nodes in both sexes were observed. At 1000 mg/kg/day, grossly, gray/green discoloration of kidneys and liver in both sexes, green discoloration of lungs, mandibular lymph nodes and mesenteric lymph node in both sexes, green intestinal content in both sexes, gray discoloration of testes in males, green content/green discoloration of non-glandular mucosa in stomach in females were observed. Microscopically pigment deposits in lamina propria of jejunum and macrophages in lungs, mandibular lymph nodes, mesenteric lymph nodes in both sexes, pigment deposit in tubular epithelial cells in kidneys, hepatocytes in liver and lamina propria of ileum in females were observed.
All the above gross and microscopic changes were attributed to the physical appearance of test item and considered as test item-related non-adverse change in absence of inflammatory/degenerative changes in any of the organs examined microscopically.
No test item-related changes were observed in organ weights, gross pathology and histopathology of thyroid gland in pups and in adult male and female rats.
No Observed Adverse Effect Level
Daily oral (gavage) administration of Sanodal-Schwarz 2LW to male and female Wistar rats at dose levels of 100, 300 and 1000 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and 2 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery did not induce any adverse effects on fertility and reproductive performance. The No Observed Adverse Effect Level (NOAEL) of Sanodal-Schwarz 2LW for systemic and reproductive toxicity in parental rats as well as offspring is considered to be 1000 mg/kg Bwt/day.

Effect levels (P0)

Key result

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

organ weights and organ / body weight ratios

gross pathology

neuropathology

reproductive function (oestrous cycle)

reproductive performance

other: histopathology

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

To summarize, daily oral (gavage) administration of test item to Wistar rats at the dose levels 100, 300 and 1000 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and 2 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) had no effects on general health, body weights, food intake, pre-coital time, gestation length, mating and fertility parameters. The survival indices were not altered by the treatment. There were no test- item related changes in the terminal body weights, organ weights and organs weight ratios in both males and females. Gross examination of pups on LD 13 did not reveal any gross changes. The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item-administration.

Grossly, green discoloration of mandibular lymph nodes and mesenteric lymph node in both sexes, and green intestinal content in both sexes were observed at 100 mg/kg/day. At 300 mg/kg/day, grossly gray/green discoloration of kidneys and liver in both sexes, green discoloration mandibular lymph nodes and mesenteric lymph node in both sexes, green intestinal content in both sexes; gray discoloration of testes in males, green discoloration of lungs in females; green content/green discoloration of non-glandular mucosa in stomach in females were observed. Microscopically pigment deposits in tubular epithelial cells in kidneys and macrophages in lungs in females, pigment deposits in macrophages in mandibular and mesenteric lymph nodes in both sexes were observed. At 1000 mg/kg/day, grossly, gray/green discoloration of kidneys and liver in both sexes, green discoloration of lungs, mandibular lymph nodes and mesenteric lymph node in both sexes, green intestinal content in both sexes, gray discoloration of testes in males, green content/green discoloration of non-glandular mucosa in stomach in females were observed. Microscopically pigment deposits in lamina propria of jejunum and macrophages in lungs, mandibular lymph nodes, mesenteric lymph nodes in both sexes, pigment deposit in tubular epithelial cells in kidneys, hepatocytes in liver and lamina propria of ileum in females were observed.

All the above gross and microscopic changes were attributed to the physical appearance of test item and considered as test item-related non-adverse changes in absence of inflammatory/degenerative changes in any of the organs examined microscopically.

No test item-related changes were observed in organ weights, gross pathology and histopathology of thyroid gland in pups and adult male and female rats.

Hence, the evaluated No Observed Adverse Effect Level (NOAEL) of the test item for systemic and reproductive toxicity in parental rats as well as offspring is considered to be 1000 mg/kg Bwt/day.

Executive summary:

The purpose of this study in Wistar Rats was to generate limited information concerning the effects of the test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

The test item was suspended in vehicle i.e.,0.1% Carboxymethylcellulose Sodium salt (low viscosity) in Milli-Q^®water and administered by oral gavage at the dose levels of 100 (G2), 300 (G3) and 1000 (G4) mg/kg Bwt/day to male and female Wistar rats, at the dose volume of 10 mL/kg Bwt/day. Similarly, vehicle was administered to rats in the vehicle control group. Each group consisted of 10 male and 10 female rats. The prepared dose formulations were administered once daily to specific group of rats prior to mating, during mating and post-mating periods (for males), prior to mating, during mating and pregnancy and after delivery up to Lactation Day (LD) 13 (for females).

