Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat is the standard laboratory rodent species used for toxicity assessment and 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 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) 13-14 weeks
- Weight at study initiation: (P) Males: 295.18 to 359.10 g; Females: 196.40 to 230.62 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 (GD 20).
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 once a week.

- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet (Certified) manufactured by 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 Limited., Mumbai 400 001, India, was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: Start: 11 August 2017, End: 15 August 2017

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21−24°C
- Humidity (%): 60 – 68 %.
- 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: 16 August 2017 To: 06 November 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 small volume of vehicle [0.5 % (w/v) of carboxymethyl cellulose in Milli-Q® water] was added and mixed using glass rod. The volume was made up with the vehicle to attain desired concentrations of 11, 33 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): 0.5% carboxymethyl cellulose sodium salt (medium viscosity) in Milli-Q® water was used to prepare the dose formulations as the same vehicle was used in acute oral toxicity study (Study No.G12734),14-day repeated dose oral toxicity study (Study No.N3254) and 28 Day repeated dose oral toxicity study (G12741).
- Concentration in vehicle: G2: 11; G3: 33 and G4: 100 mg/mL
- Amount of vehicle (if gavage): 10.0 g of Sodium carboxy methyl cellulose was added to 1800 mL of Milli-Q® water in a 2000 mL pre-marked beaker and stirred on a magnetic stirrer.
- Lot/batch no. (if required): MKBQ8416V
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 (Day 38) of the treatment period and 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.: G12742. One set of samples were analysed for concentration (a.i) analysis.
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 period of 41 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 (Day 51-69). 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 (for males 41 days and
for females 51-69 days).
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: [15 to 16] weeks
Doses / concentrationsopen allclose all
Dose / conc.:
110 mg/kg bw/day (nominal)
Dose / conc.:
330 mg/kg bw/day (nominal)
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 110 (G2), 330 (G3) and 1000 (G4) mg/kg/day were selected for this by the Sponsor based on the outcome of a 28-day repeated dose study.
In addition to the test doses, vehicle control group was included. Animals inthe 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 weight recorded was 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 once

