Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Jul 2018 to 12 Apr 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to other study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
27 Feb 2018 to 23 May 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 Jul 2016
Deviations:
yes
Remarks:
Clinical chemistry including hormone determinations were not performed
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No. of test material: 0000091655
- Expiration date of the lot/batch: 31 August 2018, The test item expired during the experimental phase of the study as per the declared expiry date on the CoA (Certificate of Analysis). To establish the stability of the test item, the active ingredient content will be analysed concurrently with the dosing period i.e., within one week before declared expiry and within one week after completion of dosing following the validated analytical method. The CoA and comparison of analysed purity were reported in the final report.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: As per the instruction received from the Sponsor on storage of the test item, the test item will be stored:
- Storage Temperature: Room Temperature
- Storage Container: In original container as supplied by the Sponsor
- Stability under test conditions: The test item is stable up to 14 days at room temperature based on the results of the method validation study. The test diet will be prepared at least once every 8 days.
Species:
rat
Strain:
Wistar
Remarks:
RccHan
Details on species / strain selection:
Rat is the preferred test system because it is a readily available laboratory animal and is historically shown as acceptable animal for the repeated dose toxicity studies and recommended by the OECD and other regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jai Research Foundation
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 12-13 wks
- Range of mean body weight per group on Day 1: (P) Males: 368.59 - 371.29 g; Females: 235.54-237.14 g. At initiation of treatment, the body weight of rats (P) was within ±20% of the mean body weight for each sex.
- Range of mean body weight per group on PND0: (F1) Males: 5.16-5.53 g; Females: 4.82-5.24 g
- Housing: Throughout the experiment period, the male and female rats were housed individually and enrichment material were provided. During the mating period, rats were housed in groups of 2 rats/cage (one male plus one female). Mated female rat were caged individually and nesting material was provided at near parturition. During study, rats were housed in solid floor polypropylene rat cages (size: 41 cm x 28.2 cm x 18 cm). Each cage was fitted with a stainless steel top grill having provision for polypropylene water bottle with stainless steel drinking nozzle. Separate feed hoppers were attached to the cages for diet. The bottom of cages was layered with clean sterilised rice (paddy) husk as the bedding material. Cages were placed on 5/6 tier racks. Cages were changed at least twice a week. Cages were arranged in a such way that possible effects due to cage placement are minimised. Contaminant analysis of samples of the bedding material is being performed half yearly intervals.
- Diet: ad libitum with standard rodent diet.
- Water: unlimited supply of clean and filtered drinking water in polypropylene bottles.
- Acclimation period: A total of 36 male and 36 female rats were selected for acclimatisation, after due consideration. Rats were received into the experimental room and acclimatised for a minimum period of 5 days prior to oestrous cycle evaluation. During acclimatisation period, rats were observed daily, at least once, for clinical signs.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN LIFE DATE:
27 Feb 2018 to 23 May 2018
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
The test item was mixed with the 10% of total diet or 500 g of food (whichever is higher from the total quantity of total diet) in order to prepare the pre-mix. To this pre-mix diet, remaining amount of diet was added and mixed. This whole amount of diet, thus prepared, was mixed in blender and served to animals ad libitum throughout the study.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until mating occurs or 2 weeks have elapsed
- Proof of pregnancy: ‘Day 0’ of pregnancy was defined as the day on which sperms were observed in the vaginal smears of rats.
- After 2 weeks of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged individually and nesting material was provided at near parturition.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The active ingredient concentration and homogeneity of the test item in diet was analysed once before initiation of treatment and twice during treatment period. Triplicate samples from different locations (i.e., top, middle, and bottom of container) of the test diet from each group was taken to determine the homogeneity and concentration of the test item in the test diets. Duplicate samples were collected and one set was sent for analysis and second set were stored at 2-8 °C. Stored samples were used, if re-analysis was required. Every time, the mean concentration will be determined and compared with the nominal value. During the study, the mean percent recovery (MR) and coefficient of variation (CV) obtained for different test diet concentrations given to rats during the treatment period at different intervals are mentioned in Table 1 in "Any other information on results, incl. tables".
Duration of treatment / exposure:
During the treatment period, rats were offered the control or test diets continuously on a 7-days/week basis, till necropsy. Exposure of rats, belonging to both sexes, were initiated at least 2 weeks prior to mating and continued during the mating period. After mating, male rats were further exposed up to and including the day before scheduled sacrifice (until approximately 80% of the females have delivered). Female rats were further exposed during gestation and at least up to post-partum day 13.
Frequency of treatment:
Continuous
Dose / conc.:
150 ppm
Remarks:
Dietary equivalent to 8.59 and 18.78 mg/kg bw/day for males and females, respectively
Dose / conc.:
300 ppm
Remarks:
Dietary equivalent to 17.59 and 33.92 mg/kg bw/day for males and females, respectively
Dose / conc.:
600 ppm
Remarks:
Dietary equivalent to 33.53 and 66.91 mg/kg bw/day for males and females, respectively
No. of animals per sex per dose:
8
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Based on the results of dose range finding study.
- Other: On the postnatal day four, the size of each F1 litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four male and four female pups per litter. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (e.g., six males and two females) was performed. If possible, the two pups/litter were female pups and male pups were reserved for nipple retention observation. Adjustments were not done for litters of eight pups or less.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: daily, at least twice for mortality and morbidity and once for clinical signs
- Observations: mortality and morbidity. Rats, which died or sacrificed during moribund condition in the course of study, were weighed and subjected to post-mortem examination. Female rats, which show marked signs of reaction to treatment or premature delivery, were sacrificed and examined.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily, at least once, throughout the study
-Clinical observations: Visible clinical signs, such as changes in skin, fur, eye, mucous membranes as well as behaviour pattern and abortion.

BODY WEIGHT:
- Time schedule for examinations: Body weight of all male animals was recorded on the first day of treatment and at weekly intervals thereafter. Body weight of all female animals was recorded on the first day of treatment and at weekly intervals during pre-mating and mating periods. During the gestation period, female rats were weighed on GDs 0, 7, 14, and 20. During the lactation period, female animals were weighed within 24 hours of parturition (day '0' post-partum/lactation day), and on post-partum days 4, 7, 14 and on day of terminal sacrifice. Parturition day '0' is defined as the day on which the female littered. On day of fasting, body weights of all surviving rats were recorded. Body weights of all the rats were also recorded on the day of sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as food input (g) - Food leftover (g)
- The food consumption was determined by differentiating the weight of feed input and leftover. Food weights of male rats were measured weekly, at least once, during pre-mating and postmating period. In female rats, during pre-mating period food weights were recorded weekly, at least once. During gestation period, food weights were measured at least on days 0, 7, 14, and 20. During lactation period, food weights were measured at least on lactation days 0, 4, 7, 14 and on day of terminal sacrifice. Additional food were offered as and when required. Food consumption was not measured during mating period. Food spillage/waste may not have been estimated during the period. However, evidence of excessive spillage was recorded in the raw data.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data
Oestrous cyclicity (parental animals):
Vaginal smear were monitored daily from the beginning of the treatment period until evidence of mating. Vaginal smear, from each pregnant animal, were also observed on the day of terminal sacrifice. Care was taken to avoid disturbance of mucosa while obtaining vaginal cells.
Litter observations:
Each litter was examined after delivery to establish the number of pups, sex of pups, stillbirths, live birth, runts, and the presence of gross anomalies. Ano-genital distance (AGD) of each pup was measured on PND 0. Male pups were observed for the retention of nipples/areolae on PND 13. Individual pup body weight was recorded at least on lactation days 0, 4, 7, and 14.
Postmortem examinations (parental animals):
- Surviving rats were sacrificed by using an over dose of carbon dioxide. Gross necropsy was conducted under direct supervision of a veterinary pathologist. Rats were examined carefully for external abnormalities. After opening the abdominal cavity, rats were exsanguinated by cutting the abdominal aorta or posterior vena cava to drain out blood from the rat. Care was taken to avoid any damage to the visceral organs while opening the body cavities. The thoracic and abdominal cavities were cut, open, and a thorough examination of organs were carried out to detect abnormalities. Special attention was paid to organs of the reproductive system. Gross examination of animals were conducted. Male rats were sacrificed after approximately 80% of females have delivered. Female rats were sacrificed on post-partum day 15. Females which did not mate were sacrificed during 24-26 days after the last day of mating period. Females which have not delivered by day 25 post-coitum were sacrificed on post-coitum day 25.
- At the time of sacrifice or death during the study, adult rats were examined macroscopically for any structural abnormalities or pathological changes. Particular attention were paid to the external reproductive genitals which were examined for signs of altered development. Number of implantation sites were recorded for each dam (including rats subjected to moribund sacrifice or found dead). Special attention was given to the organs of the reproductive system.
- The liver, kidney, brain, and spleen were weighed from the surviving parental rats. The testes, epididymis, prostate and seminal vesicles with coagulating glands as a whole, levator ani plus bulbocavernosus muscle complex, Cowper’s glands, and glans penis of all parental male and paired ovaries (wet weight) and uterus (including cervix) in females rats were weighed. The thyroid with parathyroid weight were determined after fixation from all surviving parental rats.
Postmortem examinations (offspring):
Surviving rats including pups of PND 14 were humanely sacrificed by using an over dose of carbon dioxide. Culled pups on PND 4 were sacrificed through intraperitoneal injection of thiopentone sodium. Pups which died during lactation were weighed and subjected to post-mortem examination.
Gross necropsy was conducted under direct supervision of a veterinary pathologist. Rats were examined carefully for external abnormalities. After opening the abdominal cavity, rats were exsanguinated by cutting the abdominal aorta or posterior vena cava to drain out blood from the rat. Care was taken to avoid any damage to the visceral organs while opening the body cavities. The thoracic and abdominal cavities were cut, opened, and a thorough examination of organs was carried out to detect abnormalities. Special attention was paid to organs of the reproductive system. Gross examination of pups was conducted. At the time of sacrifice, pups were examined macroscopically for any structural abnormalities or pathological changes. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development. Pups were sacrificed on post-partum day 14. Particular attention were paid to the external reproductive genitals which were examined for signs of altered development. Special attention was given to the organs of the reproductive system. Thyroid with parathyroid from 1 male and 1 female pup per litter per group were weighed after fixation.
Statistics:
The statistical analysis was carried out for the parameters using validated software developed at JRF. Non-pregnant animals were excluded from statistical analysis. Data such as body weight, body weight gain, food consumption, food efficiency, litter size, organ weight, organ weight ratio, number of implantation, pre-natal loss, post-natal loss, and litter parameters [male pups, female pups, total pups (male + female) counts and pups weight] were analysed using Bartlett's test of homogeneity of variance. When the result was not significant then an analysis of variance (ANOVA) was carried out. When the ANOVA was significant then a Dunnett's multiple comparison test was carried out. When the data did not meet the homogeneity of variance then a t-test was performed to calculate significance at 95 and 99% confidence levels. AGD was normalised (the ratio of AGD to the cube root of body weight) and then subjected for statistical analysis. Non-parametric data such as mortality rate, gestation index, parturition index, pregnancy rate, pup survival index, live birth index and fertility index were analysed using a Chi-Square test. Flags for significant difference between control and treated groups (single arrow for p<0.05 and double arrows for p<0.01) were given in the tables along with the footnote.
Reproductive indices:
In detail all indices are displayed in the field 'Any other information on materials and methods, inlc. tables'.
Offspring viability indices:
In detail all indices are displayed in the field 'Any other information on materials and methods, inlc. tables'.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight gain of male rats, belonging to the 600 ppm dose group, was statistically significant decreased during treatment days 1-8, 29-36, and 1-43, when compared with that of the control group. The mean body weight gain of female rats, belonging to the 600 ppm dose group, was also statistically significant decreased during pre-mating days 1-8 and 1-15, when compared with that of the control group. The mean body weight gain of female rats, belonging to the 600 ppm dose group, was also statistically significant decreased during Lactation Day 4-7 and 0-14, when compared with that of the control group. The mean body weight of female rats, belonging to the 600 ppm dose group, was statistically significant decreased on Lactation Day 14, when compared with that of the control group. These decreases in mean body weight and body weight gain could be due to a decrease in mean food consumption of rats belonging to the 600 ppm dose groups. These effects could be considered as treatment related adverse effects of the test item.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- The mean food consumption of male rats, belonging to the 600 ppm dose group, were statistically significant decreased during the treatment days 1-8, 28-36, 1-43, and 1-8, 1-43, respectively, when compared with that of the control group. The mean food consumption of male rats, belonging to the 300 ppm dose group, were statistically significant decreased during the treatment days 28-36, when compared with that of the control group. The mean food consumption of male rats, belonging to the 150 ppm dose group, were statistically significant decreased during the treatment days 1-8, 28-36, and 1-43, when compared with that of the control group. The mean food consumption of female rats, belonging to the 600 ppm dose group, were statistically significant decreased during the premating days 1-8, 8-15, 1-15, and 1-8 and 1-15, respectively, when compared with that of the control group. The mean food consumption of female rats, belonging to the 600 ppm dose group, were statistically significant decreased during the Lactation Days 4-7 and 0-14, and 4-7, 7-14, 0-14, respectively, when compared with that of the control group. The mean food consumption of female rats, belonging to the 300 ppm dose group, were statistically significant decreased during the Lactation Days 4-7, when compared with that of the control group. The mean food consumption of female rats, belonging to the 150 ppm dose group, were statistically significant decreased during the pre-mating days 1-8, when compared with that of the control group. These decreases in mean food consumption of male and female rats, belonging to the 600 ppm dose groups, could be due to an adverse effect of the test item on the liver. An occasional decrease in mean food consumption without significant effect on body weight and body weight gain of male and female rats, belonging to the 150 and 300 ppm dose groups, could be considered as an incidental incidence without any toxicological relevance.
- The overall mean test item intake of male rats, belonging to the 0, 150, 300, and 600 ppm dose groups was 0, 8.59, 17.59, 33.53 mg/kg b. wt./day, respectively. The overall mean test item intake of female rats during pre-mating, belonging to the 0, 150, 300, and 600 ppm dose groups was 0, 10.86, 22.46, 39.85 mg/kg b. wt./day, respectively. The overall mean test item intake of female rats during gestation, belonging to the 0, 150, 300, and 600 ppm dose groups was 0, 10.47, 20.96, 42.19 mg/kg b. wt./day, respectively. The overall mean test item intake of female rats during lactation, belonging to the 0, 150, 300, and 600 ppm dose groups was 0, 18.78, 33.92, 66.91 mg/kg b. wt./day, respectively.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
The mean food efficiency of male rats, belonging to the 600 ppm dose group, were statistically significant decreased during the treatment days 1-8, 28-36, 1-43, and 1-8, 1-43, respectively, when compared with that of the control group. The mean food efficiency of female rats, belonging to the 600 ppm dose group, were statistically significant decreased during the premating days 1-8, 8-15, 1-15, and 1-8 and 1-15, respectively, when compared with that of the control group. The mean food efficiency of female rats, belonging to the 600 ppm dose group, were statistically significant decreased during the Lactation Days 4-7 and 0-14, and 4-7, 7-14, 0-14, respectively, when compared with that of the control group. These decreases in mean food efficiency of male and female rats, belonging to the 600 ppm dose groups, could be due to an adverse effect of the test item on the liver. An occasional decrease in mean food efficiency without significant effect on body weight and body weight gain of male and female rats, belonging to the 150 and 300 ppm dose groups, could be considered as an incidental incidence without any toxicological relevance.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Mean estrous cycle length and pattern of estrous cycle were comparable with the control group.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related differences were observed in sex ratio and mean counts of male pups, female pups, and pups of both sexes combined when compared with that of the control group.
The live birth index of female pups and pups of both sexes combined, belonging to the 300 ppm dose group, was statistically significant decreased, when compared with that of the control group. However, no dose dependency was observed. Therefore this could be considered incidental without toxicological relevance.
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
150 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
Remarks on result:
other:
Remarks:
Dietary equivalent to 8.59 and 18.78 mg/kg bw/day for males and females, respectively
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
> 600 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Dietary equivalent to 33.53 and 66.91 mg/kg bw/day for males and females, respectively
Critical effects observed:
yes
Lowest effective dose / conc.:
300 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
All pups belonging to control and test item treated groups were normal throughout the study period.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The mortality of male pups, belonging to the 600 ppm dose group, were statistically significant increased during the PNDs 0-4, when compared with that of the control group. The mortality of male pups and pups of both sexes combined, belonging to the 300 ppm dose group, were statistically significant increased during the PNDs 0-4, when compared with that of the control group. The mortality without significant effect on body weight (PNDs 0 and 4) and body weight gain (PNDs 0-4) of pups, belonging to the 600 ppm dose group, could be considered as an incidental finding without any toxicological relevance. The mortality of pups, belonging to the 300 ppm dose group, was attributed to two litters in which all pups were cannibalised, and was not considered as treatment related.
The survival index of male pups, belonging to the 600 ppm dose group, were statistically significant decreased on PND 4, when compared with that of the control group. The survival index of male pups and pups of both sexes combined, belonging to the 300 ppm dose group, were statistically significant decreased on PND 4, when compared with that of the control group. The decrease in survival index without significant effect on body weight and body weight gain of pups, belonging to the 600 ppm dose group, could be considered as incidental without any toxicological relevance. The decreased survival index of pups, belonging to the 300 ppm dose group, was attributed to two litters in which all pups were cannibalised, and was not considered as treatment related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight of male pups, female pups, and pups of both sexes combined, belonging to the 600 ppm dose group, was statistically significant decreased on PND 14, when compared with that of the control group. The mean body weight gain of male pups, female pups, and pups of both sexes combined, belonging to the 600 ppm dose group, was statistically significant decreased on PNDs 7-14, when compared with that of the control group. The mean body weight gain of female pups and pups of both sexes combined, belonging to the 600 ppm dose group, was statistically significant decreased on PNDs 4R-7, when compared with that of the control group. These decreases in mean pups body weight and body weight gain could be due to a decrease in mean food consumption and food efficiency of female rats of the 600 ppm dose group which subsequently might not produce sufficient milk for the pups. This effect could be considered as a treatment related adverse effect of the test item on pups growth.
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
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Ano-genital distance measured on PND 0 of the test item treated groups was comparable with that of the control group.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
None of the male pups belonging to either control or the test item treatment groups showed retention of nipples.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease was observed in mean terminal body weight of male and females pups, belonging to the 600 ppm dose group. The effect was considered as a treatment related effect of the test item. A statistically significant increase in relative weights of the thyroid with parathyroid was observed in male and female pups, belonging to the 600 ppm dose group. This effect without statistical significance was also observed in absolute weights and was considered as a treatement related effect of the test item. See results in Table 3 in "Any other information on results, incl. tables'.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
External and internal examination of pups (found dead and terminally sacrificed on PNDs 4 and 14) did not reveal any abnormality. One found dead pup was autolysed on internal examination and the milk band was absent in all the found dead pups.
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEL
Remarks:
Developmental Toxicity
Generation:
F1
Effect level:
300 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
other:
Remarks:
Dietary equivalent to 17.59 and 33.92 mg/kg bw/day for males and females, respectively
Critical effects observed:
yes
Lowest effective dose / conc.:
600 ppm
System:
endocrine system
Organ:
parathyroid gland
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Reproductive effects observed:
no