The identity of the test item was provided by the study sponsor by a Certificate of Analysis (CoA). The stability and homogeneity of test item in the vehicle was carried out under Advinus Study No.G11857. Based on the results, the test item was found to be stable for up to 48 hours at 1 and 100 mg/mL concentrations, when stored at room temperature. The dose formulations were analysed for active ingredient (a.i.) concentration on Day 1 and during 2^ndmonth of the treatment period. The results indicated that the analysed concentrations were within ± 15 % of variations from the theoretical concentrations.

All rats were observed for clinical signs once daily. Body weight was recorded at the beginning of the treatment, weekly thereafter and at termination. Food consumption was recorded weekly except during the cohabitation period.Female rats were also weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4, 7 and 13. The number, weight, survival and mortality of pups were observed during the lactation period. The ano-genital distance of each pup was measured on LD 4. All the survived male pups were examined for the appearance of nipples/areolae on post-natal day (PND 13). The blood samples were collected for thyroid hormone analysis prior to necropsy from males, on LD 4 and on LD 13 from available pups. The animals were subjected to detailed necropsy at sacrifice and study plan specified tissues were collected. From female rats (dams), the blood was collected on LD 13 prior to necropsy. Gross necropsy was performed on all dams on LD 14 and study plan specified tissues were collected. All the surviving pups were sacrificed on LD 13 and thyroid gland from available one male and one female pup from each litter was collected for histopathological examination.

Tissues collected from all animals in the control and high dose groups were examined microscopically for histopathological changes. Histopathological examination of the testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. All gross lesions were examined in all the groups. The reproductive organs of animals failing to mate were examined in all the dose groups.

Under the experimental conditions employed, the following results were obtained:

· There were no treatment-related clinical signs or mortality observed at any of the doses tested.

· The body weights and food consumption were unaffected by the treatment at all the doses tested.

· The maternal body weight and food consumption during gestation and lactation periods were unaffected by the treatment at all the doses tested.

· Treatment had no effect on pre-coital time or gestation length, oestrous cycle length at all the tested doses. No treatment-related changes were observed in the fertility indices of sires and dams at all the doses tested. The survival indices were not altered by the treatment at all the doses tested.

· There were no treatment-related effects on the uterine/implantation data, mean litter size and mean viable litter size. There were no external abnormalities in live or dead pups in any of the groups.

· No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses tested when compared to the vehicle control group.

·  The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

·  There were no test item related changes in the terminal body weights, organ weights and organs weight ratios in both males and females. Gross examination of pups on LD 13 did not reveal any gross changes.

· The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item-administration.

·  Gross and histopathology: Grossly, green discoloration of mandibular lymph nodes and mesenteric lymph node in both sexes, green intestinal content in both sexes was observed at 100 mg/kg/day. At 300 mg/kg/day, grossly gray/green discoloration of kidneys and liver in both sexes, green discoloration mandibular lymph nodes and mesenteric lymph node in both sexes, green intestinal content in both sexes; gray discoloration of testes in males, green discoloration of lungs in females; green content/green discoloration of non-glandular mucosa in stomach in females were observed. Microscopically pigment deposits in tubular epithelial cells in kidneys and macrophages in lungs in females, pigment deposits in macrophages in mandibular and mesenteric lymph nodes in both sexes were observed. At 1000 mg/kg/day, grossly, gray/green discoloration of kidneys and liver in both sexes, green discoloration of lungs, mandibular lymph nodes and mesenteric lymph node in both sexes, green intestinal content in both sexes, gray discoloration of testes in males, green content/green discoloration of non-glandular mucosa in stomach in females were observed. Microscopically pigment deposits in lamina propria of jejunum and macrophages in lungs, mandibular lymph nodes, mesenteric lymph nodes in both sexes, pigment deposit in tubular epithelial cells in kidneys, hepatocytes in liver and lamina propria of ileum in females were observed.

All the above gross and microscopic changes were attributed to the physical appearance of test item and considered as test item-related non-adverse change in absence of inflammatory/degenerative changes in any of the organs examined microscopically. 

No test item-related changes were observed in organ weights, gross pathology and histopathology of thyroid gland in pups and in adult male and female rats.

No Observed Adverse Effect Level

Daily oral (gavage) administration of the test item to male and female Wistar rats at dose levels of 100, 300 and 1000 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and 2 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery did not induce any adverse effects on fertility and reproductive performance. The No Observed Adverse Effect Level (NOAEL) of Sanodal-Schwarz 2LW for systemic and reproductive toxicity in parental rats as well as offspring is considered to be 1000 mg/kg Bwt/day.