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly once

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 females with regular 4-5 days cyclicity for the study. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating to determine the Day 0 of pregnancy/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.
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 in 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 [Section 9.9.2 in Main 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 [Section 9.9.3 in Main 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, ano-genital index 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 is found significant.
Post implantation loss (%), 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 found significant.
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 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 animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related clinical signs were observed at any of the doses tested in both sexes.
Slight bluish discolouration of the skin (Cyanosis) was observed at 110 and 330 mg/kg Bwt/day doses after test item administration in both sexes. Moderate bluish discolouration of the skin (Cyanosis) was observed at 1000 mg/kg Bwt/day dose after test item administration and became slight prior to next dose (pre-dose) on each day of observation. Light bluish coloured faeces were observed at 110 mg/kg Bwt/day dose, whereas at 330 and 1000 mg/kg Bwt/day doses, dark bluish coloured feces were observed in both sexes. The bluish discolouration of the skin/faeces observed could be due to physical nature of the test item.
The clinical sign of sparse hair loss was observed in one female (Ru2946) at 110 mg/kg Bwt/day dose and considered as spontaneous finding and not related to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortalities were observed in parental males and females at any of the tested doses.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights and body weight gains were unaffected throughout the treatment period in males and two weeks pre-mating period in females at 110 and 330 mg/kg Bwt/day doses and in females at 1000 mg/kg Bwt/day doses. An incidence of significantly lower mean net body weight gain at 110 mg/kg Bwt/day dose in males was considered incidental finding as the mean body weights were not altered by the treatment.
At 1000 mg/kg Bwt/day dose males, the mean body weights from week 3 to 6 were apparently lower (-4.7 to -5.8%). The net body weight gain was significantly lower at the end of six weeks treatment period.
Thus, the treatment resulted in decreased the body weight at 1000 mg/kg/day dose in males.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The mean food consumption was significantly lower on week 6 in males at all the treated groups when compared to vehicle control. This significant difference was considered incidental due to lack of dose correlation. The food consumption was not altered for two weeks pre-mating period in females at all the tested doses when compared to vehicle control. Sporadic incidence of significantly lower food consumption was observed on week 1 in females at 1000 mg/kg Bwt/day dose. This significant difference was considered incidental as the mean body weights were not altered by the treatment.
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:
effects observed, treatment-related
Description (incidence and severity):
Thyroid hormone profile (TSH and T4) was not affected in adult males (at termination), adult females (on lactation Day 13) and pups (on lactation Days 4 and 13) across the treated groups when compared to the concurrent control group. Increased thyroid stimulating hormone (TSH) level noted in adult males (128%) at 1000 mg/kg Bwt/day dose was considered to be non-adverse effects of the test item in the absence of any alterations in T4 levels and associated microscopic findings in thyroids.
Decreased thyroxin (T4) level noted in adult females (25%) at 1000 mg/kg Bwt/day was considered incidental, as the variation in individual animal values of T4 was larger with fewer animals available in this group. Further, it was not accompanied by any changes in thyroid stimulating hormone (TSH) and/or in the absence of correlating microscopic changes in thyroid gland.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes in terminal fasting body weights in adult male and female rats of all groups compared to the control group. Increased relative testes weight (15%) at 1000 mg/kg Bwt/day dose was considered as toxicologically insignificant change in the absence of any associated gross and microscopic changes in testes.
Decrease in absolute and relative thyroid gland weight at ≥ 330 mg/kg Bwt/day in adult female rats considered as incidental change in absence of correlating microscopic changes in thyroid gland at 1000 mg/kg Bwt/day.
A statistically significant decrease in the terminal body weight was observed in male pups (14-17%) at ≥ 330 mg/kg Bwt/day and in female pups (18%) at 1000 mg/kg Bwt/day dose. This finding was considered as toxicologically insignificant change as these alterations in terminal body weights fall within the in-house historical control range (HD range: males – 14 to 43% and females -12 to 36%). In addition, there was no apparent intergroup difference in the pup body weights on lactation Days 0 and 4.
Statistically significant increase in relative thyroid weight (29%) at 1000 mg/kg Bwt/day in male pups considered as test item-related non adverse change in absence of correlating microscopic changes in thyroid gland.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no test item-related adverse gross pathological changes observed (including reproductive organs) in adult male and female rats. Gross pathological examination of dead pups and pups sacrificed on LD 13 did not reveal any abnormalities at all the dose levels tested.
The following test item-related discoloration (bluish and/or bluish black) was observed in liver, skin-hair coat and gastro-intestinal content in male adult rats at ≥ 330 mg/kg Bwt/day and in female adult rats at ≥ 110 mg/kg Bwt/day. It was attributed to the physical nature of test item-Sanolin Lave Blue A and considered as test item-related non-adverse change. Further, bluish discoloration of liver surface did not result in any microscopic changes. In pups grossly greenish discoloration of stomach and /or intestinal content was observed.
Single incidence of small sized thymus in 330 mg/kg Bwt/day dose adult female was histologically associated with minimal lymphoid depletion and was considered incidental and not related to test item administration.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In liver test item-related minimal to mild diffuse hepatocellular hypertrophy was observed in male and female adult rats dosed at ≥ 330 mg/kg Bwt/day. There were no test item-related histopathological changes noted in any other tissues (including reproductive tissues) in any of the adult male and female rats.
The staging of spermatogenesis did not reveal any stage specific changes. In all treated males, the spermatogenic cycles observed in the different seminiferous tubules of testes were complete.
The qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular structures did not reveal any test item associated findings in parental rats.
There were no test item-related microscopic changes observed in thyroid gland of parents and pups at all the dose levels tested. Few randomly distributed microscopic findings observed in some of the rats were considered as incidental background findings commonly noted in this age group.
The cause of infertility in the two female rats (Ru2985 and Ru2988) and one male rat (Ru2971) from 1000 mg/kg Bwt/day dose group could not be ascertained as no corresponding gross and microscopic changes were observed in reproductive organs.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

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 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 calculated mean oestrous cycle length was 4.24, 4.25, 4.32 and 4.24 days in vehicle control, 110, 330 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.
Pre-coital time was determined (in days) from oestrous cycle evaluation from the day of cohabitation to the evidence of mating.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Histopathological examination of the testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure.
Reproductive performance:
no effects observed
Description (incidence and severity):
The duration of gestation (gestation length) and pre-coital time were not affected at any of the doses tested, when compared to vehicle control group. Significantly lower male and female mating (90%) and fertility indices (80 %) were observed at 1000 mg/kg Bwt/day dose. This significant difference could be due to one male (Ru2971) which did not impregnate a female and two non-littering females (Ru2985 and Ru2988) at this dose. Further, this significant difference was within the historical control data (HD range: 80-100%). Hence these significant differences were considered toxicologically not relevant.