Table 1. Homogeneity and Active Ingredient Concentration Analysis of the Test Item in the Diet

Group N°

G1

G2

G3

G4

Dose (ppm)

0

150

300

600

Interval

MR
CA)

% CV

MR (%)

% CV

MR (%)

% CV

MR (%)

% CV

Before treatment

-

-

99.98

2.95

96.68

4.10

105.54

0.15

During treatment

-

-

84.09

1.56

97.84

1.93

88.18

1.89

During treatment

-

-

85.61

4.15

81.20

0.67

96.00

0.07

 Key: - = Not applicable, MR = Mean recovery, % = Percent, CV = Coefficient of variation

The mean percent recovery obtained for the test diet was within the acceptance level of ±20% of the nominal concentration demonstrating that the exposure concentrations were as intended by the study plan and the %CV was less than 20, suggesting that the test diet were homogeneously mixed.

Table 2. Liver weight result.

Sex

Liver

Groups

% increase/decrease
than control

G1

G2

G3

G4

G2

G3

G4

Male

Absolute weight

(g) SD)

(Mean ±

12.090 ±

1.622

12.493 ±

0.653

13.958 ±

0.88411'

14.840 ±

0.93211'

3

15

23

Relative weight (g) (Mean ± SD)

2.862 ±

0.297

2.991 ±

0.141

3.335 ±

0.23311'

3.683 ±

0.26711'

5

17

29

 Key: 1r= significantly higher than control (1)0.01)

PND 14 Pups:

Table 3: Terminal body weights and thyroid with parathyroid (relative and absolute)

Sex

Organ

Groups (Mean ± SD)

% increase/ decrease than control

G1

G2

G3

G4

G2

G3

G4

 

 

24.800 ±

23.375 ±

22.383 ±

18.283 ±

 

 

 

 

B. wt. (TS)

 

 

 

 

-6

-10

-26

 

 

1.698

4.335

4.868

2.282 J.

 

 

 

 

Thyroid with

 

 

 

 

 

 

 

 

 

0.0283 ±

0.0295 ±

0.0319 ±

0.0453 ±

 

 

 

 

parathyroid-

 

 

 

 

4

13

60

M

relative

0.0036

0.0129

0.0095

0.0118T

 

 

 

 

Thyroid with

 

 

 

 

 

 

 

 

 

0.0070 ±

0.0066 ±

0.0071 ±

0.0082 ±

 

 

 

 

parathyroid-

 

 

 

 

-6

1

17

 

absolute

0.0009

0.0022

0.0023

0.0016

 

 

 

 

 

23.667 ±

23.563 ±

21.200 ±

18.843 ±

 

 

 

 

B. wt. (TS)

 

 

 

 

0

-10

-20

 

 

2.884

2.170

3.278

3.637

 

 

 

 

Thyroid with

 

 

 

 

 

 

 

 

 

0.0276 ±

0.0284 ±

0.0276 ±

0.0481 ±

 

 

 

 

parathyroid-

 

 

 

 

3

0

74

F

relative

0.0069

0.0072

0.0068

0.0153 T T

 

 

 

 

Thyroid with

 

 

 

 

 

 

 

 

 

0.0065 ±

0.0066 ±

0.0058 ±

0.0087 ±

 

 

 

 

parathyroid-

 

 

 

 

2

-11

34

 

absolute

0.0017

0.0013

0.0013

0.0020

 

 

 

 

Keys: M = Male, F = Female, T = significantly higher than control (p0.05), J. = significantly lower than control (p0.05) T T= significantly higher than control (p0.01)

A statistically significant decrease in terminal body weights (20 to 26 % decrease compared to control) was noted in male and female pups of the high dose group. The effect was related to treatment of the test item.

A statistically significant increase in relative weights of thyroid with parathyroid (60 to 74 % increase compared to control) was noted in pups of the high dose group of both sexes. The effect without statistical significance was also noted in absolute weights (17 to 34% increase compared to control) and was considered as related to test item treatment.

Conclusions:
- Based on the results of the present study, the following dose levels are suggested for the extended one-generation reproductive toxicity study (cohort-1) of the test item in Wistar rats: 75, 150, and 300 ppm.
- NOAEL systemic toxicity: 150 ppm, based on the gross pathology abd greyish discoloration of the liver, both sexes (Dietary equivalent to 8.59 and 18.78 mg/kg bw/day for males and females, respectively)
- NOAEL reproductive toxicity: >600ppm, based on no adverse toxic effect observed, both sexes (Dietary equivalent to 33.53 and 66.91 mg/kg bw/day for males and females, respectively)
- NOAEL developmental toxicity: 300 ppm, based on the effect on the thyroid and parathyroid gland weight and the body weight ratios, both sexes (Dietary equivalent to 17.59 and 33.92 mg/kg bw/day for males and females, respectively)
Executive summary:

The study was conducted under GLP conditions to determine the initial information on toxic characteristic, systemic and reproduction/developmental toxicity, occurring as a result of repeated daily dietary administration of the test item during pre-mating, mating, gestation, and lactation period of rats to select the dose levels for definitive study. A total of 36 male and 36 female Wistar rats were randomly divided into four groups (8 rats/sex/group). Test item was given through diet, in graduated doses of 150, 300, and 600 ppm, to three groups of male and female rats. The control group received basal diet. Male rats were treated for 43 days. Female rats were treated for two weeks prior to mating, variable time to conception, the duration of pregnancy and 14 days after delivery. The active ingredient concentration and homogeneity of the test item in diet were analysed once before the initiation of treatment and twice during the treatment period. Oestrous cycle length and pattern of female rats were evaluated by vaginal smears during pre-treatment of two weeks and daily from the beginning of the treatment period until evidence of mating. Rats were observed daily, twice, for mortality and morbidity, and once for visible clinical signs throughout the study period. Body weight of rats was recorded on the first day of treatment and the day of sacrifice. Body weight of male rats were recorded weekly throughout the treatment period. Body weights of female rats were recorded weekly during pre-mating and mating periods. Pregnant females were weighed on gestation days (GDs) 0, 7, 14, 20, within 24 hours of parturition, and post-coitum on day 25. Body weights of dams and pups were recorded on lactation days 0, 4, 7, and 14. Food consumption and test item intake was calculated during the treatment period. Pups were observed for sex, stillbirths, live birth, runts, gross anomalies, and measured for ano-genital distance (AGD) on post-natal day (PND) 0. Each litter was standardised on PND 4 to obtain nearly 4 male and 4 female pups. Rats were terminally sacrificed by carbon dioxide asphyxiation and subjected to gross pathological examination. Absolute organ weights were recorded and relative organ weights were calculated.

The results of diet formulation analyses were within the acceptable range of ±20% of the nominal concentration and the %CV was less than 20 which suggests that the prepared diet had an acceptable a.i. concentration and it was homogeneously prepared. Parents: No morality, morbidity, and clinical signs of toxicity were observed in male and female rats up to the 600 ppm dose level. The mean body weight gain, food consumption, and food efficiency of male rats, belonging to the 600 ppm dose group, was statistically significant decreased during treatment, when compared with that of the control group. The mean terminal body weight of female rats, belonging to the 600 ppm dose group, was statistically significant decreased, when compared with that of the control group. The mean body weight gain, food consumption, and food efficiency of female rats, belonging to the 600 ppm dose group, was statistically significant decreased during the pre-mating and lactation period, when compared with that of the control group. These decreases in mean body weight gain, food consumption, and food efficiency of male and female rats, belonging to the 600 ppm dose groups, could be due to an adverse effect of the test item on the liver. Occational decreases in mean food consumption and food efficiency without significant effect on body weight and body weight gain of male and female rats, belonging to the 150 and 300 ppm dose groups, could be considered as an incidental incidence without any toxicological relevance. External examination of terminally sacrificed male and female rats, belonging to the 0, 150, 300, and 600 ppm dose groups did not reveal any abnormality. Greyish discolouration of liver of male and female rats, belonging to the 300 and 600 ppm dose groups, was observed during necropsy. Treatment related increases in mean absolute and relative liver weights of male rats, belonging to the 300 and 600 ppm dose groups was observed. Pups: Pup mortality was observed in litters, belonging to the 300 and 600 ppm dose groups. However, the body weight and body weight gain of pups, belonging to the 600 ppm dose group was comparable. Therefore, this could be considered as an incidental change. The mortality of pups, belonging to the 300 ppm dose group, was attributed to two litters in which all pups were cannibalised and was not considered as treatment related. Pups mortality was also reflected in live birth and survival indices. The mean body weight and body weight gain of pups, belonging to the 600 ppm dose group, was statistically significant decreased during PNDs 7-14, when compared with that of the control group. This could be due to a decrease in mean food consumption and food efficiency of female rats of the 600 ppm dose group which might not produce sufficient milk for the pups. No treatment related differences were observed in litter size, sex ratio, AGD, and nipple retention of the pups. External and internal examination of the pups did not reveal any abnormality. A statistically significant increase in relative weights of thyroid with parathyroid was observed in male and female pups, belonging to the 600 ppm dose group.

Based on the results of the present study, the following dose levels are suggested for the extended one-generation reproductive toxicity study (cohort-1) of the test item in Wistar rats: 75, 150, and 300 ppm. The following NOAELs were determined based on the study:

- NOAEL systemic toxicity: 150 ppm, based on the gross pathology abd greyish discoloration of the liver, both sexes (Dietary equivalent to 8.59 and 18.78 mg/kg bw/day for males and females, respectively)

- NOAEL reproductive toxicity: >600ppm, based on no adverse toxic effect observed, both sexes (Dietary equivalent to 33.53 and 66.91 mg/kg bw/day for males and females, respectively)

