7-(diethylamino)-4-methyl-2-benzopyrone

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

No Observed Adverse Effect Level (NOAEL) of the test chemical is considered 1000-1500 mg/kg body weight when Wistar male and female rats orally by gavage for Approx 63 days.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Weight of evidence approach based on various test chemicals

Justification for type of information:

Weight of evidence approach based on various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

WoE report is based on reproductive toxicity studies on rats.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: 2. Wistar; 3. Sprague Dawley

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

other: 1. corn oil; 2. water

Details on exposure:

2.PREPARATION OF DOSING SOLUTIONS: The test item was weighed and dissolved in a vehicle (corn oil) to achieve desired concentration of test item. Dose formulation was freshly prepared daily. At the time of dosing, dose formulation was kept on the magnetic stirrer to maintain the homogeneity of test item.
3.PREPARATION OF DOSING SOLUTIONS: The test chemical was mixed with vehicle at dose levels of 0, 100, 500 or 1500 mg/kg body weight and prepared daily
VEHICLE
- Justification for use and choice of vehicle (if other than water):No data
- Concentration in vehicle:0, 100, 500 or 1500 mg/kgbw
- Amount of vehicle (if gavage):10 mL/Kg
- Lot/batch no. (if required):No data
- Purity:No data

Details on mating procedure:

Details on mating procedure
2.- M/F ratio per cage:1:1
- Length of cohabitation: until pregnancy occurs or two weeks elapsed.
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: Sperm positive vaginal smear was considered as day "0" of gestation.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.; yes
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): : housed individually
- Any other deviations from standard protocol:
3.- M/F ratio per cage: 1: 1.
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy Copulatory plug or presence of sperm in vaginal washings was designated as day 0 of gestation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Specificity, Precision (%RSD), Accuracy (% Recovery) and Homogeneity by HPLC-UV

Duration of treatment / exposure:

2.Approx 63 days
3.14 days (days 6-19 of gestation)

Frequency of treatment:

daily

Remarks:

Study 2:
0,250,500,1000 mg/kgbw/day

Remarks:

Study 3: 0,100,500,1500 mg/kgbw/day

No. of animals per sex per dose:

2.Total:124
0 mg/kg bw: 13 male, 13 female
250 mg/kg bw: 13 male, 13 female
500 mg/kg bw: 13 male, 13 female
1000 mg/kg bw: 13 male, 13 female
Recovery Group
0 mg/kg bw: 5 male, 5 female
1000 mg/kg bw: 5 male, 5 female
3.Total: 100 females
0 mg/Kg bw: 25 females
100 mg/Kg bw: 25 females
500 mg/Kg bw: 25 females
1500 mg/Kg bw: 25 females

Control animals:

yes, concurrent vehicle

Details on study design:

2.- Dose selection rationale: The dose levels 250, 500 and 1000 mg/kg body weight were selected for the Main Study based on the results of Dose Range Finding (DRF).
- Rationale for animal assignment (if not random): Randomization was done based on recent body weight, before first dosing.

Parental animals: Observations and examinations:

2.Mortality and Morbidity, General clinical sign, motor Activity and Behavioural, Body Weight and Fe
ed Consumption were examined.
3.Survival, clinical sign and body weights were examined.

Oestrous cyclicity (parental animals):

2.Estrous cycle were monitored daily

Sperm parameters (parental animals):

not specified

Litter observations:

2.Live pups, Number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller
than corresponding control pups) and body weight were examined.
3.sex and body weight were examined

Postmortem examinations (parental animals):

2.Hematology, clinical biochemistry, Organ Weight, gross Pathology and Histopathology were examined.
3.Gross pathology were examined

Postmortem examinations (offspring):

2.Gross Pathology were examined.
3.Gross external malformations and variations were examined.

