[[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data from experimental study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Principles of method if other than guideline:

Reproduction/Developmental toxicity screening test of test chemical by oral gavage in sprague dawley rats.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

[[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile

EC Number:

258-964-5

EC Name:

[[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile

Cas Number:

54079-53-7

Molecular formula:

C27H31N3O

IUPAC Name:

2-[(4-{[2-(4-cyclohexylphenoxy)ethyl](ethyl)amino}-2-methylphenyl)methylidene]propanedinitrile

Details on test material:

- Name of test material (as cited in study report): [[4-[[2-(4-cyclohexylphenoxy)ethyl]ethylamino]-2-methylphenyl]methylene]malononitrile
- Molecular formula: C27H31N3O
- Molecular weight: 413.562 g/mol
- Substance type: Organic
- Physical state: Solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Details on test animal
TEST ANIMALS
- Source:
- Age at study initiation: 10 to 13 weeks
- Weight at study initiation: Males: 230.23 g to 279.99 g
Females: 200.03 g to 247.48 g
- Fasting period before study:
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.

- Diet (e.g. ad libitum): Altromin Maintenance diet for rats and mice 1324 manufactured by AltrominSpezialfutter GmbH & Co. KG was provided ad libitum
- Water (e.g. ad libitum): Water was provided ad libitum
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 to 23.9oC
- Humidity (%):41 to 69%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% w/v carboxy methyl cellulose

Details on exposure:

Details on exposure
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were freshly prepared before dose administration on each day. The required quantity of test item was weighed in an aluminum foil, transferred in to a mortar and grind well in a mortar and pestle by adding small quantity of 0.5% w/v Carboxy Methyl Cellulose until a clear suspension is formed. Thereafter, the entire quantity of the formulation was transferred into a measuring cylinder. Again, a small quantity of the vehicle was added to rinse the mortar and pestle and this was transferred into the measuring cylinder. The rinsing of mortar and pestle was repeated to ensure the complete transfer of the contents to the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get a desired concentration .
VEHICLE
- Justification for use and choice of vehicle (if other than water): 0, 250, 500 and 1000 mg/kg body weight
- Concentration in vehicle: - Amount of vehicle (if gavage): 10 mL/kg body weight.
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

Details of mating
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy)
- After … days of unsuccessful pairing replacement of first male by another male with proven fertility. No data available.
- Further matings after two unsuccessful attempts: [no / yes (explain)] No data available.
- After successful mating each pregnant female was caged (how): Males were housed with their former cage mates while females were housed individually.

Duration of treatment / exposure:

Male : 33 days
Female : two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13

Frequency of treatment:

Daily

Duration of test:

Approx 64 days

Doses / concentrations

Remarks:

0, 250, 500 and 1000 mg/kg body weight

No. of animals per sex per dose:

96 (48 males + 48 females)
0 mg/kg body weight : 12 male and 12 female
250 mg/kg body weight : 12 male and 12 female
500mg/kg body weight : 12 male and 12 female
1000 mg/kg body weight : 12 male and 12 female

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

Parental animals observation and examinations
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed for clinical signs once daily, mortality and morbidity twice daily,
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes , weekly once,

BODY WEIGHT: Yes
- Time schedule for examinations: weekly once.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly once. Feed consumption was not measured during mating period for both males and females. Thereafter, feed consumption for females was recorded during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4, 4 to 7 and 7 to 13.

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: No data
- Skeletal examinations: No data
- Head examinations: No data

Statistics:

The raw data was subjected to computer statistical processing. The computer printout of the data (in the form of appendix) was verified with the raw data.After verification, the data was subjected to various statistical analyses using SPSS software version 22.
All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05), indicated by the aforementioned tests designated by the superscripts throughout the report as stated below: * Statistically significant (P<0.05) change than the vehicle control group.
Type of Analysis Parametric - One-way ANOVA with Dunnett’s post test
Non Parametric - Kruskal-Wallis followed by the Mann-Whitney test if differences were indicated
Cross Tabs -Chi-square test/ Fischer's Exact Test
Note: Data of females mated but not littered was excluded from statistical analysis.