Details on results (P0)

• There were no treatment-related clinical signs observed at any of the doses tested in both sexes.
• Bluish discolouration of the skin (slight to moderate) and light bluish coloured faeces were observed at 110 mg/kg Bwt/day dose, whereas at 330 and 1000 mg/kg Bwt/day doses, the faeces colour was dark bluish in both sexes. The bluish coloured skin/faeces observed could be due to physical nature of the test item. No mortalities were observed at any of the tested doses in both sexes.
• The treatment decreased the body weights and net body weight gains in males at 1000 mg/kg Bwt/day dose.
• 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, fertility indices of sires and dams and survival indices at all the tested doses.
• Mean number and weight of male, female and combined sex pups in treated groups were comparable to control group.
• 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 were observed in the ano-genital distance and ratio to ano-genital distance to cube root of body weight at any of the doses tested when compared to the vehicle control group.
• No areola/nipple retention in male pups on PND 13 at any of the doses tested.
• The test item Sanolin Lave Blue A resulted in increased TSH level at 1000 mg/kg Bwt/day dose male adult rats. This increase in TSH level was considered as non-adverse effect as there were no alterations in T4 levels and no microscopic findings were observed in thyroid gland. Decreased thyroxin (T4) level noted in adult females at 1000 mg/kg Bwt/day dose was considered incidental as it was not accompanied by any changes in thyroid stimulating hormone (TSH) and/or in the absence of correlating microscopic changes in thyroid gland. Thyroid hormone profile (TSH and T4) was not affected in pups (on lactation Days 4 and 13) across the treated groups when
compared to the concurrent control group.
• There were no test item related changes in the terminal body weights, organ weights and organs weight ratios in males and females and in pups at all the tested doses.
• Grossly, discoloration (bluish and/or bluish black) of liver, skin-hair coat and gastro-intestinal content in male adult rats at ≥ 330 mg/kg Bwt/day and in female adult rats at ≥ 110 mg/kg Bwt/day was observed. It was attributed to the physical nature of test item and considered as test item-related nonadverse change. Further, bluish discoloration of liver surface did not result in any microscopic changes. In pups, grossly greenish discoloration of stomach and /or intestinal content was observed at all the tested doses. This could be due to exposure of pups to test item via milk and hence, considered as test item-related non-adverse change. There were no test item related microscopic changes observed in the reproductive organs and/or thyroid glands in male/female parental rats. In liver, test item-related minimal to
mild diffuse hepatocellular hypertrophy was observed in male and female adult rats dosed at ≥ 330 mg/kg Bwt/day dose groups. This hypertrophy was considered as adaptive, non-adverse change.

No Observed Adverse Effect Level
Daily oral (gavage) administration of Sanolin Lave Blue A to male and female Wistar rats at dose levels of 110, 330 and 1000 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and post mating in males or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery in females did not induce any adverse effects on fertility, reproductive performance and on the offspring parameters. The No Observed Adverse Effect Level (NOAEL) of the test item for reproductive and developmental toxicity 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
food consumption and compound intake
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

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 specified
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:
no effects observed
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed

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 (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed at highest dose

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
To summarize, daily oral (gavage) administration of test item to Wistar rats at the dose levels 110, 330 and 1000 mg/kg Bwt/day for 2 weeks prior to mating, during mating, post mating in males or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery in females at all the tested doses had no effects on general health, body weights, food consumption, pre-coital time, gestation length, mating and fertility parameters, survival indices, terminal body weights, organ weights and organs weight ratios. The mean body weights and net body weight gains were lower in males at 1000 mg/kg Bwt/day dose.
The test item resulted in increased TSH level at 1000 mg/kg Bwt/day dose male adult rats. This increase in TSH level was considered as non-adverse effect as there were no alterations in T4 levels and microscopic findings in thyroid gland.
Decreased thyroxin (T4) level noted in adult females at 1000 mg/kg Bwt/day dose was considered incidental as it was not accompanied by any changes in thyroid stimulating hormone (TSH) and/or in the absence of correlating microscopic changes in thyroid gland. Thyroid hormone profile (TSH and T4) was not affected in pups (on lactation Days 4 and 13) across the treated groups when compared to the concurrent control group.
There were no test item related changes in the terminal body weights, organ weights and organs weight ratios in males and females and in pups at all the tested doses.
Grossly, discoloration (bluish and/or bluish black) of liver, skin-hair coat and gastro-intestinal content in male adult rats at ≥ 330 mg/kg Bwt/day and in
female adult rats at ≥ 110 mg/kg Bwt/day was observed. It was attributed to the physical nature of test item and considered as test item-related non-adverse change. Further, bluish discoloration of liver surface did not result in any microscopic changes. In pups, grossly greenish discoloration of stomach and /or intestinal content was observed at all the tested doses. This could be due to exposure of pups to test item via milk and hence, considered as test item-related non-adverse change. There were no test item related microscopic changes observed in the reproductive organs and/or thyroid glands in male/female parental rats. In liver test item-related minimal to mild diffuse hepatocellular hypertrophy was observed in male and female adult rats dosed at ≥ 330 mg/kg Bwt/day dose groups.
No Observed Adverse Effect Level
Daily oral (gavage) administration of the test item to male and female Wistar rats at dose levels of 110, 330 and 1000 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and post mating in males or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery in females did not induce any adverse effects on fertility, reproductive performance and on the offspring parameters.
The No Observed Adverse Effect Level (NOAEL) of the test item for reproductive and developmental toxicity 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 weighed and suspended in vehicle, i.e. 0.5% Carboxy methylcellulose Sodium salt (medium viscosity) in Milli-Q Water, and administered by oral gavage at the dose levels of 110 (G2), 330 (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 consists of 10 males and 10 female rats. The prepared dose formulations were administered once daily to specific groups of rats prior to mating, during mating and post-mating periods for males, prior to mating, during conception and pregnancy and after delivery up to Lactation Day (LD) 13 for females.