Statistics:

2.Raw data was analysed using statistical software “Sigma Plot 11.0” (Supplied by Cranes Software International Ltd. Bangalore). The mean and standard deviation was calculated using the software and all data was summarized in tabular form. All continuous data (body weight, feed consumption, Functional Observational Battery parameters, hematology, clinical chemistry, absolute and relative organ weights, maternal and pup parameters etc.) were checked for normality using Shapiro Wilk test. All homogenous data was analysed using ANOVA and data showing significance in their variances was subjected to Dunnett’s t-test. All heterogeneous data was analysed using F test and Student’s t-test, Dunn’s Test, Kruskal-Wallis, ANOVA on ranks.
3.Differences in the fetal sex distribution and the number of litters with malformations between control and treated groups wcre compared using the Chi-square test criterion with Yates" correction for 2 x 2 contingency lables and, or Fisher's exact probability test as described by Sicgel (1956). The numbers of early and late resorptions, dead foetuses and post-implantation losses were compared betweengroups by the Mann Whitney U test as described by Siegel (1956) and Well (1970). The mean numbers of viable foetuses, total implantations, corpora lutca and mean t\)etal weights were compared between groups by analysis of variance (oneway classification), Bartlett's test l\~r homogeneity of variances, and the appropriate t test (for equal or unequal variances) as described by Steel & Torric (1960) using Dunnctt's multiple comparison tables (1964).

Reproductive indices:

2.Gestational length, Litter size, No. of live births, Post-implantation loss and Pregnancy Index (%) were examined.

Offspring viability indices:

2.Fetal Survival Index at Post-natal Day 1 and 4 were examined.

Clinical signs:

no effects observed

Description (incidence and severity):

2.Statistically significant decrease were observed in number of rears (250, 500 and 1000 mg/kg bw at pre-treatment and 1000 mg/kg bw at Week 1), number of urine pools (250 mg/kg bw at Week 2) in male as compared to control. In recovery male, statistically significant increase in number of urine pools at week 4 in recovery at 1000 mg/kg bw as compared to recovery control. In female, statistically significant decrease was observed in number of fecal bolus (1000 mg/kg bw recovery at Week 1) as compared to control recovery group. The above changes observed were inconsistent/ biologically insignificant and not dose dependant hence considered as incidental and not attributed to the effect of test item administration.
3.Orange discoloration of the urine was observed in all the treated rats as compared to control.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

2.One female was found dead on day 41 at 1000 mg/kg bw due to gavaging error
3.When treated with 1500 mg/kg bw, Six rats died during the dosing period as compared to control.
Survival was 100% in the controls and the groups receiving 100 and 500 mg/kg of dye

Body weight and weight changes:

no effects observed

Description (incidence and severity):

2.Statistically significant decrease was observed in percent body weight change of 250, 500 and 1000 mg/kg bw on day 1-20 as compared to control group.
Body weight and Percent body weight changes in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective
control group. These changes observed were inconsistent, hence not considered as effect of the test item administration.
3.When treated with 1500 mg/kg bw, slight reductions in body-weight gains as compared to controls, throughout the dosing period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

2.Statistically significant decrease in feed consumption was observed in 250, 500 and 1000 mg/kg bw female on gestation day 14-20 as compared to the control. Feed consumption in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group.
Changes observed in feed consumption were inconsistent, hence not considered as effect of the test item administration.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

2.At the end of treatment period revealed statistically significant decreased of aPTT in male rats at 1000 mg/kg, statistically significant increased of aPTT in female rats at 250 mg/kg while statistically significant decreased in Lymphocyte Count in female rats at 500 mg/kg. At the end of recovery period, all of the above altered parameters had returned to normal level similar to control group rats in both sexes.
During treatment free recovery period, statistically significant increase was observed in PT of recovery male rats treated at 1000 mg/kg and statistically significant decreased was observed Hematocrit and Hemoglobin in recovery male rats treated at 1000 mg/kg, while statistically significant decreased of Monocyte in female recovery rats at 1000 mg/kg when compared to respective control group. The observed variations in PT, Hematocrit, Hemoglobin and monocyte were considered to be of no toxicological significance, minimal in nature and occurred in the absence of clear dose related response