- NOAEL developmental toxicity: 300 ppm, based on the effect on the thyroid and parathyroid gland weight and the body weight ratios,  both sexes (Dietary equivalent to 17.59 and 33.92 mg/kg bw/day for males and females, respectively)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals : The male and female rats of the parental (P) generation were treated for at least ten weeks prior to mating. In this specific case ten weeks exposure duration is supported by the lipophilicity of the substance to ensure that the steady state in parental animals has been reached before mating.
- Basis for dose level selection : The dose levels were selected based on results of a dose range finding study (496-1-04-19024).
- Inclusion of extension of Cohort 1B, because the uses of the registered substance is leading to significant exposure of professionals and consumers, and there are indications that the internal dose for the registered substance will reach a steady state in the test animals only after an extended exposure.
- Termination time for F2 : F2 pups were sacrificed on postnatal day (PND) 21
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B, no triggers for the inclusion were identified.
- Exclusion of developmental immunotoxicity Cohort 3 , no triggers for the inclusion were identified.
- Route of administration : Oral, through diet, Since the substance to be tested is a crystalline solid, ECHA concludes that testing should be performed by the oral route.
- Other considerations, on choice of species: The rat is the preferred test system because it is a readily available laboratory animal. The rat has been historically shown to be an acceptable animal for reproduction toxicity testing and is recommended by the OECD and other regulatory authorities.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Purity: 98%
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0000091655
- Expiration date of the lot/batch: 31 August 2018, The test item was expired during the experimental phase of the study as per the declared expiry date on the CoA (Certificate of Analysis) provided by the Sponsor. To establish the stability of the test item, the active ingredient content was analysed concurrently with the dosing period i.e., within one week before declared expiry and within one week after completion of dosing following the validated analytical method. The CoA and comparison of analysed purity are reported in the final report. Analysis of the active ingredient demonstrated that the content of the test item was within experimental variation of previously established purity of 98.00% and that the purity of the test item was unchanged.
- Purity: 98% (analysed purity); 97.83% (Verification of purity before dosing); 97.79% (Verification of purity after dosing)
- Appearance: White to off-white solid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: As per the instruction received from the Sponsor on storage of the test item, the test item was stored: Storage Temperature : Room Temperature; Storage Container : In original container as supplied by the Sponsor
- Stability under test conditions: The test item is stable up to 14 days at room temperature based on the results of the method validation study.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan:WIST
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jai Research Foundation
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 5-6 weeks
- Housing: During the experiment period, rats were housed in groups of 2/3 rats/cage/sex and enrichment material was provided. During the mating period, rats were housed in groups of two rats/cage (one male and one female). Mated female rats were housed individually and nesting material were provided near parturition. During the study, rats were housed in solid floor polypropylene rat cages (size: 41 cm x 28.2 cm x 18 cm). Each cage was fitted with a stainless-steel top grille having provision for a polypropylene water bottle with stainless steel drinking nozzle. Separate food hoppers were attached to the cages for diet. The bottom of the cages was layered with clean sterilised rice (paddy) husk as the bedding material. Cages were placed on 5 and 6 tier racks. Cages were changed at a minimum of twice a week. Cages were arranged in such way that possible effects due to cage placement are minimised. Contaminant analysis (chemical and microbial) of samples of the bedding material (paddy husk) were performed at six months intervals and the most recent results were included in the final report.
- Diet: The experimental rats were fed ad libitum with standard rodent diet. The standard pellet food was powdered (using a grinder) before offering to animals or mixing with the test item. Every food consignment received was accompanied with a certificate of analysis of nutrient content from the food supplier. The most recent results of microbial contaminant analyses were included in the final report.
- Water: unlimited supply of clean and filtered drinking water (filtered through reverse osmosis water filtration system) in polypropylene bottles. The most recent results of microbial and chemical contaminant analyses were included in the final report.
- Acclimation period: 6 days (prior to commencement of randomisation).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 57-67
- Air changes (per hr): 16-17
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet: test diet was prepared at least once a week.
- Mixing appropriate amounts with powder diet: The test item was mixed with the 10% of total diet or 500 g of food (whichever was higher from the total quantity of diet prepared) in a blender in order to prepare the pre-mix. To this pre-mix diet, the remaining amount of diet was added and mixed. This whole amount of diet, thus prepared, was mixed in a blender and made available to the animals ad libitum throughout the study.
- Storage temperature of food: The prepared test diet was stored in labelled stainless steel containers covered inside with polythene bags, at the experimental room condition.
Details on mating procedure:
- M/F ratio per cage: 1:1 pairing (unrelated, same dose group)
- Length of cohabitation: until evidence of copulation was observed or 2 weeks had elapsed
- Proof of pregnancy: presence of sperm or copulatory plug referred to as day 0 of pregnancy;
- If mating had not occurred after 2 weeks, the rats were separated without further opportunity for mating and considered as a presumed mated.
- After successful mating each pregnant female was caged: individually
- To produce the F2 generation, 1 male and 1 female rat from the F1 (Cohort 1B) generation were randomly selected and subsequently mated (14-15 weeks of age) in a 1:1 ratio. Care was taken to avoid sibling mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The active ingredient (a.i.) concentration and homogeneity of the test item in the test diet was analysed once before initiation of treatment and at one-month intervals during the treatment period. Duplicate samples of control from middle and triplicate samples from different locations (i.e., top, middle and bottom of the container) of the test diet from each group were taken to determine the homogeneity and concentration of the test item in the treated diet. On each occasion of test diet analysis, the mean concentration was determined and compared to the nominal value. The acceptance criteria were ± 20% deviation from nominal value and %CV < 20. The samples were analysed using a validated analytical method. Following instrumental parameters will be used for analyses: Instrument : HPLC; Column : 250 mm x 4.6 mm (i.d), 5 µm particle size; Wavelength (nm) : 210; Injection volume (µL) : 20; Flow rate (mL/minute) : 1.0; Mobile phase : Solvent (A) - Acetonitrile (80%), Solvent (B) - 0.1 % Orthophosphoric acid in Milli-Q Water (20%)
Duration of treatment / exposure:
- Parental (P) and F1 (selected for Cohort 1A and 1B) rats were offered the control or test diet continuously on a 7-days/week basis, till the scheduled necropsy. Exposure to the test item to both P sexes were commenced ten weeks prior to mating and continued during the two-weeks mating period. After mating, P male rats were further exposed, up to, and including the day before scheduled sacrifice (i.e. till delivery of P females). P female rats were further exposed during gestation and up to weaning of F1 pups. Unless already initiated during the lactation period, direct treatment of the selected F1 male and female pups began at weaning and continue, until the scheduled necropsy.
- Cohort 1B rats was maintained on treatment beyond PND 90 and bred to obtain a F2 generation. Male rats and female rats were 14-15 weeks old at the time of mating. Procedure of mating was the same as described for the P animals. F2 generation pups were evaluated up to PND 21.
Frequency of treatment:
Continuous
Details on study schedule:
- F1 parental animals not mated until Postnatal day 90 after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age (PND 22).
- Age at mating of the mated animals in the study: 14-15 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
50 ppm (analytical)
Remarks:
G2: Low Dose; P Generation, F1 Generation (Cohort 1A) and F1 Generation (Cohort 1B)
Dose / conc.:
100 ppm (analytical)
Remarks:
G3: Mid Dose; P Generation, F1 Generation (Cohort 1A) and F1 Generation (Cohort 1B)
Dose / conc.:
300 ppm (analytical)
Remarks:
G4: High Dose; P Generation, F1 Generation (Cohort 1A) and F1 Generation (Cohort 1B)
No. of animals per sex per dose:
P: 25 rats/sex/group
F1 (Cohort 1A/1B): 20 rats/sex/group
Control animals:
yes, plain diet
Details on study design:
- Dose Justification: The dose levels were selected based on results of a dose range finding study (496-1-04-19024)
- Selection of Pups for Post-Weaning Evaluations on Post-natal Day: A) Cohort 1A: One male and one female F1 pup per litter (20 pups/sex/group; wherever possible) were randomly assigned for primary assessment of effects on reproductive systems and general toxicity. , B) Cohort 1B: One male and one female F1 pup per litter (20 pups/sex/group; wherever possible) were randomly assigned for follow-up assessment of reproductive performance by mating F1 rats.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
Rats were observed daily, twice, for mortality and morbidity. All visible clinical signs were noted and recorded daily, once, throughout the study.

DETAILED CLINICAL OBSERVATIONS:
Detailed clinical examination of all P and F1 rats (after weaning) was performed on a weekly basis, on the day of the body weight recording. Signs noted included, but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture, response to handling, as well as the presence of the clonic or tonic movements, stereotypy (e.g., excessive grooming, repetitive circling) or bizarre behaviour (e.g., self-mutilation, walking backwards) were also recorded.

BODY WEIGHT:
Body weight of P male rats was recorded on the first day of treatment and weekly thereafter. Body weight of P female rats was recorded on the first day of treatment and weekly thereafter during the pre-mating and mating period. Body weight of P female rats was recorded on gestation day 0, 7, 14 and 20, and lactation days 0, 4, 7, 14, and 21.
On the day of fasting, body weight of respective rats was recorded. Body weight of all rats was also recorded on the day of sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The food consumption was determined by weighing of food input and leftover. Food weight of all P male rats was measured weekly during pre-mating and post-mating period. In P female rats, during pre-mating period, food weight was recorded, at weekly intervals. During gestation period, food weight was recorded, on gestation days 0, 7, 14, and 20. During lactation period, food weight was recorded, on lactation days 0, 4, 7, 14, and 21. Additional food was offered as and when required. Food consumption was not measured during mating period. After weaning, the cage wise food weight was recorded at weekly interval, till necropsy.
Compound intake was calculated as time-weighted averages from the consumption and body weight gain data.

BLOOD AND URINE COLLECTION
At the time of the terminal sacrifice, blood (maximum 3.5 mL) was collected from P and F1 (Cohort 1A) rats (except female rats sacrificed on GD 25) under anaesthesia (isoflurane) by orbital plexus puncture. Rats were deprived of food overnight (water provided), prior to the blood collection. Blood samples were collected for haematology (in vials containing 4% EDTA), coagulation parameters PT and APTT (in vials containing 3.2% sodium citrate), clinical chemistry, and thyroid hormone analysis (in vials without anticoagulant). Samples were collected before 13:00 hours per day. Serum samples were preserved at -70 ± 10 °C, till their analysis. Any residual/retained serum samples will be discarded after confirmation for the finalisation of the study report.
Urine samples were collected overnight from 10 rats/sex/group (P and cohort 1A) in rat metabolic cages using graduated collecting tubes.

CLINICAL PATHOLOGY OBSERVATIONS
The haematology, clinical chemistry, and urine parameters as described in Tables 2, 3 and 4 in 'Any other information on materials and methods, incl. tabels' were analysed from 10 rats/sex/group (from P and Cohort 1A).

THYROID HORMONE ANALYSIS
Serum thyroid hormones T3, T4, and TSH were analysed. Level of T3 and T4 in serum was analysed using a validated bioanalytical method. Level of TSH in serum was analysed through ELISA methods provided in the kit literature. Serum thyroid hormones T3, T4, and TSH levels were analysed as specified in Tables 5 and 7 in 'Any other information on materials and methods, incl. tables'.

LYMPHOCYTE SUBPOPULATION ANALYSIS
From randomly selected 10 F1 rats (cohort 1A)/group/sex, spleen was cut half (transverse section) and used for lymphocyte (phenotypic) subpopulation analysis. From the collected spleen, homogenate was prepared and used for T lymphocytes (CD4+ and CD8+), B lymphocyte, and natural killer cell evaluation using flow cytometer. The other half part of the spleen was preserved for the histopathological examination.
Oestrous cyclicity (parental animals):
Oestrous cycle length and pattern was evaluated by the vaginal smears for all P female rats for two weeks period, prior to the mating and throughout the cohabitation/mating period. It was ensured to avoid disturbance of mucosa while obtaining vaginal smear.
Vaginal smear was taken for F1 (Cohort 1A) female pups since the day of vaginal opening till the first oestrus phase was observed. Oestrous cycle length and pattern was evaluated for, two weeks period prior to the terminal sacrifice by vaginal smears for all F1 female rats belonging to the cohort 1A. Vaginal smear was evaluated in cohort 1B since the time of pairing until the mating evidence was observed.
At the time of necropsy, the vaginal smear was examined (from P and F1 rats) to determine the stage of the oestrous cycle.
Sperm parameters (parental animals):
SPERM PARAMETERS
Sperm count was performed in the control and high dose male rats belonging to P and F1 (cohort 1A) generation. Immediately after the sacrifice, right cauda epididymis, and right testis were excised, weighed, and stored at -20 ± 3 °C for enumeration of cauda epididymal sperm reserves and homogenisation - resistant spermatids, respectively. Vas deferens was used for the assessment of sperm motility (performed in all-male rats of P and F1 generation - cohort 1A) and sperm morphology (performed in control and high dose male rats belonging to P and F1 generation – cohort 1A).
Sample of right cauda epididymis and right testis belonging to the low and mid-dose groups will be discarded at the time of report finalisation.

SPERM MOTILITY
Immediately after sacrifice, right vas deferens was placed in a petri dish containing 3 mL of Dulbecco's Phosphate Buffered Saline (maintained at 36-38 °C) on a slide warming table. The vas deferens was pressed from approximately the mid-point with gentle pressure to allow the sperm ‘swim out”. After completion of this step, the mixture of the sperm and medium was pipetted (one drop) directly onto a pre-warmed slide for motility assessment under a light microscope at 40x magnification. Motility assessment of the sperm was determined based on the movement of the sperm and respective motility grades were assigned.

SPERM MORPHOLOGY
After the assessment of sperm motility, about 1 mL of sperm suspension was transferred to a test tube (10 x 75 mm) and diluted to 4 mL with Dulbecco's Phosphate Buffered Saline, followed by an addition of 1 drop of 5% eosin Y. The contents of the test tube were gently agitated by “flicking” the bottom of the test tube with a fingertip. These samples were then incubated at the room temperature for approximately 30 minutes to allow staining. At the end of the staining period, sperm was re-suspended gently with a pasture pipette. Using pasture pipette, 1 or 2 drops of stained solution was spread uniformly on the glass slide. Two slides were prepared for each rat. These slides were placed at an angle of 35 - 40° on a tray whose surface was covered with absorbent tissue paper to remove excess stained suspension. These slides were air-dried and mounted with DPX. 200 sperms/slide were evaluated for morphological abnormalities from the control and high dose rats belonging to P and F1 generation (cohort 1A).

SPERM COUNT
Frozen right cauda epididymis and testis were removed from the freezer and maintained at the room temperature for approximately 10 to 15 minutes for thawing. A known volume of 15 mL saline merthiolate-triton (SMT) for cauda epididymis and 25 mL SMT for testis was used. The tissue was homogenised for 2 to 3 minutes and allowed the froth to dissipate for a few minutes. The samples were loaded on the chamber of the haemocytometer using a micropipette. The sperms were left undisturbed for approximately 5 minutes, to settle down, prior to their count. When the mean count of the five squares in the first chamber varied by more than 15% on either side of the mean value, the second chamber was loaded, and sperm were counted in a similar manner to that of the first chamber. In this case, the final mean value was calculated from all squares counted. The number of sperm head per gram of tissue was calculated.
Litter observations:
STANDARDISATION OF LITTERS
On postnatal day (PND) four, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, five male pups and five female pups per litter. Wherever the number of male pups or female pups prevents having five of each sex per litter, partial adjustment (e.g., six male and four female) was performed. Adjustments were not done for litters of ten pups or less.

PUPS OBSERVATIONS
Each litter was examined as soon as possible, after parturition to establish the number and sex of pups, stillbirths, runts, live birth, and the presence of gross anomalies. Found dead pups, on the day of littering, which were not macerated were examined for possible defects and cause of death. Pups which died during study were weighed and subjected to the post-mortem examination. Each pup was observed for the presence of milk band in the stomach from PND 0 to 4, as a part of the nursing care.
In addition, all live pups on day of delivery were observed for the first clinical examination of the pups which included a qualitative assessment of the body temperature, state of activity, and reaction to handling.

BODY WEIGHT
Live pups (F1 and F2) were counted and weighed individually at birth or soon thereafter and on postnatal day 4, 7, 14, 21, and 22.

ANOGENITAL DISTANCE (AGD)
The anogenital distance (AGD) of each pup (F1 and F2) was measured on PND 0.

NIPPLE RETENTION
On PND 13, nipples/areolae in male pups was observed. As no nipple presence was observed, hence counting was not performed.

PHYSICAL AND SEXUAL DEVELOPMENT LANDMARKS
The day for unfolding of pinna, ear-opening, eye-opening, tooth eruption, and hair growth were recorded on their respective day of occurrence.
The age at the vaginal opening or balano-preputial separation was determined for the F1 weanlings selected for the subsequent evaluations. F1 pups were also weighed on the day of the vaginal opening or balano-preputial separation attended.

BLOOD AND URINE COLLECTION
Blood was collected from F1 surplus pups on PND 4 and F1 weanling pups on PND 22, through decapitation and orbital plexus puncture, respectively for the serum thyroid hormone analysis. Blood was collected from the minimum 1 randomly selected male and female pup per litter, wherever feasible. To increase the sample volume, blood from the additional pups was also collected in some dams where pups were available.

THYROID HORMONE ANALYSIS
Serum thyroid hormones T3, T4, and TSH were analysed. Level of T3 and T4 in serum was analysed using a validated bioanalytical method. Level of TSH in serum was analysed through ELISA methods provided in the kit literature. Serum thyroid hormones T3, T4, and TSH levels were analysed as specified in Table 6 in 'Any other information on materials and methods, incl. tables'.
Postmortem examinations (parental animals):
SACRIFICE
Schedule for sacrifice: P male rats were sacrificed after the delivery of P female rats. P female rats were sacrificed on lactation day 22. Cohort 1A rats were sacrificed during the 14-weeks of the treatment period. Cohort 1B male rats were sacrificed, following during the 21 weeks of the treatment period. Cohort 1B female rats were sacrificed on lactation day 22. Females, which have not delivered by post-coitum day 25, were sacrificed on the same day. Female rats with no live pups were kept fasting on the same day when the observation was made and were sacrificed on the next day.