Indices:

Fertility index for dams, sires and pup live birth index, mean litter size per group, survival index and sex ratio at birth were calculated.

Historical control data:

No data available.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity was observed at any of the tested dose group animals of either sex during the experimental period.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

There were no mortality/morbidity observed at any of the tested dose group animals of either sex during the experimental period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes observed in mean body weight and percent change in body weight with respect to day 1 at all the tested group animals of either sex during the experimental period. There were no changes observed in the gestation body weight and percent change in gestation body weight during gestation period at any of the tested dose group animals when compared with vehicle control group animals.
There were no changes observed in the lactation body weight and percent change in lactation body weight at any of the tested dose group animals when compared with vehicle control group animals.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no changes observed in feed consumption at any of the tested dose group animals of either sex during the experimental period. There were no changes observed in the feed consumption during gestation period at any of the tested dose group animals when compared with vehicle control group animals.

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:

no effects observed

Description (incidence and severity):

There were no effects observed in absolute and relative organ weights at any of the tested dose group animals of either sex when compared with vehicle control group

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes (both external and internal) observed at all the tested dose group adult animals and pups of either sex at all the tested dose groups examined at termination. However, one male from G3 group observed with bilateral reduction of testes and epididymides and one male from G4 group observed with unilateral reduced size testes and epididymides.This change is considered as incidental but not treatment related

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment related histopathological findings noticed in the study.
Lesions considered spontaneous and incidental were observed in treated and control rats. These lesions consisted of mononuclear cell (MNC) infiltration at ovarian bursa (one female from G1 group and one female from G4 group).
Minimal degeneration of seminiferous tubules of testes and reduction in sperm content in epididymis unilaterally was observed in one male animal of G4 group. This lesion considered as incidental lesion because of unilateral occurrence and lack of consistency in the group.
One male animal from G3 group showed marked degeneration of seminiferous tubules, bilaterally and epididymis showed reduction in sperm content bilaterally. This lesion considered as incidental because G4 group (high dose) did not showed consistency in findings only one animal reveled minimal seminiferous degenerationthat to be unilateral in nature.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

There were no changes observed in the gestation length

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

mortality

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Remarks on result:

other: No toxic effects were observed

Maternal abnormalities

Abnormalities:

not specified

Localisation:

not specified

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no changes observed in mean pup (male and female) weight on lactation day 1, 4, 7 and 13 at any of the tested dose groups when compared with vehicle control group dams.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no treatment related changes observed in the number of pups of each litter at any of the tested dose group animals during lactation period when compared with vehicle control group animals.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no treatment related changes observed in the sex ratio of each litter at any of the tested dose group animals during lactation period when compared with vehicle control group animals.

Changes in litter size and weights:

not specified

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

There were no treatment related changes observed in the pup survival index of each litter at any of the tested dose group animals during lactation period when compared with vehicle control group animals.

External malformations:

no effects observed

Description (incidence and severity):

There was no external anomalies observed in any of the pups of tested dose group animals during lactation period.

Skeletal malformations:

not specified

Visceral malformations:

not specified

Other effects:

no effects observed

Description (incidence and severity):

There were no changes observed in ano-genital distance ratio of both male and female pups recorded on lactation day 4 at any of the tested dose group litters when compared with vehicle control group litters.
There were no occurrences of nipples in male pups of dams at any of the tested dose group litters and vehicle control group litters observed on lactation day 13.
There were no treatment related changes observed in serum T4 levels in adult males and lactation day 13 pups at any of the tested dose groups when compared with vehicle control group.
However, statistically significant decrease in serum T4 levels of lactation day 13 pups in G2 and G4 was noted when compared with vehicle control group lactation day 13 pups. This change is considered as incidental but not treatment related as there were no dose dependency was noted and also the values are within the biological range of the species.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

changes in sex ratio

fetal/pup body weight changes

changes in postnatal survival

external malformations

Remarks on result:

other: No developmental toxic effects were observed

Fetal abnormalities

Abnormalities:

not specified

Localisation:

other: not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Treatment related:

not specified

Any other information on results incl. tables

SUMMARY OF THE STUDY

Parameters ↓		Group & Dose (mg/kg body weight/day)
		G1 & 0	G2 &100	G3 &300	G4 &1000
Reproductive Indices
Mating Indices
Pairs started (No.)	12		12	12	12
Males showing evidence of mating (No.) within 14 days	12		12	12	12
Females showing evidence of copulation (No.)	12		12	12	12
Male Mating Index (%) within 14 days	100.00		100.00	100.00	1000.00
Female Mating Index (%)	100.00		100.00	100.00	100.00
Fertility Indices
Females achieving pregnancy (No.)	10		11	10	10
Male Fertility Index (%)	83.33		91.67	83.33	83.33
Female Fertility Index (%)	83.33		91.67	83.33	83.33
Copulatory Indices
Conceiving days 1 to 5 (No.)	6		7	8	8
Conceiving days >6 (No.)	6		5	4	4
Mean Precoital Interval (Days)	7.00		4.33	4.75	6.17
Gestation Indices
Pregnancy≤21 days (No.)	1		1	0	1
Pregnancy = 22 days (No.)	6		6	6	5
Pregnancy≥23 days (No.)	3		4	4	4
Mean Gestation length (Days)	22.50		22.36	22.50	22.60
Gestation Index (%)	100.00		100.00	100.00	100.00
Dams with live young born (No.)	10		11	10	10
Dams with live young at day 4 post-partum (No.)	10		11	10	10
Dams with live young at day 13 post-partum (No.)	10		11	10	10
Implantation Index and Pre and Post implantation Losses
Implants/dam (Mean)	10.90		10.91	12.00	10.60
Corpora luetea/dam (Mean)	10.90		10.91	12.00	10.60
Implantation Index (%)	100.00		100.00	100.00	100.00
Pre-Implantation Loss (%)	0.00		0.00	0.00	0.00
Post-Implantation Loss (%)	0.00		0.00	0.00	0.00
Offspring Viability Indices
Live Birth Indices and Sex Ratio at Birth
Live pups/dam at birth (mean)	10.80		10.91	12.00	9.20
Litter Size (Total No. of pups born/dam) at birth (mean)	10.90		10.91	12.00	9.20
Mean Live Birth Index/dam (%)	99.29		100.00	100.00	100.00
Male Live pups/dam at birth (mean)	5.30		5.45	5.90	5.20
Female Live pups/dam at birth (mean)	5.50		5.45	6.10	4.00
Sex Ratio (male/female)	1.08		1.31	1.11	1.98

 

SUMMARY OF THE STUDY (Contd..,).

Parameters ↓	Group & Dose (mg/kg body weight/day)
	G1 & 0	G2 & 100	G3 & 300	G4 & 1000
Pup Survival Indices and Sex Ratio during lactation
Mean No. of Pups survived per dam ( LD1 to 4)	10.80	10.91	12.00	9.20
Mean No. of Pups dead per dam ( LD1 to 4)	0.00	0.00	0.00	0.00
Mean Pup Survival Index (%) per dam (LD1 to 4)	100.00	100.00	100.00	100.00
Sex Ratio (male/female) per dam at LD 4	1.08	1.31	1.11	1.98
Mean No. of Pups Sacrificed for Blood Collection on LD4	0.80	1.09	1.60	0.40
Mean No. of Pups survived per dam (LD4 to 7)	5.30	5.45	5.90	5.20
Mean No. of Pups dead per dam (LD4 to 7)	0.00	0.00	0.00	0.00
Mean Pup Survival Index (%) per dam (LD4 to 7)	100.00	100.00	100.00	100.00
Sex Ratio (male/female) per dam at LD 7	1.24	1.52	1.63	2.14
Mean No. of Pups survived per dam (LD7 to 13)	10.00	9.82	10.40	8.80
Mean No. of Pups dead per dam (LD7 to 13)	0.00	0.00	0.00	0.00
Mean Pup Survival Index (%) per dam ( LD7 to 13)	100.00	100.00	100.00	100.00
Sex Ratio (male/female) per dam at LD 13	1.24	1.52	1.63	2.14
Pre and Postnatal loss
Mean Pre-natal (implantations minus live births) (No.)	0.10	0.00	0.00	1.40
Females with 0 (No.)	9	11	10	10
Females with 1 (No.)	1	0	0	0
Post-natal (live births minus alive at post natal day 13)
Females with 0 (No.)	10	11	10	10
Females with ≥ 1 (No.)	0	0	0	0
Litter Observations
Male Pup weight at birth (mean) in gram	6.93	7.02	6.49	6.82
Female Pup weight at birth (mean) in gram	6.12	6.11	5.68	6.31
Male Pup weight on LD4 (mean) in gram	11.63	11.46	11.31	11.23
Female Pup weight on LD4 (mean) in gram	10.51	10.38	10.23	10.53
Male Pup weight on LD7 (mean) in gram	15.65	15.49	15.40	15.43
Female Pup weight on LD7 (mean) in gram	14.95	14.68	14.50	14.92
Male Pup weight on LD13 (mean) in gram	25.46	25.89	26.39	25.74
Female Pup weight on LD13 (mean) in gram	24.64	24.57	25.18	24.80