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

2.decrease in bile acid of male rats treated at 250 mg/kg and 1000 mg/kg respectively while statistically significant decrease in Triglyceride of female rats treated at 500 mg/kg. During treatment-free recovery period revealed statistically significant increase in Potassium in female rats treated at 1000 mg/kg while statistically decrease in Bile acid in female rats treated at 1000 mg/kg was observed when compared to respective control group.
The observed variations in Potassium and Bile acid was considered incidental and not test item related as it was observed only in single sex, inconsistent, not dose dependent and further is not evidenced by histopathological observations.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

3.Orange discoloration of the urine was noted in all treated rats during the treatment period.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

2.The sensory reactivity measurements were comparable and no changes were revealed in any of the animals of all treated groups in both the sexes.
Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups.
Motor activity measurements were comparable and no changes were revealed in any of the animals from all treated groups of both the sexes as compare to control group.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

2.Visceral examination of the rats of control and other treated groups did not reveal any pathological abnormality. focal mild to multifocal mild mononuclear cell infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration and minimal chronic changes and hydropic degeneration in female liver, Unilateral: focal minimal to multifocal mild mononuclear cells infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration in female Kidneys, focal mild to multifocal mild mononuclear cells infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration in female Lungs and focal minimal to multifocal mild GALT hyperplasia in male and focal minimal to multifocal minimal GALT hyperplasia in female intestin at 250 and 1000 mg/kg bw, Vacuolar, degeneration multifocal, mild in male and diffuse vacuolations in female Adrenals at 250 mg/kg bw, minimal cyst in male and mild serosanginous fluid cavity in female Pituitary Gland, focal minimal to multifocal moderate Retention of sperm, focal minimal to focal mild Exfoliation of round spermatid, focal minimal to multifocal mild Seminiferous tubules Vacuolar degeneration, marked diffuse Necrosis of Seminiferous tubules, minimal diffuse Atrophy Seminiferous tubules, minimal to mild Vacuolar degeneration of Leydig cell, marked diffuse Necrosis of Epididymis, marked diffuse decrease in Epididymis sperm and Epididymis of Epididymis in male rat and segmental minimal Vacuolar degeneration of Ovary and minimal Perimetrium and myometrium vacuolations of Uterus in female rats were observed at different doses.
Lesions observed in liver, kidneys, lungs, adrenals, intestine, pituitary gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies (Boorman et al., 1990, Greaves, 2007, Greaves and Faccini, 1992; Haschek et al., 2002). Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item

Histopathological findings: neoplastic:

not specified

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

2.One females showed prolonged diestrous i.e more than 3 days with total estrous cycle period of 6 days or more before mating period at 250 and 1000 mg/kg bw.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

2.No statistically significant effect on Gestational length, Litter size, No. of live births, Post-implantation loss, Post-natal loss, Pups sex ratio and Pregnancy index of treated rats were observed as compa
red to control.
3.No effect on Viable and non-viable foetuses, early and late resorptions, total implantations and corpora lutea and sex ratio of fetuses were observed as compared to control.

Dose descriptor:

NOAEL

Effect level:

> 1 000 - <= 1 500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive performance

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

2.No effect on Survival of pups were observed as compared to control.
3.No effect on Viable and non-viable fetuses were observed as compared to control

Body weight and weight changes:

no effects observed

Description (incidence and severity):

2.No effect on weight gain for pups at PND4 were observed as compared to control.
3.No effect on Body weight of fetuses were observed as compared to control.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not examined

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

2.Pups died during course of study revealed various lesions among the control and treated groups. Cannibalism in male pups at 250 and 500 mg/kg bw and in female at 250 mg/kg bw, absence of milk in stomach in male at 250 and 500 mg/kg bw and in female at 250 mg/kg bw, reddish discoloration of lungs in female at 250 mg/kg bw and brain haemorrhage in male rat at 250 mg/kg bw. No gross patholoical changes were observed at 1000 mg/kg bw. Hence, occurrence of these lesi ons could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item.
3.When treated with 1500 mg/kg bw, A slight increase in the number of litters with unossified sternebrae (sternebrae nos 1-6) and rudimentary 14th rib(s) was observed however, these values fell within the ranges of historical control data. No biologically meaningful or statistically significant differences in the number of litters and number of fetuses with malformations and number of fetuses or litters with developmental variations were observed in treated rats as compared to control.