GROSS NECROPSY
Surviving rats (including weanling pups) were sacrificed, by using an overdose of carbon dioxide. Gross pathological observations were made for all adult rats (P and F1). Gross necropsy was conducted under the direct supervision of a veterinary pathologist. Rats were examined carefully for the external abnormalities. After opening the abdominal cavity, rats were exsanguinated by cutting the abdominal aorta or posterior vena cava to drain out blood from the rat. It was ensured to avoid any damage to the visceral organs while opening the body cavities. The thoracic and abdominal cavities were cut, opened, and a thorough examination of the organs was carried out to detect abnormalities. Special attention was paid to the organs of the reproductive systems during the gross examination of all rats. The uteri of all cohabited female rats were examined for the presence and number of implantation sites. Number of corpora lutea were recorded from those female rats in which implants were observed on the gestation day 25.

ORGAN WEIGHTS
On the day of the terminal sacrifice, wet weight of the below-mentioned organs (see Table 1 in 'Any other information on materials and methods incl. tables') from all adult rats (P and F1) were determined as soon as possible after dissection to avoid drying. Paired organs were weighed individually except four rats of GD 25. The organs were weighed and preserved in 10% NBF except testes and eyes in a modified Davidson’s fixatives and Davidson’s fixatives, respectively.

HISTOPATHOLOGY
Histopathology of the organs was performed on all high dose and control rats. Organs (liver – target organ and thyroid) demonstrating treatment-related changes were examined in the low and mid-dose groups from Parent and cohort 1A rats.
Parent animals: Histopathology was performed for the organs listed in Table 1 in 'Any other information on materials and methods incl. tables'. Additionally, histopathology of the reproductive organs of rats suspected of reduced fertility was performed in dams sacrificed on GD 25 and their respective male rats and also in dams in which oestrous cycle was affected. Gross lesions were subjected to histopathological evaluation.

F1 (Cohort 1A) animals: Histopathology was performed for the organs listed in Table 1 in 'Any other information on materials and methods incl. tables'. Quantitative evaluation of primordial and small growing follicles, as well as corpora lutea (from 10 rats of each group G1 and G4), oviduct, uterus, and vagina were examined for appropriate organ-typic development. Detailed testicular histopathology examinations were conducted on the F1 male rats in order to identify treatment-related effects on testes differentiation and development and on spermatogenesis. Periodic Acid Schiff (PAS) and hematoxylin and eosin staining were used for examination of the testes. Caput, corpus, and cauda of the epididymis and the vas deferens were examined for appropriate organ-typic development.

F1 (Cohort 1B) animals: Organs/tissues of cohort 1B rats were processed to the block stage.
Postmortem examinations (offspring):
SACRIFICE
Schedule for sacrifice: Unselected weanlings of F1 were sacrificed on PND 22. F2 pups were sacrificed on the PND 21.

GROSS NECROPSY
Surviving rats (including weanling pups) were sacrificed, by using an overdose of carbon dioxide. Culled pups on PND 4 (not subjected for hormone analysis) were sacrificed, through intraperitoneal administration of thiopentone sodium. Gross pathological observations were made for all adult rats (P and F1) and all pups (F1 and F2). Gross necropsy was conducted under the direct supervision of a veterinary pathologist. Rats were examined carefully for the external abnormalities. After opening the abdominal cavity, rats were exsanguinated by cutting the abdominal aorta or posterior vena cava to drain out blood from the rat. It was ensured to avoid any damage to the visceral organs while opening the body cavities. The thoracic and abdominal cavities were cut, opened, and a thorough examination of the organs was carried out to detect abnormalities. Special attention was paid to the organs of the reproductive systems during the gross examination of all rats including pups.
Pups, which were found dead on the day of delivery (i.e., PND 0), were subjected to a gross examination and a portion of the lung was immersed in water for confirmation of the status (stillbirth or dead). Pups which were found dead were observed for the presence of milk band.
Pups, which died before scheduled sacrifice (during PND 0 to 21) were examined, for gross lesions and the cause of death or condition, as soon as possible, after the observation is made. In case of a pup with gross lesions (PND 0 to 21), the whole pup was kept in Bouin’s fluid. Pups without gross lesions were discarded, after examination.

ORGAN WEIGTHS
See Table 1 in 'Any other information on materials and methods incl. tables'.

HISTOPATHOLOGY
Microscopic examination of liver (target organ) from F1 and F2 pups was carried out from control and all dose group rats (10 pups/sex/group).
Statistics:
The statistical analysis was carried out for the parameters using validated statistical software. Non-pregnant rats were excluded from analysis. Assumed pregnant rats were excluded from analysis of gestation phase parameters. Pups data (litter size, AGD, pups body weight, and physical development landmark) was evaluated using the litter as the unit for data analysis. All parametric data were analysed to calculate the significance at 95% and 99% whereas non-parametric data was analysed to calculate significance at 95%.
Data such as body weight (gain), food consumption, food efficiency, physical & sexual development landmark, organ weight (ratio), % pre-natal loss, % post-natal loss, male sex ratio, oestrous cycle length, sperm count, % sperm morphology, haematology, clinical chemistry, urinalysis, thyroid hormone, and litter parameters (pups body weight (gain), body temperature, and AGD) were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. When the data did not meet the homogeneity of variance, statistical analysis was extended following a decision tree as provided by Gad, S.C., 2007. AGD was normalised (the ratio of AGD to the cube root of body weight) and then subjected for statistical analysis.
Count data (viz., litter size, number of implants, pre-coital interval, duration of gestation, number of oestrous cycles; pre-natal loss, post-natal loss) were subjected to non-parametric test i.e., Kruskal-Wallis test. The non-parametric data (sperm morphology - number and ovarian follicular count) was analysed using the Mann-Whitney test.
Non-parametric indices such as mating, fertility, gestation, parturition, pregnancy rate, survival, mortality, live birth and lactation were analysed using a Chi-Square test.
Flags for significant difference between control and treated groups (single arrow for p≤0.05 and double arrows for p≤0.01) were given in the table along with the footnote.
Reproductive indices:
Mating index = Number of mated animals / Number of paired animals x 100
Male fertility index = Number of males impregnating female / Number of males used for cohabitation x 100
Female fertility index = Number of females with confirmed mating / Number of female cohabited x 100
Gestation index = Number of females with live-born pups / Total N° of female with evidence of mating x 100
Sperm count = (Mean number of sperm head counted x Square factor x Haemocytometer factor x Dilution factor) / Tissue (Testis or Epididymis) weight x 100
Offspring viability indices:
Post-implantation loss = (Number of implants - Number of viable pups) / Number of implants x 100
Post-natal loss = Number of live pups born – pups alive at postnatal day 4
Pup survival index = (N° of live pups on lactation day 0 or 4 or 7 or 14 or 21) / (N° of pups born or N° of pups retained on lactation day 4) x 100
Live birth index = Number of live-born pups / Number of delivered pups x 100
Male sex ratio = Number of male pups on lactation day 0 / (Total number of males + female pups on lactation day 0) x 100
Lactation index = Number of pups alive on lactation day 21 / Number of pups retained on lactation Day 4 x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
All rats belonging to the control and the test item treated groups were normal, throughout the study period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality and morbidity were observed during the study period in the control and the test item treated groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
BODY WEIGHT
MALE
The mean body weight of male rats of the test item treated groups was comparable with that of the control group.

FEMALE
- Pre-mating: The mean body weight of female rats of the test item treated groups was comparable with that of the control group.
- Gestation: The mean body weight of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower during gestation on days 0, 7, 14, and 20 when compared with that of the control group.
- Lactation: The mean body weight of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower during lactation on days 0, 4, 7, 14, and 21 when compared with that of the control group.
This lower mean body weight during gestation and lactation days could be due to a lower mean food consumption of female rats, belonging to the 300 ppm dose group. This effect could be considered as a treatment-related adverse effect of the test item.

BODY WEIGHT GAIN
MALE
The mean body weight gain of male rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 43-50 and 64-71 of treatment when compared with that of the control group.
These occasional lower mean body weight gain without significant effect on food consumption could be considered incidental and without any toxicological relevance.

FEMALE
- Pre-mating: The mean body weight gain of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 15-22, 43-50, and 50-57 of treatment when compared with that of the control group. The mean body weight gain of female rats, belonging to the 100 ppm dose group, was statistically and significantly lower over days 29-36 of treatment when compared with that of the control group.
- Gestation: The mean body weight gain of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 0-7 of gestation when compared with that of the control group.
- Lactation: The mean body weight gain of female rats, belonging to the 50 ppm dose group, was statistically and significantly lower over days 4-7 of lactation when compared with that of the control group.
This decrease in the mean body weight gain during the pre-mating and gestation days could be due to a lower mean food consumption of female rats, belonging to the 300 ppm dose group. This effect could be considered as a treatment-related adverse effects of the test item. However, an occasional lower mean body weight gain without a significant effect on the food consumption and dose dependency in the 50 and 100 ppm dose groups, could be considered incidental, and without any toxicological relevance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
FOOD CONSUMPTION
MALE
The mean food consumption of male rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 85-92 and 92-TS (Terminal Sacrifice) of treatment when compared with that of the control group. These occasional lower mean food consumptions without a significant effect on body weight could be considered incidental, and without any toxicological relevance.

FEMALE
- Pre-mating: The mean food consumption of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 15-22, 29-36, 43-50, 50-57, 57-64, 64-71, and 1-71 of treatment when compared with that of the control group. The mean food consumption of female rats, belonging to the 100 ppm dose group, was statistically and significantly lower over days 15-22 and 43-50 of treatment when compared with that of the control group. The mean food consumption of female rats, belonging to the 50 ppm dose group, was statistically and significantly lower over days 15-22, 43-50, and 50-57of treatment when compared with that of the control group.
- Gestation: The mean food consumption of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 0-7, 7-14, 14-20, and 0-20 of gestation when compared with that of the control group. The mean food consumption of female rats, belonging to the 50 ppm dose group, was statistically and significantly lower over days 7-14 of gestation when compared with that of the control group.
- Lactation:
The mean food consumption of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 4-7 of lactation when compared with that of the control group. The mean food consumption of female rats, belonging to the 50 ppm dose group, was statistically and significantly lower over days 4-7 of lactation when compared with that of the control group.
The lower mean food consumption during the pre-mating, gestation, and lactation days of female rats belonging to the 300 ppm dose group, could be considered as a treatment-related adverse effect of the test item. However, an occasionally lower food consumption without significant effect on the mean body weight and dose dependency in the 50 and 100 ppm dose groups, could be considered incidental, and without any toxicological relevance.

TEST ITEM INTAKE
Test item intake, with respect to the dose level, was consistent throughout the treatment period (i.e., the test item intake for G3 was approximately twice that of G2, and the same for G4 was approximately thrice of G3). Overall test item intake is given in Table 1 in 'Any other information on results, incl. tables'.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
MALE
The mean food efficiency of male rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 43-50 and 64-71 of treatment when compared with that of the control group.

FEMALE
- Pre-mating: The mean food efficiency of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 50-57 treatment when compared with that of the control group. The mean food efficiency of female rats, belonging to the 100 ppm dose group, was statistically and significantly lower over days 29-36 of treatment when compared with that of the control group. These occasional lower in the mean food efficiency without a significant effect on the body weight could be considered incidental, and without any toxicological relevance.
- Gestation and Lactation: The mean food efficiency of female rats of the test item treated groups was comparable with that of the control group.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant lower haematocrit value was noted in the males of the 300 ppm dose group. This effect was considered to be related to the test item treatment and considered a mild effect as the reduction was less than 6% when compared to the control group.
A statistically significant higher value was observed in the reticulocyte count of males of the 300 ppm dose group, which was considered related to the lower haematocrit value, hence was considered related to the test item treatment.
A statistically significant higher value in the reticulocyte count of males of the 50 and 100 ppm dose groups was not considered related to the treatment as a relevant effect was not observed in any of the red cell mass parameters (RBC, haemoglobin or haematocrit).
A statistically significant lower MCV was noted in females of the 50 ppm dose group. This alteration was not related to treatment in the absence of dose dependency.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant lower globulin level in males (and apparent decrease in females) of the 300 ppm dose group caused a significantly higher albumin: globulin ratio. It was considered to be related to the test item treatment.
A statistically significant lower value was observed for triglycerides of females of the 300 ppm dose group. Statistically significant higher values were noted for ALT and AST of females of the 300 ppm dose group. These effects were considered to be related to the test item treatment and more specifically to be related to alterations in the liver.
Statistically significant higher values were noted for urea, BUN, and potassium of females of the 300 ppm dose group. These alterations were not considered to be related to test item treatment as it lacked consistency between generation/sexes and support from histopathology. Statistically significant lower values were observed for bile acids of males and total bilirubin and creatinine of females of the 300 ppm dose group which were considered as toxicologically insignificant.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis did not reveal any treatment-related effect. Count/values of treated groups were comparable with those of the control group.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examination revealed treatment-related lesions in the liver (hepatocyte hypertrophy) and in the thyroid (follicular cell hypertrophy).
Hepatocyte hypertrophy in liver and follicular cell hypertrophy in thyroid were observed with higher number of incidences in 300 ppm dose rats of both sexes. Both lesions were also present in 100 ppm dose rats of both sexes with marked lower incidence than that of the 300 ppm dose. Incidences noted in 50 ppm in male rats were comparable to that of the control. In female rats at 50 ppm incidences were significantly lower compared to that of 300 ppm. Lesions observed in liver were well comparable with increased weight of the liver.
Histopathology of the reproductive organs of female rats suspected of reduced fertility, their respective male rats and a dam in which the oestrous cycle was affected, did not reveal lesions definitive for the cause.
Microscopic lesions observed in other organs in male and female rats were at a lower rate of occurrence with almost comparable incidence amongst the control and the 300 ppm dose. Further, these lesions were mostly minimal to mild in nature, non-specific, insignificant, and correlated to the respective gross finding. Hence, these lesions were spontaneous or incidental and not related to the test item treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
THYROID HORMONES
Serum T3, T4, and TSH levels of male and female rats were comparable amongst the control and the test item treated groups.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The mean oestrous cycle length, number of normal oestrous cycle, and pre-coital interval prior to mating were comparable with that of the control group.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm Motility: Sperm motility of male rats was comparable with that of the control group.
Sperm Count: Cauda epididymis sperm and testicular spermatid counts were comparable amongst the control and the high dose groups.
Sperm Morphology: No test item treatment-related change was observed in sperm morphology (number and percent) amongst the control and the high dose groups.
Reproductive performance:
no effects observed
Description (incidence and severity):
Male fertility index, female fertility index, gestation index, parturition index, percentage of pregnant rats, and mating index were comparable between the control and the test item treated groups. The duration of the gestation was comparable amongst the control and the test item treated groups.

PRE AND POSTNATAL DATA
No test item related effect on mean number of implants was observed when compared with that of the control group. A statistically significant lower mean percentage prenatal loss was observed in the 300 ppm dose group when compared with that of the control group. The lower mean percentage prenatal loss could be considered as an incident and of no toxicological importance.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
SYSTEMIC
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: Dietary equivalent to 7.44 and 11.83 mg/kg bw/day, for males and females, respectively
Key result
Dose descriptor:
NOAEL
Remarks:
FERTILTY
Effect level:
>= 300 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Dietary equivalent to 22.11 and 27.43 mg/kg bw/day, for males and females, respectively.
Key result
Dose descriptor:
NOAEL
Remarks:
REPRODUCTION
Effect level:
>= 300 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Dietary equivalent for 22.11 and 25.46 mg/kg bw/day, for mailes and females, respectively.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Description (incidence and severity):
COHORT 1B:
All rats (except the one found dead) belonging to the control and the test item treated groups were normal throughout the study period.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
COHORT 1B:
A female belonging to the 50 ppm dose group was found dead on gestation day 10. All remaining rats belonging to the control and the test item treated groups were normal throughout the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
COHORT 1B:
BODY WEIGHT
MALE
The mean body weight of male rats, belonging to the 300 ppm dose group, was statistically and significantly lower during treatment days 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85 when compared with that of the control group.  