The identity of Sanolin Lave Blue A was provided by the study Sponsor by a Certificate of Analysis. The stability and homogeneity of the test item in the vehicle was established at 1 and 250mg/mL under Advinus Study No. G12742.

Based on the results, the test item was stable and homogenous in the vehicle (0.5 % Carboxy methylcellulose sodium salt (medium viscosity) in Milli-Q® water) up to 4 days when stored at room temperature. The dose formulations were analysed for active ingredient (a.i.) concentration on Day 1 and during 2nd month (Day 38) 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 where

food consumption was not measured. 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 recorded during the lactation period. The ano-genital distance of each pup was measured on LD 0.

All the survived male pups were examined for the appearance of nipples/areolae on post-natal day 13 (PND 13). Blood samples were collected for thyroid hormone analysis prior to necropsy from males and females (LD 13), on LDs 4 and 13 from available pups. The animals were subjected to detailed necropsy at sacrifice and study plan specified tissues were collected. A 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 glands from available one male and one female pup from each litter were 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. Livers showing grossly bluish discoloration were examined in all the groups. The reproductive organs of one male failing to mate (male –Ru2971) and two not littered females (Ru2985 and Ru2988) were examined in the 1000 mg/kg Bwt/day dose group.

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

• There were no treatment-related clinical signs observed at any of the doses tested in both sexes.

• Bluish discolouration of the skin (slight to moderate) and light bluish coloured faeces were observed at 110 mg/kg Bwt/day dose, whereas at 330 and 1000 mg/kg Bwt/day doses, the faeces colour was dark bluish in both sexes. The bluish coloured skin/faeces observed could be due to physical nature of the test item. No mortalities were observed at any of the tested doses in both sexes.

• The treatment decreased the body weights and net body weight gains in males at 1000 mg/kg Bwt/day dose.

• 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, fertility indices of sires and dams and survival indices at all the tested doses.

• Mean number and weight of male, female and combined sex pups in treated groups were comparable to control group.

• 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 were observed in the ano-genital distance and ratio to ano-genital distance to cube root of body weight at any of the doses tested when compared to the vehicle control group.

• No areola/nipple retention in male pups on PND 13 at any of the doses tested.

• The test item Sanolin Lave Blue A resulted in increased TSH level at 1000 mg/kg Bwt/day dose male adult rats. This increase in TSH level was considered as non-adverse effect as there were no alterations in T4 levels and no microscopic findings were observed in thyroid gland. Decreased thyroxin (T4) level noted in adult females at 1000 mg/kg Bwt/day dose was considered incidental as it was not accompanied by any changes in thyroid stimulating hormone (TSH) and/or in the absence of correlating microscopic changes in thyroid gland. Thyroid hormone profile (TSH and T4) was not affected in pups (on lactation Days 4 and 13) across the treated groups when compared to the concurrent control group.

• There were no test item related changes in the terminal body weights, organ weights and organs weight ratios in males and females and in pups at all the tested doses.

• Grossly, discoloration (bluish and/or bluish black) of liver, skin-hair coat and gastro-intestinal content in male adult rats at ≥ 330 mg/kg Bwt/day and in female adult rats at ≥ 110 mg/kg Bwt/day was observed. It was attributed to the physical nature of test item and considered as test item-related nonadverse change. Further, bluish discoloration of liver surface did not result in any microscopic changes. In pups, grossly greenish discoloration of stomach and /or intestinal content was observed at all the tested doses. This could be due to exposure of pups to test item via milk and hence, considered as test item-related non-adverse change. There were no test item related microscopic changes observed in the reproductive organs and/or thyroid glands in male/female parental rats. In liver, test item-related minimal to mild diffuse hepatocellular hypertrophy was observed in male and female adult rats dosed at ≥ 330 mg/kg Bwt/day dose groups. This hypertrophy was considered as adaptive, non-adverse change.

No Observed Adverse Effect Level

Daily oral (gavage) administration of Sanolin Lave Blue A to male and female Wistar rats at dose levels of 110, 330 and 1000 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and post mating in males or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery in females did not induce any adverse effects on fertility, reproductive performance and on the offspring parameters. The No Observed Adverse Effect Level (NOAEL) of Sanolin Lave Blue A for reproductive and developmental toxicity is considered to be 1000 mg/kg Bwt/day.