Histopathological findings:

no effects observed

Description (incidence and severity):

2.There were no biologically meaningful or statistically significant differences in number of fetuses with malformations, number of foetuses or litters with developmental variations in any of the treated groups.

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 - <= 1 500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

gross pathology

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Reproductive effects observed:

not specified

Treatment related:

no

Conclusions:

No Observed Adverse Effect Level (NOAEL) of the test chemical is considered 1000-1500 mg/kg body weight when Wistar male and female rats orally by gavage for Approx 63 days.

Executive summary:

Data available from different studies were reviewed to determine the reproductive toxicity of test chemical.The studies are as mentioned below:

Study 1

In a Repeated Dose Oral Toxicity Study in combination with Reproduction/ Developmental Toxicity study, Wistar male and female rats were treated with test chemical in the concentration of 0, 250, 500 and 1000 mg/kg bw orally by gavage for aprrox 63 days. Statistically significant decrease were observed in number of rears (250, 500 and 1000 mg/kg bw at pre-treatment and 1000 mg/kg bw at Week 1), number of urine pools (250 mg/kg bw at Week 2) in male as compared to control. In recovery male, statistically significant increase in number of urine pools at week 4 in recovery at 1000 mg/kg bw as compared to recovery control. In female, statistically significant decrease was observed in number of fecal bolus (1000 mg/kg bw recovery at Week 1) as compared to control recovery group. The above changes observed were inconsistent/ biologically insignificant and not dose dependant hence considered as incidental and not attributed to the effect of test item administration. One female was found dead on day 41 at 1000 mg/kg bw due to gavaging error. Statistically significant decrease was observed in percent body weight change of 250, 500 and 1000 mg/kg bw on day 1-20 as compared to control group. Body weight and Percent body weight changes in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. These changes observed were inconsistent, hence not considered as effect of the test item administration. Statistically significant decrease in feed consumption was observed in 250, 500 and 1000 mg/kg bw female on gestation day 14-20 as compared to the control. Feed consumption in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. Changes observed in feed consumption were inconsistent, hence not considered as effect of the test item administration. No effect on Eye was observed in treated rats as compared to control. Similarly, At the end of treatment period revealed statistically significant decreased of aPTT in male rats at 1000 mg/kg, statistically significant increased of aPTT in female rats at 250 mg/kg while statistically significant decreased in Lymphocyte Count in female rats at 500 mg/kg. At the end of recovery period, all of the above altered parameters had returned to normal level similar to control group rats in both sexes. During treatment free recovery period, statistically significant increase was observed in PT of recovery male rats treated at 1000 mg/kg and statistically significant decreased was observed Hematocrit and Hemoglobin in recovery male rats treated at 1000 mg/kg, while statistically significant decreased of Monocyte in female recovery rats at 1000 mg/kg when compared to respective control group. The observed variations in PT, Hematocrit, Hemoglobin and monocyte were considered to be of no toxicological significance, minimal in nature and occurred in the absence of clear dose related response. At the end of treatment period revealed, statistically significant increase in Chloride of male rats treated at 500 mg/kg, Sodium of male rats treated at 1000 mg/kg. Statistically significant decrease in bile acid of male rats treated at 250 mg/kg and 1000 mg/kg respectively while statistically significant decrease in Triglyceride of female rats treated at 500 mg/kg. During treatment-free recovery period revealed statistically significant increase in Potassium in female rats treated at 1000 mg/kg while statistically decrease in Bile acid in female rats treated at 1000 mg/kg was observed when compared to respective control group. The observed variations in Potassium and Bile acid was considered incidental and not test item related as it was observed only in single sex, inconsistent, not dose dependent and further is not evidenced by histopathological observations. The sensory reactivity measurements were comparable and no changes were revealed in any of the animals of all treated groups in both the sexes. Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups. Motor activity measurements were comparable and no changes were revealed in any of the animals from all treated groups of both the sexes as compare to control group. The above changes observed were inconsistent, hence considered as incidental and not attributed to the effect of test item administration. At the end of treatment and recovery period, absolute and relative weight of organs of treated group rats of either sex did not differ significantly when compared to respective control group except, statistical significant increase absolute and relative adrenal weight in male rat at 250 mg/kg bw as compared to main control group. Statistical significant decrease relative testes and Epididymis weight in male rat at 1000 mg/kg bw was observed when compared to control rats. Statistical significant increased relative heart weight in male recovery rats was observed when compared to control rats. External examination of all male and female rats of control and all treated groups including recovery groups did not reveal any abnormality of pathological significance. Visceral examination of the rats of control and other treated groups did not reveal any pathological abnormality. Focal mild to multifocal mild mononuclear cell infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration and minimal chronic changes and hydropic degeneration in female liver, Unilateral: focal minimal to multifocal mild mononuclear cells infiltration