FEMALE
- Pre-mating: The mean body weight of female rats, belonging to the 300 ppm dose group, was statistically significantly lower throughout the treatment period when compared with that of the control group.
- Gestation: The mean body weight of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower during gestation day, 0, 7, 14, and 20 when compared with that of the control group.
- Lactation: The mean body weight of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower during lactation day, 0, 4, 7, 14, and 21, when compared with that of the control group. This lower mean body weight of male and female rats could be due to a decrease in mean food consumption of rats belonging to the 300 ppm dose group. This effect could be considered as a treatment-related adverse effects of the test item.

BODY WEIGHT GAIN
MALE
The mean body weight gain of male rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 1-8, 8-15, 15-22, 22-29, 36-43, and 106-113 of treatment, when compared with that of the control group. The mean body weight gain of male rats, belonging to the 300 ppm dose group, was statistically significantly higher over days 50-57, 71-78, and 92-99 of treatment, when compared with that of the control group. The mean body weight gain of male rats, belonging to the 100 ppm dose group, was statistically significantly higher over days 50-57, 71-78, and 78-85 of treatment, when compared with that of the control group. The mean body weight gain of male rats, belonging to the 50 ppm dose group, was statistically and significantly lower over days 15-22 and 36-43 of treatment when compared with that of the control group. The mean body weight gain of male rats, belonging to the 50 ppm dose group, was statistically significantly higher over days 71-78, 78-85, and 127-134 of treatment, when compared with that of the control group.

FEMALE
- Pre-mating: The mean body weight gain of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower during treatment days 1-8, 8-15, and 1-78 when compared with that of the control group. The mean body weight gain of female rats, belonging to the 50 ppm dose group, was statistically and significantly lower during treatment days 8-15 when compared with that of the control group.
- Gestation: The mean body weight gain of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 0-7, 14-20, and 0-20 of gestation when compared with that of the control group.
The mean body weight gain of female rats, belonging to the 100 ppm dose group, was statistically and significantly lower over days 0-7 of gestation when compared with that of the control group.
This lower mean body weight gain during pre-mating and gestation days could be due to a decrease in mean food consumption of female rats, belonging to the 300 ppm dose group. This effect could be considered to be a treatment-related adverse effect of the test item. However, an occasionally lower mean body weight gain without a significant effect on food consumption and dose dependency in the 50 and 100 ppm dose groups, could be considered to be incidental and without any toxicological relevance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
COHORT 1B:
FOOD CONSUMPTION
Male
The mean food consumption of male rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 1-8, 8-15, 15-22, 22-29, 29-36, 36-43, and 43-50 of treatment when compared with that of the control group.
The mean food consumption of male rats, belonging to the 100 ppm dose group, was statistically significantly higher over days 92-99 of treatment when compared with that of the control group. It could be considered incidental and without any toxicological relevance.
This lower mean food consumption during treatment days of male rats, belonging to the 300 ppm dose group, could be considered as a treatment-related adverse effects of the test item.

FEMALE
- Pre-mating: The mean food consumption of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 1-8, 8-15, 15-22, 22-29, and 1-78 of treatment when compared with that of the control group.
The mean food consumption of female rats, belonging to the 100 ppm dose group, was statistically and significantly lower over days 8-15 of treatment when compared with that of the control group.
- Gestation: The mean food consumption of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 7-14 of gestation when compared with that of the control group.
- Lactation: The mean food consumption of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 7-14 of lactation when compared with that of the control group.
This lower mean food consumption during pre-mating, gestation and lactation days of female rats, belonging to the 300 ppm dose group, could be considered to be a treatment-related adverse effect of the test item.

TEST ITEM INTAKE
Test item intake, with respect to the dose level, was consistent throughout the treatment period (i.e., the test item intake for G3 was approximately twice that of G2, and the same for G4 was approximately thrice of G3).
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
COHORT 1B:
MALE
The mean food efficiency of male rats, belonging to the 300 ppm dose group, was statistically and significantly lower during treatment days 8-15 and 106-113 when compared with that of the control group. The mean food efficiency of male rats, belonging to the 300 ppm dose group, was statistically significant higher during treatment days 50-57, 71-78, and 92-99, when compared with that of the control group. The mean food efficiency of male rats, belonging to the 100 ppm dose group, was statistically significant higher during treatment days 50-57 when compared with that of the control group. The mean food efficiency of male rats, belonging to the 50 ppm dose group, was statistically and significantly lower during treatment days 8-15 when compared with that of the control group. The mean food efficiency of male rats, belonging to the 50 ppm dose group, was statistically significant higher during treatment days 71-78 when compared with that of the control group.

FEMALE
- Pre-mating: The mean food efficiency of female rats, belonging to the 300 ppm dose group, was statistically significant higher during treatment days 15-22 when compared with that of the control group. The mean food efficiency of female rats, belonging to the 50 ppm dose group, was statistically and significantly lower during treatment days 8-15 when compared with that of the control group.
- Gestation: The mean food efficiency of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower during gestation days 0-7 when compared with that of the control group. The mean food efficiency of female rats, belonging to the 100 ppm dose group, was statistically and significantly lower during gestation days 0-7 when compared with that of the control group.
- Lactation: The mean food efficiency of female rats was comparable with that of the control group. This lower mean food efficiency during pre-mating and gestation days of rats, belonging to the 300 ppm dose group, is correlated with lower food consumption and considered as a treatment-related adverse effects of the test item. However, an occasional decrease in mean food efficiency without significant effect on body weight gain and dose dependency in the 50 and 100 ppm dose groups, could be considered incidental and without any toxicological relevance.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
COHORT 1B:
Statistically significant lower terminal body weight was noted in female rats. Statistically significant higher relative liver weight was noted in male and female rats of the 300 ppm dose, while higher absolute liver weight was also observed in the 300 ppm dose males. Moreover, three female rats of the 300 ppm dose group had values above historical range for relative weight of liver, while none of the control rat had values above historical range (Female: 3.382 to 5.097%, central 95 percentile, n = 138). These findings were considered related to the test item treatment as similar effects were observed in parental and cohort 1A rats.
Statistically significant higher relative weights were noted in the thyroid of female rats of the 300 ppm dose which was not considered related to the test item treatment as values of female rats of the 300 ppm dose group were below upper limit of historical range (0.0051 to 0.0110%, central 95 percentile, n = 138).
Statistically significant lower values were observed in absolute weights of left epididymis (males) and pituitary (females) of rats of the 300 ppm dose, while lower values were noted in absolute and relative weights of uterus of females of the 300 ppm dose. These changes were considered unrelated to the test item treatment due to lack of consistency between sexes and lack of similar effect in other generation (parent and cohort 1A).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
COHORT 1B:
Macroscopic examination revealed treatment-related lesions in liver (greyish discolouration).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
COHORT 1B:
PRE-COITAL INTERVAL
The pre-coital interval was comparable with that of the control group.

FERTILITY
Male fertility index, female fertility index, gestation index, parturition index, percentage of pregnant rats, and mating index were comparable between the control and the test item treated groups. The duration of the gestation days was comparable among all the test item treated groups with that of the control group.

PRE AND POSTNATAL DATA:
A statistically significant lower mean number of implants was observed in the 50 and 300 ppm dose groups when compared with that of the control group. However, pre- and postnatal data of cohort 1B female rats were comparable with that of the control group. The lower mean number of implants did not show a dose dependency. Histopathology of uterus and ovarian follicular counts of cohort 1A females, belonging to 50 and 300 ppm dose groups, were also comparable with that of the control group. Therefore, it could be considered as incidental and without toxicological relevance.

Effect levels (P1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
COHORT 1B (SYSTEMIC)
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
Remarks on result:
other: Dietary equivalent to 10.11 and 11.94 mg/kg bw/day, for males and females, respectively.
Key result
Dose descriptor:
NOAEL
Remarks:
COHORT 1B (FERTILITY)
Effect level:
>= 300 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Dietary equivalent to 30.05 and 25.14 mg/kg bw/day, for males and females, respectively.
Key result
Dose descriptor:
NOAEL
Remarks:
COHORT 1B (REPRODUCTION)
Effect level:
>= 300 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Dietary equivalent to 30.05 and 26.34 mg/kg bw/day, for males and females, respectively.

Target system / organ toxicity (P1)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
F1 PUPS: Effects observed, non-treatment-related: No treatment-related clinical signs were observed in all pups. However, weakness was occasionally observed in following F1 pups.

COHORT 1A: No effects observed: All rats belonging to the control and the test item treated groups were normal throughout the study period.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
F1 PUPS: Mortality observed, non-treatment-related: During the lactation period, no treatment-related mortality was observed in the test item treated groups.
The mortality index of male pups, belonging to the 100 ppm dose group, was statistically and significantly lower on the postnatal day 7, when compared with that of the control group. It was reflected as significantly higher survival of male pups on the postnatal day 7. This effect could be considered incidental to treatment and without any toxicological relevance.

COHORT 1A: No mortality observed: No mortality and morbidity were observed during the study period in the control and the test item treated groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F1 PUPS: Effects observed, treatment-related:
BODY WEIGHT
MALE PUPS
The mean body weight of male pups, belonging to the 300 ppm dose group, was statistically and significantly lower on postnatal day 0 when compared with that of the control group.

FEMALE PUPS
The mean body weight of female pups, belonging to the 300 ppm dose group, was statistically and significantly lower on postnatal days 14 and 21 when compared with that of the control group.

COMPOSITE OF MALE AND FEMALE PUPS
The mean body weight of pups, belonging to the 300 ppm dose group, was statistically and significantly lower on postnatal day 0 when compared with that of the control group. These lower body weights could be due to the lower gestation body weight of pregnant rats and considered as a growth retardation effect of the test item on developing pups.

BODY WEIGHT GAIN
MALE PUPS
The mean body weight gain of male pups, belonging to the 300 ppm dose group, was statistically and significantly lower during postnatal days 7-14 when compared with that of the control group.

FEMALE PUPS
The mean body weight gain of female pups, belonging to the 300 ppm dose group, was statistically and significantly lower during postnatal days 7-14 and 14-21 when compared with that of the control group.

COMPOSITE OF MALE AND FEMALE PUPS
The mean body weight gain of pups, belonging to the 300 ppm dose group, was statistically and significantly lower during postnatal days 7-14 and 14-21 (without statistical significance) when compared with that of the control group. This lower mean body weight gain of pups, belonging to the 300 ppm dose group toward the end of the lactation period could be due to decreased milk production of lactating females and direct exposure of pups to the test item through diet. Therefore, it could be considered as an adverse effect of the test item on developing pups.

COHORT 1A: Effects observed, treatment-related:
BODY WEIGHT
MALE
The mean body weight of male rats, belonging to the 300 ppm dose group, was statistically significant lower throughout the treatment period when compared with that of the control group. The mean body weight of male rats, belonging to the 50 ppm dose group, was statistically and significantly lower during treatment days 71 and TS when compared with that of the control group.

FEMALE
The mean body weight of female rats, belonging to the 300 ppm dose group, was statistically significant lower throughout the treatment period when compared with that of the control group.

This lower mean body weight of male and female rats during the treatment period could be due to a lower mean food consumption of male and female rats, belonging to the 300 ppm dose group. This effect could be considered to be a treatment-related adverse effect of the test item. However, an occasionally lower mean body weight of male rats without significant effect on food consumption and dose dependency in the 50 ppm dose group, could be considered incidental and without any toxicological relevance.

BODY WEIGHT GAIN
MALE
The mean body weight gain of male rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 1-8, 8-15, 22-29, 50-57, 64-71, and 1-TS of treatment when compared with that of the control group. The mean body weight gain of male rats, belonging to the 300 ppm dose group, was statistically significant higher over days 71-TS of treatment, when compared with that of the control group. The mean body weight gain of male rats, belonging to the 100 ppm dose group, was statistically and significantly lower over days 29-36 and 57-64 of treatment when compared with that of the control group. The mean body weight gain of male rats, belonging to the 50 ppm dose group, was statistically and significantly lower over days 22-29, 36-43, 57-64, and 1-TS of treatment when compared with that of the control group.

FEMALE
The mean body weight gain of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 8-15, 64-TS, and 1-TS of treatment when compared with that of the control group. The mean body weight gain of female rats, belonging to the 300 ppm dose group, was statistically significantly higher over days 50-57 of treatment, when compared with that of the control group. The mean body weight gain of female rats, belonging to the 50 ppm dose group, was statistically and significantly lower over days 43-50 of treatment when compared with that of the control group.

These lower mean body weight gains of male and female rats during the treatment period could be due to a lower mean food consumption of male and female rats, belonging to the 300 ppm dose group. This effect could be considered to be a treatment-related adverse effect of the test item. However, an occasionally lower mean body weight of rats without a significant effect on food consumption and dose dependency in the 50 and 100 ppm dose groups, could be considered incidental and without any toxicological relevance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
F1 PUPS: Not examined

COHORT 1A: Effects observed, treatment-related
FOOD CONSUMPTION
MALE
The mean food consumption of male rats, belonging to the 300 ppm dose group, was statistically and significantly lower during treatment days 1-8, 8-15, 15-22, and 22-29 when compared with that of the control group.

FEMALE
The mean food consumption of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower during treatment days 1-8, 8-15, and 1-TS when compared with that of the control group. This lower mean food consumption during treatment days of male and female rats could be considered a treatment-related adverse effect of the test item.

TEST ITEM INTAKE
Test item intake, with respect to the dose level, was consistent throughout the treatment period (i.e., the test item intake for G3 was approximately twice that of G2, and the same for G4 was approximately thrice of G3).
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
F1 PUPS: Not examined

COHORT 1A: Effects observed, non-treatment-related
MALE
The mean food efficiency of male rats, belonging to the 300 ppm dose group, was statistically significantly higher during treatment days 50-57 when compared with that of the control group.
The mean food efficiency of male rats, belonging to the 100 ppm dose group, was statistically significant higher during treatment days 50-57 and lower during treatment days 29-36, 57-64, when compared with that of the control group.
The mean food efficiency of male rats, belonging to the 50 ppm dose group, was statistically and significantly lower during treatment days 15-22, 36-43, and 64-71 when compared with that of the control group.

FEMALE
The mean food efficiency of female rats, belonging to the 300 ppm dose group, was statistically significant higher during treatment days 50-57 and lower during treatment days 64-TS when compared with that of the control group. The mean food efficiency of female rats, belonging to the 50 ppm dose group, was statistically and significantly lower during treatment days, 8-15 and 43-50 when compared with that of the control group.