 

SUMMARYOF CLINICAL SIGNS OF TOXICITY, DETAILED CLINICAL EXAMINATIONAND MORTALITY RECORD

Group, Sex & Dose (mg/kg body weight/day)	No. of Animals	Clinical Signs of Toxicity/ Detailed Clinical Examination	Mortality (No. of Mortality / No. of Animals dosed)
G1, M & 0	12	N	0/12
G2, M &250	12	N	0/12
G3, M &500	12	N	0/12
G4, M &1000	12	N	0/12

M: Male; N: Normal

 

(Contd..,). SUMMARYOF CLINICAL SIGNS OF TOXICITY, DETAILED CLINICAL EXAMINATION AND MORTALITY RECORD

Group, Sex & Dose (mg/kg body weight/day)	No. of Animals [including pregnant and non-pregnant females]	Clinical Signs of Toxicity/ Detailed Clinical Examination	Mortality (No. of Mortality / No. of Animals dosed)
G1, F & 0	12	N	0/12
G2, F &250	12	N	0/12
G3, F &500	12	N	0/12
G4, F & 1000	12	N	0/12

F: Female; N: Normal

 

SUMMARY OF BODY WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Days
		1	8	15	22	29
G1, M & 0	Mean	346.37	368.27	392.04	404.31	413.00
	±SD	22.04	29.17	35.32	36.86	36.35
	n	12	12	12	12	12
G2, M & 250	Mean	346.84	382.58	411.67	432.84	440.44
	±SD	24.73	42.11	40.98	44.49	40.24
	n	12	12	12	12	12
G3, M & 500	Mean	348.81	376.72	408.42	415.61	427.93
	±SD	21.92	23.97	39.43	38.53	38.64
	n	12	12	12	12	12
G4, M & 1000	Mean	343.64	376.73	402.78	412.50	424.64
	±SD	18.00	25.27	31.81	33.30	33.94
	n	12	12	12	12	12

M: Male;SD: Standard Deviation; n: Number of Animals          

 

Contd..,). SUMMARY OF BODY WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Days
		1	8	15	22
G1, F & 0	Mean	239.53	249.54	257.03	260.06
	±SD	8.79	10.48	12.44	20.50
	n	12	12	12	4
G2, F & 250	Mean	240.00	247.37	250.47	255.36
	±SD	6.94	10.00	14.33	6.82
	n	12	12	12	2
G3, F & 500	Mean	241.14	248.50	255.06	249.42
	±SD	8.90	11.42	13.23	12.32
	n	12	12	12	2
G4, F & 1000	Mean	236.30	246.54	251.16	259.25
	±SD	6.98	9.64	10.77	9.45
	n	12	12	12	4

F: Female; SD: Standard Deviation; n: Number of Animals

#: The statistical analysis was not performed for day 21 body weight as the presented mean and standard deviations were calculated only for the females in cohabitation.