in male and focal minimal to multifocal minimal mononuclear cells infiltration in female Kidneys, focal mild to multifocal mild mononuclear cells infiltration in male and focal minimal to multifocal minimal

mononuclear cells infiltration in female Lungs and focal minimal to multifocal mild GALT hyperplasia in male and focal minimal to multifocal minimal GALT hyperplasia in female intestine at 250 and 1000 mg/kg bw, Vacuolar, degeneration multifocal, mild in male and diffuse vacuolations in female Adrenals at 250 mg/kg bw, minimal cyst in male and mild serosanginous fluid cavity in female Pituitary Gland, focal minimal to multifocal moderate Retention of sperm, focal minimal to focal mild Exfoliation of round spermatid, focal minimal to multifocal mild Seminiferous tubules Vacuolar degeneration, marked diffuse Necrosis of Seminiferous tubules, minimal diffuse Atrophy Seminiferous tubules, minimal to mild Vacuolar degeneration of Leydig cell, marked diffuse Necrosis of Epididymis, marked diffuse decrease in Epididymis sperm and Epididymis of Epididymis in male rat and segmental minimal Vacuolar degeneration of Ovary and minimal Perimetrium and myometrium vacuolations of Uterus in female rats were observed at different doses. Lesions observed in liver, kidneys, lungs, adrenals, intestine, pituitary gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies (Boorman et al., 1990, Greaves, 2007, Greaves and Faccini, 1992; Haschek et al., 2002). Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item. In addition, no reproductive toxicity were observed in treated rats such as Gestational length, Litter size, No. of live births, Post-implantation loss, Post-natal loss, Pregnancy index, Pups sex ratio, survival, body weight on PND 4 and gross pathology of treated rats were observed as compared to control. One females showed prolonged diestrous i.e more than 3 days with total estrous cycle period of 6 days or more before mating period at 250 and 1000 mg/kg bw. Therefore, No Observed Adverse Effect Level (NOAEL) of the test chemical is considered 1000 mg/kg body weight when Wistar male and female rats orally by gavage for Approx 63 days.

Study 2

In a Teratogenic Potential Test, CD Sprague-Dawley female rats treated with test material in the concentration of 0, 100, 500 or 1500 mg/kg bw orally by gavage in water for 14 days (days 6-19 of gestation). Six rats died during the dosing period at 1500 mg/kg bw as compared to control. Survival was 100% in the controls and the groups receiving 100 and 500 mg/kg of dye. Slight reductions in body-weight gains at 1500 mg/kg bw and Orange discoloration of the urine was noted in all treated rats during the treatment period. No effect on reproductive parameters such as Viable and non-viable fetuses, early and late resorptions, total implantations and corpora lutea and sex ratio of fetuses were observed in treated female rats as compared to control. Green discoloration of the amniotic fluid was observed in 1, 10 and 16 rats at 100, 500 and 1500 mg/kg/day groups, respectively, and the small intestines were green in colour in many rats at 500 mg/Kg group. In addition, No effect on Body weight offetuses were observed as compared to control. A slight increase in the number of litters with unossified sternebrae (sternebrae nos 1-6) and rudimentary 14th rib(s) was observed at 1500 mg/kg bw however, these values fell within the ranges of historical control data. No biologically meaningful or statistically significant differences in the number of litters and number of fetuses with malformations and number of fetuses or litters with developmental variations were observed in treated rats as compared to control. Therefore, NOAEL was considered to be 1500 mg/kg/day for P and F1 generation when CD Sprague- Dawley female rats treated with test material orally by gavage for 14 days.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