These occasional higher and lower mean food efficiencies without dose dependency could be considered incidental and without any toxicological relevance.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
F1 PUPS: Not examined

COHORT 1A: Effects observed, treatment-related: Statistically significant lower values were noted for haematocrit, RBC, and haemoglobin of males of the 300 ppm dose. These effects were considered to be related to the test item treatment and considered a mild effect as the decrease compared to control was below 6%. A statistically significant higher value was observed for PT of males of the 100 and 300 ppm doses, and WBC and lymphocyte of females of the 300 ppm dose which were not considered to be an effect of the test item treatment due to inconsistencies between sexes and/or generations. A statistically significant lower MCV was noted in females of the 50 and 300 ppm dose groups, and APTT of females of the 50 and 100 ppm dose groups. These alterations were not related to treatment in the absence of dose dependency.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
F1 PUPS: Not examined

COHORT 1A: Effects observed, treatment-related: Statistically significant lower globulin was noted in male and female rats of the 300 ppm dose. This effect lead to a statistically significant higher albumin: globulin ratio of male and female rats of the 300 ppm dose group. The effect was also observed in males of the 100 ppm dose. It was considered to be related to the test item treatment. A statistically significant higher albumin: globulin ratio of female rats of the 100 ppm group was observed whereas no changes were observed in globulin or albumin level. Statistically significant lower total cholesterol was observed in male and female rats of the 300 ppm dose group. These effects were considered to be related to the test item treatment and more specifically to be related to alterations in the liver.
Urinalysis findings:
no effects observed
Description (incidence and severity):
F1 PUPS: Not examined

COHORT 1A: No effects observed: Urinalysis did not reveal any treatment-related effect. Count/values of treated groups were comparable with that of the control group.
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
F1 PUPS: Effects observed, treatment-related
MALE
The mean balano-preputial separation (BPS) of male pups (cohort 1A and 1B), belonging to the 300 ppm dose group, was statistically and significantly increased when compared with that of the control group. The mean balano-preputial separation of male pups (cohort 1B), belonging to the 50 and 100 ppm dose groups, was statistically and significantly increased when compared with that of the control group. The mean body weight of male pups (cohort 1A and 1B) on the day of balano-preputial separation was comparable between the control and the test item treated groups. This increase in balano-preputial separation in the 300 ppm dose group could be due to a lower preweaning body weight. This effect could be considered a treatment-related growth retardation effect of the test item on developing pups. The mean balano-preputial separation of male pups (cohort 1B), belonging to the 50 and 100 ppm dose groups, were within historical control data range (44.27 to 49.05) of the performing laboratory. Therefore, it was considered to be a biological variation without any toxicological importance.

FEMALE
Vaginal opening of female pups was comparable between the control and the test item treated groups. The mean body weight on the day vaginal opening of female pups (cohort 1A and 1B), belonging to the 300 ppm dose groups, was statistically and significantly decreased when compared with that of the control group. It could be considered a growth retardation effect of the test item on developing pups.
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
F1 PUPS: Effects observed, non-treatment--related: The mean anogenital distance of female pups, belonging to the 300 ppm dose group, was statistically and significantly increased on postnatal day 0 when compared with that of the control group. However, anogenital distance of F2 pups was comparable with that of the control group. Therefore, this effect could be considered incidental to treatment and without any toxicological relevance.
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
F1 PUPS: Effects observed, treatment-related: Statistically significant lower values were noted for terminal body weight of male and female pups. A statistically significant lower value was noted for the absolute weight of the liver in 300 ppm dose females. This effect was also noted in 300 ppm dosed males and although it did not reach statistical significance in males, it was considered related to treatment as it was supported by histopathology. Statistically significant higher values were noted in relative weights of the brain in 300 ppm dosed male and female pups, which was caused by a lower body weight. The lower value noted for the absolute weight of the brain in 300 ppm dosed females was considered not related to treatment.

COHORT 1A: Effects observed, treatment-related: Statistically significant lower terminal body weights were noted in male and female rats. Statistically significant higher relative liver weight was noted in the male and female rats of the 300 ppm dose. Moreover, six male rats of the 300 ppm dose group had values above historical range for relative weight of liver, while none of the control male rat had values above historical range (Male: 2.409 to 3.541 %, central 95 percentile, n = 155; Female: 3.382 to 5.097%, central 95 percentile, n = 138). The effect was considered related to the test item treatment and was supported by hypertrophy observed with histopathology. Statistically significant higher relative weights were also noted in the thyroid of males of the all three dose levels. Values of male rats of the 300 ppm dose group were below upper limit of historical range, while only 1 male rat each of low dose and mid dose level had values above historical range (0.0033 to 0.0077 %, central 95 percentile, n = 155). Hence, effect was not related to the test item treatment. Higher relative weight of the kidneys was noted in males of the 300 ppm dose. Effects on relative kidney weights were likely to be due to a decrease in body weight.
A statistically significant reduction was observed in the absolute weight of the heart and spleen of females of the 300 ppm dose, which could be owed to lower body weight. A statistically significant lower value was noted in the absolute weight of the brain of males and an increase was observed in the relative weight of the thymus of females of the 300 ppm. These effects were not related to the test item treatment as it lacked histopathological lesions. Statistically significant lower values were noted in terminal body weight, absolute weights of liver, heart, epididymis (left), and epididymis cauda (right) of males of the 50 ppm dose, which was unrelated to treatment in the absence of a dose dependency. Similarly, statistically significant lower values noted in whole epididymis and cauda (left) of males of the 300 ppm dose were not related to treatment in the absence of consistency between the two sides. Statistically significant lower values observed in absolute weight of the adrenals of male rats of the 50 ppm dose (left and right), 300 ppm dose (left) and 50 ppm dose (right) were not associated with the test item treatment as it lacked a dose-dependency/consistency between sexes and histopathological examination did not reveal any changes.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
F1 PUPS: Effects observed, non-treatment-related: Macroscopic examination of F1 weanling pups did not reveal any pathologic lesions. One of the pups which was found dead in the 100 ppm dose group revealed neck oedema upon gross examination.

COHORT 1A: Effects observed, treatment-related: Macroscopic examination revealed treatment-related lesions in the liver (greyish discolouration).
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
F1 PUPS: Effects observed, treatment-related: Glycogen depletion in the liver was observed in the 300 ppm dose group pups. It could be due to nutritional insufficiency and it was considered as an effect of the test item.

COHORT 1A: Effects observed, treatment-related: Histopathological examination revealed treatment-related lesions in the liver (hepatocyte hypertrophy) and in the thyroid (follicular cell hypertrophy). Hepatocyte hypertrophy in liver and follicular cell hypertrophy in thyroid were observed with higher number of incidences in the 300 ppm dose rats of both sexes. Incidences noted in the 50 and 100 ppm dose were almost comparable to that of the control except for a higher number of incidences of liver hypertrophy found in male rats of the 100 ppm dose group. Lesions observed in liver were well comparable with increased weight of the liver.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
F1 PUPS: Effects observed, treatment-related

LIVE BIRTH, SURVIVAL, AND LACTATION INDICES
Live birth, survival, and lactation indices were comparable amongst the control group and the test item treated groups. Survival index of male pups, belonging to the 100 ppm dose group, was statistically significant higher on the postnatal day 7, when compared with that of the control group. It was reflected as significantly lower mortality of male pups on the postnatal day 7. This effect could be considered incidental and without any toxicological relevance.

LITTER SIZE AND MALE SEX RATIO
Litter size and male sex ratio of F1 pups were comparable with that of the control group.

BODY TEMPERATURE
The mean body temperature of male, female, and a combination of both sexes’ pups, belonging to the 300 ppm dose group, was statistically significantly lower on postnatal day 0 when compared with that of the control group. This lower body temperature could be due to a decrease in mean body weight of the pups on postnatal day 0. This effect could be considered a treatment-related adverse effect of the test item on developing pups.

PHYSICAL DEVELOPMENT LANDMARK
Physical development landmark of male and female pups was comparable between the control and the test item treated groups.

THYROID HORMONES
Serum T3 level of female rat pups (F1) on PND 22, belonging to the 100 ppm dose group, was statistically and significantly higher when compared with that of the control group. However, serum T3 level of male rat pups (F1) on PND 22 was comparable with that of the control group. The changes observed at 100 ppm lack dose dependency, hence, they are considered as incidental in nature. Serum T4 and TSH levels of male and female pups (F1) on PND 22 were comparable between the control and the test item treated groups. Serum T4 and T3 levels of pups (F1) on PND 4 were comparable between the control and the test item treated groups.

COHORT 1A: Effects observed, treatment-related
OESTROUS CYCLE
The mean oestrous cycle length of female rats was comparable between the control and the test item treated groups.

SPERM PARAMETERS
- Sperm motility: Sperm motility of male rats was comparable with that of the control group.
- Sperm Count: Cauda epididymis sperm and testicular spermatid counts were comparable between the control and the high dose groups.
- Sperm Morphology: No test item treatment-related changes were observed in sperm morphology (number and percentage) between the control and the high dose groups.

SPLENIC LYMPHOCYTE SUBPOPULATION
Splenic lymphocyte subpopulation of male and female rats was comparable with that of the control group.

THYROID HORMONES
Serum TSH and T4 levels of cohort 1A female rats, belonging to the 300 ppm dose group, were statistically significantly higher when compared with that of the control group. Increased serum thyroid levels of female rats could be due to hypertrophy of the thyroid.

OVARIAN FOLLICULAR QUANTITATION
The total number and ovarian follicles of different types i.e. primordial, growing and antral follicles in the 300 ppm dose group of F1 generation females were found comparable with that of the control group.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
FETAL TOXICITY
Generation:
F1
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
histopathology: non-neoplastic
other: lower body temperature
Remarks on result:
other: Dietary equivalent to 7.44 and 13.1 mg/kg bw/day, for males and females, respectively.
Key result
Dose descriptor:
NOAEL
Remarks:
SYSTEMIC
Generation:
F1 (cohort 1A)
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: Dietary equivalent to 11.13 and 13.42 mg/kg bw/day, for males and females, respectively.
Key result
Dose descriptor:
NOAEL
Remarks:
REPRODUCTION
Generation:
F1 (cohort 1A)
Effect level:
>= 300 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Dietary equivalent to 34.63 and 40.86 mg/kg bw/day, for males and females, respectively.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results: F2 generation

General toxicity (F2)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were observed in all pups. However, weakness was occasionally observed in some F2 pups( G1, 2; G2, 1; G3, 2; G4, 2).
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
The mortality index of F2 pups was comparable between the control and the test item treated groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
BODY WEIGHT
Male Pups
The mean body weight of male pups, belonging to the 300 ppm dose group, was statistically and significantly lower on postnatal days 14 and 21 when compared with that of the control group.

Female Pups
The mean body weight of female pups, belonging to the 300 ppm dose group, was statistically and significantly lower on postnatal days 14 and 21 when compared with that of the control group.

Composite of Male and Female Pups
The mean body weight of pups, belonging to the 300 ppm dose group, was statistically and significantly lower on postnatal day 14 and 21 when compared with that of the control group. This lower mean body weight of pups, belonging to the 300 ppm dose group toward the end of the lactation period could be due to decreased milk production of lactating females and direct exposure of pups to the test item through diet during 3rd week of lactation (i.e., approximately from postnatal day 14 onwards). Therefore, it could be considered as an adverse effect of the test item on developing pups.

BODY WEIGHT GAIN
Male Pups
The mean body weight gain of male pups, belonging to the 300 ppm dose group, was statistically and significantly lower during postnatal days 4-7, 7-14, and 14-21 when compared with that of the control group.
The mean body weight gain of male pups, belonging to the 100 ppm dose group, was statistically and significantly lower during postnatal days 7-14 when compared with that of the control group.

Female Pups
The mean body weight gain of female pups, belonging to the 300 ppm dose group, was statistically and significantly lower during postnatal days 4-7, 7-14, and 14-21 when compared with that of the control group. The mean body weight gain of female pups, belonging to the 100 ppm dose group, was statistically and significantly lower during postnatal days 7-14 when compared with that of the control group.

Composite of Male and Female Pups
The mean body weight gain of pups, belonging to the 300 ppm dose group, was statistically and significantly lower during postnatal days 4-7, 7-14, and 14-21 when compared with that of the control group. The mean body weight gain of pups, belonging to the 100 ppm dose group, was statistically and significantly lower during postnatal days 7-14 when compared with that of the control group.
This lower mean body weight gain of pups, belonging to the 100 and 300 ppm dose groups toward the end of the lactation period could be due to decreased milk production of lactating females and direct exposure of pups to the test item through diet. Therefore, it could be considered as an adverse effect of the test item on developing pups.
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
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The mean anogenital distance of F2 pups was comparable between the control and the test item treated groups.
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant lower terminal body weight was noted in male and female pups. In F2 weanling pups, statistically significant lower values were noted for absolute weight of spleen and thymus in the 300 ppm dose of both sexes and also of the thymus in the 100 ppm dose males, while higher values were noted for relative weights of brain in the 300 ppm dose of both sexes. Effects were considered related to a lower body weight. A statistically significant higher relative liver weight was noted in the 300 ppm dosed females which was not related to treatment due to the absence of consistency between sexes and the lack of histopathological support.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of F2 weanling pups did not reveal any pathologic lesions.
Histopathological findings:
no effects observed
Description (incidence and severity):
Microscopic findings in the liver were comparable between the control and the test item treated groups of F2 pups.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
LIVE BIRTH, SURVIVAL AND LACTATION INDICES
The live birth index of pups, belonging to the 50 ppm dose group, was statistically and significantly lower, when compared with that of the control group. The lower live birth index without any dose dependency could be considered as incidental and without any toxicological relevance. Survival and lactation indices of F2 pups were comparable between the control and the test item treated groups.

LITTER SIZE AND MALE SEX RATIO
Litter size and male sex ratio of F2 pups was comparable with that of the control group.

BODY TEMPERATURE
The mean body temperature of male and female pups, belonging to the 100 ppm dose group, was statistically and significantly lower on postnatal day 0 when compared with that of the control group. This lower body temperature without any dose dependency could be considered as incidental.

PHYSICAL DEVELOPMENT LANDMARK
Physical development landmark of male and female pups was comparable amongst the control and the test item treated groups.

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Remarks:
FETAL TOXICITY
Generation:
F2 (cohort 1B)
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: Dietary equivalent to 10.11 and 13.71 mg/kg bw/day, for males and females, respectively.

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Verification of homogeneity and concentration of the test item in the test diet

The mean percent recovery obtained for the test diet was within the acceptance level of ±20% of the nominal concentration demonstrating that the exposure concentrations were as intended by the study plan and the %CV was less than 20, suggesting that the test diets were homogeneously mixed.