Klimisch Rating 1

Additional information

Data available from different studies were reviewed to determine the reproductive toxicity of test chemical.The studies are as mentioned below:

Study 1

In a Repeated Dose Oral Toxicity Study in combination with Reproduction/ Developmental Toxicity study, Wistar male and female rats were treated with test chemical in the concentration of 0, 250, 500 and 1000 mg/kg bw orally by gavage for aprrox 63 days. Statistically significant decrease were observed in number of rears (250, 500 and 1000 mg/kg bw at pre-treatment and 1000 mg/kg bw at Week 1), number of urine pools (250 mg/kg bw at Week 2) in male as compared to control. In recovery male, statistically significant increase in number of urine pools at week 4 in recovery at 1000 mg/kg bw as compared to recovery control. In female, statistically significant decrease was observed in number of fecal bolus (1000 mg/kg bw recovery at Week 1) as compared to control recovery group. The above changes observed were inconsistent/ biologically insignificant and not dose dependant hence considered as incidental and not attributed to the effect of test item administration. One female was found dead on day 41 at 1000 mg/kg bw due to gavaging error. Statistically significant decrease was observed in percent body weight change of 250, 500 and 1000 mg/kg bw on day 1-20 as compared to control group. Body weight and Percent body weight changes in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. These changes observed were inconsistent, hence not considered as effect of the test item administration. Statistically significant decrease in feed consumption was observed in 250, 500 and 1000 mg/kg bw female on gestation day 14-20 as compared to the control. Feed consumption in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. Changes observed in feed consumption were inconsistent, hence not considered as effect of the test item administration. No effect on Eye was observed in treated rats as compared to control. Similarly, At the end of treatment period revealed statistically significant decreased of aPTT in male rats at 1000 mg/kg, statistically significant increased of aPTT in female rats at 250 mg/kg while statistically significant decreased in Lymphocyte Count in female rats at 500 mg/kg. At the end of recovery period, all of the above altered parameters had returned to normal level similar to control group rats in both sexes. During treatment free recovery period, statistically significant increase was observed in PT of recovery male rats treated at 1000 mg/kg and statistically significant decreased was observed Hematocrit and Hemoglobin in recovery male rats treated at 1000 mg/kg, while statistically significant decreased of Monocyte in female recovery rats at 1000 mg/kg when compared to respective control group. The observed variations in PT, Hematocrit, Hemoglobin and monocyte were considered to be of no toxicological significance, minimal in nature and occurred in the absence of clear dose related response. At the end of treatment period revealed, statistically significant increase in Chloride of male rats treated at 500 mg/kg, Sodium of male rats treated at 1000 mg/kg. Statistically significant decrease in bile acid of male rats treated at 250 mg/kg and 1000 mg/kg respectively while statistically significant decrease in Triglyceride of female rats treated at 500 mg/kg. During treatment-free recovery period revealed statistically significant increase in Potassium in female rats treated at 1000 mg/kg while statistically decrease in Bile acid in female rats treated at 1000 mg/kg was observed when compared to respective control group. The observed variations in Potassium and Bile acid was considered incidental and not test item related as it was observed only in single sex, inconsistent, not dose dependent and further is not evidenced by histopathological observations. The sensory reactivity measurements were comparable and no changes were revealed in any of the animals of all treated groups in both the sexes. Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups. Motor activity measurements were comparable and no changes were revealed in any of the animals from all treated groups of both the sexes as compare to control group. The above changes observed were inconsistent, hence considered as incidental and not attributed to the effect of test item administration. At the end of treatment and recovery period, absolute and relative weight of organs of treated group rats of either sex did not differ significantly when compared to respective control group except, statistical significant increase absolute and relative adrenal weight in male rat at 250 mg/kg bw as compared to main control group. Statistical significant decrease relative testes and Epididymis weight in male rat at 1000 mg/kg bw was observed when compared to control rats. Statistical significant increased relative heart weight in male recovery rats was observed when compared to control rats. External examination of all male and female rats of control and all treated groups including recovery groups did not reveal any abnormality of pathological significance. Visceral examination of the rats of control and other treated groups did not reveal any pathological abnormality. Focal mild to multifocal mild mononuclear cell infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration and minimal chronic changes and hydropic degeneration in female liver, Unilateral: focal minimal to multifocal mild mononuclear cells infiltration

in male and focal minimal to multifocal minimal mononuclear cells infiltration in female Kidneys, focal mild to multifocal mild mononuclear cells infiltration in male and focal minimal to multifocal minimal

mononuclear cells infiltration in female Lungs and focal minimal to multifocal mild GALT hyperplasia in male and focal minimal to multifocal minimal GALT hyperplasia in female intestine at 250 and 1000 mg/kg bw, Vacuolar, degeneration multifocal, mild in male and diffuse vacuolations in female Adrenals at 250 mg/kg bw, minimal cyst in male and mild serosanginous fluid cavity in female Pituitary Gland, focal minimal to multifocal moderate Retention of sperm, focal minimal to focal mild Exfoliation of round spermatid, focal minimal to multifocal mild Seminiferous tubules Vacuolar degeneration, marked diffuse Necrosis of Seminiferous tubules, minimal diffuse Atrophy Seminiferous tubules, minimal to mild Vacuolar degeneration of Leydig cell, marked diffuse Necrosis of Epididymis, marked diffuse decrease in Epididymis sperm and Epididymis of Epididymis in male rat and segmental minimal Vacuolar degeneration of Ovary and minimal Perimetrium and myometrium vacuolations of Uterus in female rats were observed at different doses. Lesions observed in liver, kidneys, lungs, adrenals, intestine, pituitary gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies (Boorman et al., 1990, Greaves, 2007, Greaves and Faccini, 1992; Haschek et al., 2002). Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item. In addition, no reproductive toxicity were observed in treated rats such as Gestational length, Litter size, No. of live births, Post-implantation loss, Post-natal loss, Pregnancy index, Pups sex ratio, survival, body weight on PND 4 and gross pathology of treated rats were observed as compared to control. One females showed prolonged diestrous i.e more than 3 days with total estrous cycle period of 6 days or more before mating period at 250 and 1000 mg/kg bw. Therefore, No Observed Adverse Effect Level (NOAEL) of the test chemical is considered 1000 mg/kg body weight when Wistar male and female rats orally by gavage for Approx 63 days.

Study 2

In a Teratogenic Potential Test, CD Sprague-Dawley female rats treated with test material in the concentration of 0, 100, 500 or 1500 mg/kg bw orally by gavage in water for 14 days (days 6-19 of gestation). Six rats died during the dosing period at 1500 mg/kg bw as compared to control. Survival was 100% in the controls and the groups receiving 100 and 500 mg/kg of dye. Slight reductions in body-weight gains at 1500 mg/kg bw and Orange discoloration of the urine was noted in all treated rats during the treatment period. No effect on reproductive parameters such as Viable and non-viable fetuses, early and late resorptions, total implantations and corpora lutea and sex ratio of fetuses were observed in treated female rats as compared to control. Green discoloration of the amniotic fluid was observed in 1, 10 and 16 rats at 100, 500 and 1500 mg/kg/day groups, respectively, and the small intestines were green in colour in many rats at 500 mg/Kg group. In addition, No effect on Body weight offetuses were observed as compared to control. A slight increase in the number of litters with unossified sternebrae (sternebrae nos 1-6) and rudimentary 14th rib(s) was observed at 1500 mg/kg bw however, these values fell within the ranges of historical control data. No biologically meaningful or statistically significant differences in the number of litters and number of fetuses with malformations and number of fetuses or litters with developmental variations were observed in treated rats as compared to control. Therefore, NOAEL was considered to be 1500 mg/kg/day for P and F1 generation when CD Sprague- Dawley female rats treated with test material orally by gavage for 14 days.

Justification for classification or non-classification

No Observed Adverse Effect Level (NOAEL) of the test chemical is considered 1000-1500 mg/kg body weight when Wistar male and female rats orally by gavage for Approx 63 days.