TABLE 1a. Test item intake during treatment period (P0)

Group N°

Dose Levels (ppm)

Test Item Intake (mg/kg body weight/day)

Male Rats

Female Rats

Treatment Period

Pre-mating Period

Gestation Period

Lactation Period

G1

0

0.00

0.00

0.00

0.00

G2

50

3.74

4.66

3.99

9.03

G3

100

7.44

9.30

7.95

18.25

G4

300

22.11

27.43

23.48

56.19

TABLE 1b. Test item intake during the treatment period (F1 Cohort 1A)

Group N°

Dose Levels (ppm)

Test Item Intake (mg/kg body weight/day)

Male Rats

Female Rats

G1

0

0.00

0.00

G2

50

5.74

6.98

G3

100

11.13

13.42

G4

300

34.63

40.86

 

TABLE 1c. Test item intake during the treatment period (F1 Cohort 1B)

Group N°

Dose Levels (ppm)

Test Item Intake (mg/kg body weight/day)

Male Rats

Female Rats

Treatment Period

Pre-mating Period

Gestation Period

Lactation Period

G1

0

0.00

0.00

0.00

0.00

G2

50

5.93

4.22

4.47

9.34

G3

100

10.11

8.40

8.84

18.58

G4

300

30.05

25.14

27.54

62.23

 

TABLE 2: Summary of Effect on Fertility, Reproduction, and Development – Parent Female (P)

Observations

Values

Group & Dose (ppm)

G1 (0)

G2 (50)

G3 (100)

G4 (300)

Pairs Started (N)

25

25

25

25

Females Achieving Pregnancy (N)

22

24

23

18

Conceiving Days 1 - 5 (N)

24

25

25

25

Conceiving Days 6 - 10 (N)

0

0

0

0

Conceiving Days³11 (N)

0

0

0

0

Pregnancy = 21 days (N)

0

0

0

0

Pregnancy = 22 days (N)

7

16

14

12

Pregnancy = 23 days (N)

12

7

8

6

Pregnancy³24 days (N)

2

1

0

0

Female Sacrificed on Gestation Day 25 (N)

3

1

3

7

Dams with Live Pups at Day 4 (N)

21

24

22

18

N° of Female Rats Standardized on Lactation Day 4

8

12

12

11

Dams with Live Pups at Weaning (N)

20

24

22

18

N° of Female Rats (along with Pups) Sacrificed on Lactation Day 22

20

24

22

18

N° of Female Rats Sacrificed (Without Pups)

2

0

0

0

Implants (Mean±SD)

10.18±4.43

11.17±2.76

10.96±3.40

11.33±2.74

ABNORMAL PUPS (excluding dead/cannibalised pups)

Dams with 0

20

22

19

17

Dams with 1 (Weakness)

2

2

2

1

Dams with ≥2 (Weakness)

0

0

1

0

LOSS OF OFFSPRING

Pre-natal (implantations minus live births)

Females with 0

6

10

10

10

Females with 1

8

7

9

5

Females with 2

6

7

1

2

Females with ≥ 3

2

0

3

1

Post-natal (live births minus alive at post-natal day 4)

Females with 0

16

18

17

16

Females with 1

5

5

5

1

Females with 2

1

1

0

1

Females with ≥ 3

0

0

0

0

FERTILITY DATA

Number of Males Housed with Female

25

25

25

25

Number of Males Impregnating Female

25

25

25

25

Number of Females Housed with Male

25

25

25

25

Number of Females with Sperm Positive Vaginal Smear

25

25

25

25

Number of Females Confirmed Pregnant

22

24

23

18

Number of Females Giving Birth

22

24

22

18

Number of Females Giving Birth to at least a Viable Pup

22

24

22

18

Number of Females Giving Birth to All Viable Pups

21

23

21

17

Duration of Gestation (days) (Mean±SD)

22.76±0.62

22.38±0.58

22.36±0.49

22.33±0.49

FERTILITY INDEX

Male Fertility Index

100.00

100.00

100.00

100.00

Female Fertility Index

88.00

96.00

92.00

72.00

Gestation Index

88.00

96.00

88.00

72.00

Parturition Index

88.00

96.00

88.00

72.00

Percentage of Pregnant Rats

88.00

96.00

92.00

72.00

Percentage of Non-pregnant Rats

12.00

4.00

8.00

28.00

Mating Index

100.00

100.00

100.00

100.00

Prenatal Loss (Mean±SD)

1.23±1.07

0.88±0.85

0.96±1.26

0.67±0.91

Postnatal Loss (Mean±SD)

0.32±0.57

0.29±0.55

0.23±0.43

0.17±0.51

Prenatal Loss (%) (Mean±SD)

15.52±18.87

7.72±7.52

13.25±22.94

5.42±7.09¯*

Postnatal Loss (%) (Mean±SD)

11.03±26.27

2.72±4.97

1.66±3.15

1.35±4.23

Key: N = Number of rats, SD = Standard deviation,* =Student’s t-test

Key:  ¯=Significantly lower than control (p=<0.05)

Note: Non-pregnant rats were excluded from statistical analysis of implants and pre- and postnatal losses.

Presumed mated female was not considered for calculation of conceiving and pregnancy days

TABLE 3. Summary of Effect on Fertility, Reproduction, and Development - Pup data

Number of Pups

Group N°

G1

G2

G3

G4

Sex

M

F

T

M

F

T

M

F

T

M

F

T

Total N° of Pups on Day of Littering

94

104

198

121

127

248

117

114

231

93

100

194

Cannibalized pups on day of littering

0

0

0

0

0

0

0

0

0

0

0

1

Pups Still birth on Day of Littering

1

0

1

1

0

1

1

0

1

1

0

1

Total N° of Live Pups on Day of Littering

93

104

197

120

127

247

116

114

230

92

100

192

Litter Size on PND 4

89

101

190

116

124

240

114

111

225

90

99

189

Litter Size on PND 4 (R)

83

87

170

103

112

215

97

99

196

82

85

167

Litter Size on PND 7

78

85

163

102

110

212

97

98

195

79

82

161

Litter Size on PND 14

77

82

159

100

108

208

93

96

189

77

79

156

Litter Size on PND 21

75

80

155

99

106

205

93

95

188

74

78

152

Pups Mortality Index

Group N°

G1

G2

G3

G4

Sex

M

F

T

M

F

T

M

F

T

M

F

T

1-4 (N°)

4

3

7

4

3

7

2

3

5

2

1

3

Mortality Indexon PND 4

4.30

2.88

3.55

3.33

2.36

2.83

1.72

2.63

2.17

2.17

1.00

1.56

4R -7 (N°)

5

2

7

1

2

3

0

1

1

3

3

6

Mortality Indexon PND 7

6.02

2.30

4.12

0.97

1.79

1.40

0.00¯©

1.01

0.51

3.66

3.53

3.59

4R -14 (N°)

6

5

11

3

4

7

4

3

7

5

6

11

Mortality Indexon PND 14

7.23

5.75

6.47

2.91

3.57

3.26

4.12

3.03

3.57

6.10

7.06

6.59

14R -21 (N°)

8

7

15

4

6

10

4

4

8

8

7

15

Mortality Indexon PND 21

9.64

8.05

8.82

3.88

5.36

4.65

4.12

4.04

4.08

9.76

8.24

8.98

Key:  M = Male, F = Female, T = Total, R = Retained, N° = Number,a= Cannibalized (sex not identified),© = Chi-square test

¯=Significantly lower than control (p=<0.05)


Pups survival Index

Group N°

G1

G2

G3

G4

Sex

M

F

T

M

F

T

M

F

T

M

F

T

Survival Index on PND 4

95.70

97.12

96.45

96.67

97.64

97.17

98.28

97.37

97.83

97.83

99.00

98.44

Survival Index on PND 7

93.98

97.70

95.88

99.03

98.21

98.60

100.00­©

98.99

99.49

96.34

96.47

96.41

Survival Index on PND 14

92.77

94.25

93.53

97.09

96.43

96.74

95.88

96.97

96.43

93.90

92.94

93.41

Survival Index on PND 21

90.36

91.95

91.18

96.12

94.64

95.35

95.88

95.96

95.92

90.24

91.76

91.02

 

Lactation Index, Live Birth Index and Postnatal Loss

Group N°

G1

G2

G3

G4

Sex

M

F

T

M

F

T

M

F

T

M

F

T

Lactation Index (%)

90.36

91.95

91.18

96.12

94.64

95.35

95.88

95.96

95.92

90.24

91.76

91.02

Live Birth Index (%)

98.94

100.00

99.49

99.17

100.00

99.60

99.15

100.00

99.57

98.92

100.00

98.97

Postnatal Loss (PND 0-4)

4

3

7

4

3

7

2

3

5

2

1

3

Key:    M = Male, F = Female, T = Total, R = Retained, N° = Number, PND = Postnatal Day, © = Chi-square test

­ = Significantly higher than control (p=<0.05)

TABLE 4. Organ weight percent change compared to control

Generation

Parameters

Group

Percent Change

G1

G2

G3

G4

G2

G3

G4

Parent

B. wt. (TS)-Female

247.345

235.104

237.327

218.806¯¯

-4.9

-4.1

-11.5

Liver (Male)-R

2.614

2.641

2.669

3.026­­

1.0

2.1

15.8

Liver (Female)-R

3.894

4.095

4.083

4.696­­

5.2

4.9

20.6

Thyroid (Female)-R

0.0066

0.0072

0.0072

0.0080­­

9.1

9.1

21.2

Cohort 1A

B. wt. (TS)- Male

322.685

297.62

315.23

295.965¯¯

-7.8

-2.3

-8.3

B. wt. (TS)-Female

191.63

183.54

194.27

176.300¯

-4.2

1.4

-8.0

Liver (Male)-R

3.069

2.974

3.069

3.435­­

-3.1

0.0

11.9

Liver (Female)-R

3.174

3.154

3.178

3.558­­

-0.6

0.1

12.1

Thyroid (Male)-R

0.0047

0.0053­

0.0053­

0.0058­­

12.8

12.8

23.4

Cohort 1B

B. wt. (TS)-Female

237.415

228.653

233.494

212.606¯¯

-3.7

-1.7

-10.4

Liver (Male)-R

2.593

2.570

2.628

2.946­­

-0.9

1.3

13.6

Liver (Female)-R

4.226

4.397

4.444

4.847­­

4.0

5.2

14.7

Thyroid (Female)-R

0.0054

0.0052

0.0053

0.0060­

-3.7

-1.9

11.1

F1-Weanling Pups

B. wt. (TS)-Male

42.569

40.443

38.764

35.340¯

-5.0

-8.9

-17.0

Liver (Male)-A

1.971

1.865

1.734

1.564

-5.4

-12.0

-20.6

B. wt. (TS)-Female

39.833

39.890

37.471

32.515¯

0.1

-5.9

-18.4

Liver (Female)-A

1.786

1.853

1.689

1.438¯$

3.8

-5.4

-19.5

F2-Weanling Pups

B. wt. (TS)-Male

40.780

42.018

37.589

34.871¯¯

3.0

-7.8

-14.5

B. wt. (TS)-Female

40.495

40.900

36.718

33.900¯¯

1.0

-9.3

-16.3

Key: M-Male, F-Female, TS-Terminal sacrifice

TABLE 5. Microscopic Findings of Parents, Cohort 1A and F1& F2 Pups

Lesion

Generation

Sex

Group

G1

G2

G3

G4

Liver: Hypertrophy, hepatocytes

Parent

M

1

1

3

14

F

0

2

5

14

Cohort 1A

M

0

1

3

9

F

0

0

1

8

Thyroid: Hypertrophy, follicular cell

Parent

M

3

3

6

15

F

2

6

6

18

Cohort 1A

M

4

3

4

12

F

0

0

0

8

Liver: Glycogen depletion, hepatocytes

F1 Pups

M

0

0

0

8

F

1

0

0

6

F2 Pups

M

0

1

0

1

F

1

1

1

2

Key: M-male, F-Female

TABLE 6. Summary ofSerum T3 and T4 Level in Parent Male Rats

Groups and Dose: G1 - 0; G2 - 50; G3 - 100; G4 - 300 ppm

Hormone

Group N°

G1

G2

G3

G4

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T3

406.896

85.522

10

379.680

66.411

10

354.102

47.875

10

430.092

93.733

10

T4

162884.407

25143.440

10

159581.646

22284.244

10

164727.417

30623.771

10

161425.613

35243.036

10

 

TABLE 7. Summary ofSerum T3 and T4 Level in Parent Female Rats

Hormone

Group N°

G1

G2

G3

G4

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T3

634.485

77.520

10

669.095

89.733

10

666.089

78.733

10

542.548

107.996

10

T4

52170.941

12929.265

10

59036.481

6080.748

10

59296.682

12923.489

10

49277.953

9673.531

10

 

TABLE 8. Summary of Serum T4 Level in PND 4 Rat Pups (F1)

Hormone

Group N°

G1

G2

G3

G4

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T4

166263.378

36641.300

8

151498.847

31071.914

12

183356.165

39221.749

11

175831.884

15736.875

12

T3

173.989

28.964

8

194.562

26.907

12

183.808

32.597

11

164.592

21.045

12

 

TABLE 9. Summary of Serum T3 and T4 Level in PND 22 Male Rat Pups (F1)

Hormone

Group N°

G1

G2

G3

G4

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T3

947.624

149.214

10

893.210

128.097

10

885.531

99.916

10

828.537

155.784

10

T4

32088.317

5505.246

10

30624.640

3437.663

10

32546.858

4931.690

10

38553.791

21088.103

10

Key: N = Number of animals, SD = Standard deviation, N = Number


TABLE 10. Summary of Serum T3 and T4 Level in PND 22 Female Rat Pups (F1)

Hormone

Group N°

G1

G2

G3

G4

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T3

1166.706

98.492

10

1130.596

152.894

10

1300.428­*

171.315

10

1371.200

395.309

10

T4

75886.896

12449.540

10

74471.364

8805.684

10

78445.631

13454.459

10

91869.842

25523.427

10

 

TABLE 11. Summary of Serum T3 and T4 Level i nCohort 1A Male Rats

Hormone

Group N°

G1

G2

G3

G4

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T3

608.631

105.624

10

634.192

114.392

10

673.808

92.116

10

631.297

97.648

10

T4

46283.928

4749.377

10

46933.934

4885.995

10

52575.119

5758.954

10

44885.595

6853.688

10

 

TABLE 12. Summary of Serum T3 and T4 Level in Cohort 1A Female Rats

Hormone

Group N°

G1

G2

G3

G4

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T3

657.453

131.922

10

690.129

108.068

10

691.313

68.543

10

792.604

184.617

10

T4

30867.390

6085.017

10

34932.867

7235.657

10

32105.700

5251.861

10

49568.749­­#

10927.196

10

Keys: N = Number of animals, SD = Standard deviation, N = Number

­= Significantly higher than control (p=<0.05); ­­= Significantly higher than control (p=<0.01)

* =Student’s t-test, # = Dunnett’s t-test

 

TABLE 13: Pup body temperature

Postnatal
Day 0

Group N°

G1

G2

G3

G4

 

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

F1 Male

24.95

0.48

20

24.9

0.41

24

24.82

0.52

22

24.43↓↓$

0.37

18

F1 Female

25.01

0.62

20

24.97

0.45

24

24.79

0.47

22

24.40↓↓$

0.3

18

F1 Male+ female

24.97

0.58

22

24.93

0.42

24

24.8

0.47

22

24.40↓↓$

0.31

18

F2 Male

24.94

0.57

20

24.77

0.6

17

24.39↓$

0.54

18

24.62

0.42

17

F2 Female

25.02

0.49

20

24.71

0.6

17

24.45↓↓$

0.52

17

24.65

0.43

17

F2 Male+ female

24.99

0.49

20

24.75

0.6

17

24.42↓↓$

0.53

18

24.65

0.41

17

N = Number of observations, SD = Standard deviation, N° Number, $ t-test with Bonferroni’s adjustment, ↓ = Significantly lower than control (p≤0.05), ↓↓ = Significantly lower than control (p≤0.01)

 

 

TABLE 14: Summary of Effect on Fertility, Reproduction, and Development – Parent Female (P1)

Observations

Values

Group & Dose (ppm)

G1 (0)

G2 (50)

G3 (100)

G4 (300)

Pairs Started (N)

20

20

20

20

Females Achieving Pregnancy (N)#

20

20

20

20

Conceiving Days 1 - 5 (N)

19

16

17

19

Conceiving Days 6 - 10 (N)

0

0

0

1

Conceiving Days ³ 11 (N)

0

3

2

0

Pregnancy = 21 Days (N)

0

0

2

1

Pregnancy = 22 Days (N)

16

14

15

16

Pregnancy = 23 Days (N)

3

2

0

0

Pregnancy ³ 24 Days (N)

0

1

1

0

Female Sacrificed on Gestation Day 25 (N)

0

2

2

3

Dams with Live Pups at Day 4 (N)

20

17

18

17

N° of Female Rats Standardized on Lactation day 4

6

4

6

0

Dams with Live Pups at Weaning (N)

20

17

18

17

N° of Female Rats (along with pups) Sacrificed on Lactation Day 22

20

17

18

17

N° of Female Rats Sacrificed (without pups)

0

0

0

0

Implants (Mean ± SD)

11.05 ± 1.93

9.22 ± 2.88¯β

9.47 ± 3.29

9.59 ± 1.37¯β

ABNORMAL PUPS (excluding dead/cannibalised pups)

Dams with 0

18

16

16

15

Dams with 1 (Weakness)

2

1

2

2

Dams with ≥2 (Weakness)

0

0

0

0

LOSS OF OFFSPRING

Pre-natal (implantations minus live births)

Females with 0

7

8

9

8

Females with 1

7

4

9

7

Females with 2

6

4

1

1

Females with ≥ 3

0

2

0

1

Post-natal (live births minus alive at post-natal day 4)

Females with 0

16

15

17

14

Females with 1

2

1

1

3

Females with 2

1

0

0

0

Females with ≥ 3

1

1

0

0

FERTILITY DATA

Number of Males Housed with Female

20

20

20

20

Number of Males Impregnating Female

20

20

20

20

Number of Females Housed with Male

20

20

20

20

Number of Females with Sperm Positive Vaginal Smear

19

19

19

20

Number of Females Confirmed Pregnant

20

20

20

20

Number of Females Giving Birth

20

17

18

17

Number of Females Giving Birth to at least a Viable Pup

20

17

18

17

Number of Females Giving Birth to All Viable Pups

20

15

18

17

Duration of Gestation (days) (Mean ± SD)

22.16 ± 0.37

22.24 ± 0.56

22.00 ± 0.59

21.94 ± 0.24

FERTILITY INDEX

Male Fertility Index

100

100

100

100

Female Fertility Index

100

90

95

85

Gestation Index

100

85

90

85

Parturition Index

100

85

90

85

Percentage of Pregnant Rats

100

90

95

85

Percentage of Non-pregnant Rats

0

10

5

15

Mating Index

100

100

100

100

Prenatal Loss (Mean ± SD)

0.95 ± 0.83

1.00 ± 1.08

0.58 ± 0.61

0.71 ± 0.85

Postnatal Loss (Mean ± SD)

0.45 ± 1.19

0.24 ± 0.75

0.06 ± 0.24

0.18 ± 0.39

Prenatal Loss (%) (Mean ± SD)

8.75 ±7.97

14.59 ± 24.15

12.20 ± 24.27

6.70 ± 7.33

Postnatal Loss (%) (Mean ± SD)

4.15 ± 9.44

2.38 ± 8.16

0.79 ± 3.37

1.91 ± 4.29

Key: N = Number of rats, SD = Standard deviation,* =Student’s t-test

Key:  ¯=Significantly lower than control (p=<0.05)

Note: Non-pregnant rats were excluded from statistical analysis of implants and pre- and postnatal losses.

Presumed mated female was not considered for calculation of conceiving and pregnancy days

 

 

TABLE 15. Summary of Effect on Fertility, Reproduction, and Development - Pup data

Number of Pups (F2)

Group N°

G1

G2

G3

G4

Sex

M

F

T

M

F

T

M

F

T

M

F

T

Number of Pups

Total N° of Pups on Day of Littering

93

109

202

72

77

151a

88

81

169

76

75

151

Cannibalized Pups on Day of Littering

0

0

0

0

0

2

0

0

0

0

0

0

Pups Still birth on Day of Littering

0

0

0

1

0

1

0

0

0

0

0

0

Total N° of Live Pups on Day of Littering

93

109

202

71

77

148

88

81

169

76

75

151

Litter Size on

91

102

193

70

74

144

88

80

168

74

74

148

PND 4

Litter Size on

86

95

181

65

72

137

84

75

159

74

74

148

PND 4 (R)

Litter Size on

84

94

178

65

71

136

83

74

157

73

73

146

PND 7

Litter Size on

81

93

174

65

71

136

83

73

156

73

72

145

PND 14

Litter Size on

80

93

173

64

71

135

81

73

154

73

71

144

PND 21

Pups Mortality Index

1-4 (N°)

2

7

9

1

3

4

0

1

1

2

1

3

Mortality Index on PND 4

2.15

6.42

4.46

1.41

3.9

2.7

0

1.23

0.59

2.63

1.33

1.99

4R -7 (N°)

2

1

3

0

1

1

1

1

2

1

1

2

Mortality Index on PND 7

2.33

1.05

1.66

0

1.39

0.73

1.19

1.33

1.26

1.35

1.35

1.35

4R -14 (N°)

5

2

7

0

1

1

1

2

3

1

2

3

Mortality Index on PND 14

5.81

2.11

3.87

0

1.39

0.73

1.19

2.67

1.89

1.35

2.7

2.03

14R -21 (N°)

6

2

8

1

1

2

3

2

5

1

3

4

Mortality Index on PND 21

6.98

2.11

4.42

1.54

1.39

1.46

3.57

2.67

3.14

1.35

4.05

2.7

Pups Survival Index

Survival Index on PND 4

97.85

93.58

95.54

98.59

96.1

97.3

100

98.77

99.41

97.37

98.67

98.01

Survival Index on PND 7

97.67

98.95

98.34

100

98.61

99.27

98.81

98.67

98.74

98.65

98.65

98.65

Survival Index on PND 14

94.19

97.89

96.13

100

98.61

99.27

98.81

97.33

98.11

98.65

97.3

97.97

Survival Index on PND 21

93.02

97.89

95.58

98.46

98.61

98.54

96.43

97.33

96.86

98.65

95.95

97.3

Lactation Index, Live Birth Index and Postnatal Loss

Lactation

93.02

97.89

95.58

98.46

98.61

98.54

96.43

97.33

96.86

98.65

95.95

97.3

Index (%)

Live Birth

100

100

100

98.61

100

98.01¯©

100

100

100

100

100

100

Index (%)

Postnatal

2

7

9

1

3

4

0

1

1

2

1

3

Loss (PND 0-4)

Key:    M = Male, F = Female, T = Total, R = Retained, N° = Number, PND = Postnatal Day, © = Chi-square test

­ = Significantly higher than control (p=<0.05)

 

 

 

Applicant's summary and conclusion

Conclusions:
In this GLP-compliant Extended One Generation Reproductive Toxicity Test, performed according to OECD 443, with the inclusion of Cohorts 1A and 1B (including extension to F2), the following NOAELs were found:
- Parental - systemic toxicity: 100 ppm (dietary equivalent of 7.44 (males) and 11.83 (females) mg/kg bw/day for P0 and 10.11 (males) and 11.94 (females) mg/kg bw/day for F1 Cohort 1B), based on lower body weight (gain) and food consumption, higher liver weights, greyish discolouration of the liver and hypertrophic liver and thyroid;
- Parental - fertility/reproductive toxicity: => 300 ppm (highest dose; P0: dietary equivalent of 22.11 and 27.43/25.46 mg/kg bw/day, for males and females, respectively; F1 Cohort 1B: dietary equivalent of 30.05 and 25.14/26.34 mg/kg bw/day, for males and females, respectively), based on the absence of any treatment-related adverse effects on any fertility and reproductive performance parameters;
- Offspring (F1) - developmental toxicity: 100 ppm (dietary equivalent of 7.44 and 13.1 mg/kg bw/day, for males and females, respectively), based on lower body weight (gain), lower body temperature, lower liver weight adn depleted glycogen in the liver.
- Offspring (F2) - developmental toxicity: 100 ppm (dietary equivalent of 10.11 and 13.71 mg/kg bw/day, for males and females, respectively), based on lower body weight (gain).
Executive summary:

The objective of this GLP-compliant study, performed according to OECD 443, was to provide data on the possible effects of the test item on reproductive performance of Wistar rats and the development of pups consequent to daily dietary exposure to various concentrations of the test item (intended target concentrations: 0 ppm, 50 ppm, 100 ppm and 300 ppm) to male and female rats during a premating period of 10 weeks and during mating (max. 2 weeks), gestation and lactation until postnatal day (PN) 21. At weaning on postnatal day 21 (PN 21), pups were distributed to Cohorts 1A and 1B and were exposed to the test item (intended target concentrations: 0 ppm, 50 ppm, 100 ppm and 300 ppm). To produce the F2 generation, 1 male and 1 female rat from the F1(cohort 1B) generation were randomly selected and subsequently mated (14 - 15 weeks of age). 

The mean per cent recovery obtained for diet was within the acceptance level of ± 20% of the nominal concentration demonstrating that the exposure concentrations were as intended in the study plan and the %CV was less than 20, suggesting that the diet was homogeneously mixed.

Body weights for all generations were recorded weekly and at termination. Food consumption was measured weekly. Sperm parameters (count and morphology) were assessed for all P and F1A males rates of the control and high dose group, whereas sperm motility was assessed for all groups. Oestrous cycle length and pattern was assessed for all P females and F1A female pups (from the day of vaginal opening, until first oestrus phase). Vaginal smear was examined (from P and F1 rats) at the time of necropsy to determine the stage of the oestrous cycle. Lymphonic subpopulation analysis was performed on 10 rats/sex/group belonging to cohort 1A. Necropsy of the parent generation was scheduled as follows: male rats were sacrificed after delivery of P female rats and female rates on lactation day 22. F1 weanlings were sacrificed on the lactation day 22.Cohort 1A rats were sacrificed during 13-14 weeks of the treatment period. For Cohort F1B, males were sacrificed after sacrifice of F1 female rats on lactation day 22. F2 weanlings were sacrificed on lactation day 21. Clinical pathology included: Haematology, clinical chemistry, and urine parameters were analysed from 10 rats/sex/group (from P and cohort 1A).Thyroid hormones (T3, T4, and TSH) were analysed from parent, weanling pups, and cohort 1A rats.Organs, as defined in the study plan, were excised, weighed, preserved, and subjected to the histopathological examination.From 10 F1 female rats of control and high dose groups Primordial Follicle Quantitative Evaluation was performed.

P Generation

No morality, morbidity, and clinical signs of toxicity were observed in male and female rats up to the 300 ppm dose level.

The mean body weight, body weight gain, food consumption, and food efficiency of male rats were comparable with those of the control group. The mean body weight (gestation and lactation), body weight gain (pre-mating and gestation), and food consumption (pre-mating,gestation, and lactation) of female rats, belonging to the 300 ppmdose group,werestatistically and significantly lower when compared with those of the control group.

A statistically significant lower haematocrit (HCT) value was noted in males of the 300 ppm dose group. This effect was considered to be a treatment-related adverse effect of the test item. A statistically significant higher value was observed for the reticulocyte count of males of the 300 ppm dose group, which was considered to be related to the decrease in HCT and was considered to be related to the test item treatment.

The statistically, significant lower globulin level in males (and apparent decrease in females) of the 300 ppm dose group caused a significantly higher albumin: globulin ratio.A statistically significant lower value was observed for triglycerides of females of the 300 ppm dose group. Statistically significant higher values were noted for ALT and AST of females of the 300 ppm dose group. These effects were considered to be related to the test item treatment and more specifically to be related to alterations in the liver.

Statistically significant higher values were noted for the relative weight of the liver (males and females) of rats of the 300 ppm dose group. Effects were related to the treatment, as these were supported by the histopathology.

Treatment-related greyish discolouration of the liver was observed in male and female rats of the 300 ppm dose. A higher incidence of hepatocyte hypertrophy in liver and follicular cell hypertrophy in thyroid were observed in male and female rats of the 300 ppm dose. Lesions observed in liver and thyroid were well comparable with the increased weight of the liver and thyroid.

Oestrous cycle and sperm parameters were comparable between the control and the test item treated groups.

F1 Pups

The mean body weight, body weight gain, and temperature of pups, belonging to the 300 ppm dose group,were statistically and significantly lower during the postnatal period when compared with those of the control group. These lower values could be due to a decreased gestation body weight of pregnant rats and considered as growth retardation effect of the test item on the developing pups.

A statistically significant lower value was noted for the absolute weight of liver of females of the 300 ppm dose. this effect was also noted in males of the 300 ppm dose group and although it did not reach statistical significance in males, it was considered to the related to treatment, as it was supported by histopathology. Glycogen depletion was observed upon the histopathological examination of the liver of pups of the 300 ppm dose group. It could be considered an adverse effect of the test item.

No treatment-related clinical signs were observed in pups up to the 300 ppm dose level. Mortality, survival, live birth, and lactation indices were comparable amongst the control group and the test item treated groups.Litter size, male sex ratio, thyroid hormones, and physical development landmarks of F1 pups were comparable with that of the control group. The increase in the mean balano-preputial separation of F1 pups of the 300 ppm dose group could be due to a lower preweaning body weight.

F1 Adults (Cohort 1A)

No morality, morbidity, and clinical signs of toxicity were observed either in male or female rats up to the 300 ppm dose level.

The mean body weight, body weight gain, and food consumption of male and female rats, belonging to the 300 ppm dose group, were statistically and significantly lower, throughout the treatment period, when compared with those of the control group.

Statistically significant lower haematocrit, RBC, and haemoglobin values were noted in males of the 300 ppm dose. Statistically significant lower globulin levels were noted in male and female rats of the 300 ppm dose. This effect led to a statistically significant higher albumin: globulin ratio of male and female rats of the 300 ppm dose groups.

Statistically significant higher values were noted for the relative weight of liver (male and female) of rats of the 300 ppm dose which were considered related to the test item treatment and which were corroborated by the hypertrophy observed with histopathology.

Treatment-related greyish discolouration of the liver was observed in male and female rats of the 300 ppm dose group. A higher incidence of hepatocyte hypertrophy in liver and follicular cell hypertrophy in thyroid was observed in male and female rats of the 300 ppm dose rats.

Oestrous cycle, ovarian follicular counts, sperm parameters, lymphocyte subpopulation, and thyroid hormones were comparable amongst the control and the test item treated groups.

F1 Adults (Cohort 1B)

A female belonging to the 50 ppm dose group was found dead on gestation day 10. Other remaining rats were normal, throughout the study period.

The mean body weight, body weight gain, food consumption, and food efficiency of male rats, belonging to the 300 ppm dose group, were statistically and significantly lower, throughout the treatment period, when compared with those of the control group.

The mean body weight (pre-mating, gestation, and lactation), body weight gain and food efficiency (pre-mating and gestation), and food consumption (pre-mating, gestation, and lactation) of female rats, belonging to the 300 ppm dose group, were statistically and significantly lower when compared with that of the control group.

Statistically significant higher values were noted for the relative weight of the liver (males and females) of rats of the 300 ppm dose, whereas increases in absolute weight of the liver was also observed in the 300 ppm dosed males. Treatment-related greyish discolouration of the liver was observed in male and female rats of the 300 ppm dose.

F2 Pups

No treatment-related clinical signs were observed in pups up to the 300 ppm dose level. Mortality, survival, live birth, and lactation indices were comparable amongst the control group and the test item treated groups. Litter size, male sex ratio, anogenital distance, body temperature, and physical development landmarks of F2 pups, were comparable with those of the control group.

The mean body weight and body weight gain of pups, belonging to the 300 ppm dose group, were statistically and significantly lower during the postnatal period when compared with that of the control group. These lower values for the mean body weight and the body weight gain of pups, belonging to the 300 ppm dose group towards the end of the lactation period could be due to the decreased milk production of the lactating females and direct exposure of pups to the test item during the last week of the lactation period (i.e., approximately from postnatal day 14 onwards), through their diet.

Macroscopic and microscopic findings of the liver were comparable amongst the control and the test item treated groups.

Conclusion

In conclusion, the following NOAELs were found:

- Parental - systemic toxicity: 100 ppm (dietary equivalent of 7.44 (males) and 11.83 (females) mg/kg bw/day for P0 and 10.11 (males) and 11.94 (females) mg/kg bw/day for F1 Cohort 1B), based on lower body weight (gain) and food consumption, higher liver weights, greyish discolouration of the liver and hypertrophic liver and thyroid;

- Parental - fertility/reproductive toxicity: => 300 ppm (highest dose; P0: dietary equivalent of 22.11 and 27.43/25.46 mg/kg bw/day, for males and females, respectively; F1 Cohort 1B: dietary equivalent of 30.05 and 25.14/26.34 mg/kg bw/day, for males and females, respectively), based on the absence of any treatment-related adverse effects on any fertility and reproductive performance parameters;

- Offspring (F1) - developmental toxicity: 100 ppm (dietary equivalent of 7.44 and 13.1 mg/kg bw/day, for males and females, respectively), based on lower body weight (gain), lower body temperature, lower liver weight adn depleted glycogen in the liver.

- Offspring (F2) - developmental toxicity: 100 ppm (dietary equivalent of 10.11 and 13.71 mg/kg bw/day, for males and females, respectively), based on lower body weight (gain).