 

SUMMARY OF PERCENTCHANGE IN BODY WEIGHT (%) WITH RESPECT TO DAY 1 RECORD

                                                         

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Days
		1 to 8	1 to 14	1 to 22	1 to 29
G1, M & 0	Mean	6.33	13.16	16.76	19.33
	±SD	5.27	7.08	8.39	9.23
	n	12	12	12	12
G2, M & 250	Mean	10.38	18.85	25.12	27.35
	±SD	10.63	10.67	13.62	12.87
	n	12	12	12	12
G3, M & 500	Mean	8.06	17.01	19.09	22.63
	±SD	3.91	6.73	6.69	6.75
	n	12	12	12	12
G4, M & 1000	Mean	9.64	17.17	20.02	23.60
	±SD	4.63	5.91	6.85	7.73
	n	12	12	12	12

M: Male; SD: Standard Deviation; n: Number of Animals

 

 

Contd..,). SUMMARY OF PERCENTCHANGE IN BODY WEIGHT (%) WITH RESPECT TO DAY 1 RECORD

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Days
		1 to 8	1 to 14	1 to 22^#
G1, F & 0	Mean	4.19	7.30	8.96
	±SD	2.36	3.02	3.29
	n	12	12	5
G2, F & 250	Mean	3.06	4.32	3.31
	±SD	2.03	3.83	1.23
	n	12	12	2
G3, F & 500	Mean	3.04	5.77	6.46
	±SD	2.28	3.63	4.02
	n	12	12	2
G4, F & 1000	Mean	4.37	6.36	10.41
	±SD	3.97	5.37	4.91
	n	12	12	4

F: Female; SD: Standard Deviation; n: Number of Animals

#: The statistical analysis is not performed as the presented mean and standard deviations were calculated only for the females in cohabitation.

SUMMARY OF FEED CONSUMPTION (g/rat/day)RECORD

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g/rat/day)
		Week 1	Week 2	Post Mating
G1, M & 0	Mean	21.32	22.66	24.28
	±SD	0.38	0.52	0.37
	n	12	12	12
G2, M & 250	Mean	21.22	22.50	24.30
	±SD	0.32	0.49	0.25
	n	12	12	12
G3, M & 500	Mean	21.10	22.15	24.14
	±SD	0.51	0.36	0.23
	n	12	12	12
G4, M & 1000	Mean	21.30	22.62	24.28
	±SD	0.48	0.48	0.27
	n	12	12	12

M: Male;SD: Standard Deviation; n: Number ofanimals[two animals were housed in one cage]

 

(Contd..,). SUMMARY OF FFED CONSUMPTION (g/rat/day) RECORD

Group, Sex & Dose (mg/kg body weight/day)				Feed Consumption (g/rat/day) during pre-mating
			Week 1		Week 2
G1, F & 0		Mean		14.39		15.90
		±SD		0.23		0.51
		n		12		12
G2, F & 250		Mean		14.34		15.77
		±SD		0.41		0.63
		n		12		12
G3, F & 500		Mean		14.26		15.46
		±SD		0.22		0.43
		n		12		12
G4, F & 1000		Mean		14.38		15.63
		±SD		0.36		0.37
		n		12		12

F: Female; SD: Standard Deviation;n: Number of animals [two animals were housed in one cage]

 

SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Epididymes	Testes	Prostate+Seminal vesicles with coagulating glands (PSC)	Thyroid along with parathyroid^#
G1, M & 0	Mean	1.6255	3.3629	3.7096	0.0268
	±SD	0.1841	0.2914	0.5694	0.0063
	n	12	12	12	12
G2, M &250	Mean	1.7034	3.5546	4.1689	0.0303
	±SD	0.2302	0.3057	0.9767	0.0082
	n	12	12	12	12
G3, M &500	Mean	1.5827	3.2761	3.4957	0.0275
	±SD	0.3001	0.7072	0.4025	0.0059
	n	12	12	12	12
G4, M & 1000	Mean	1.4669	3.1611	3.4799	0.0281
	±SD	0.2855	0.6820	0.5632	0.0042
	n	12	12	12	12

M: Male;SD: Standard Deviation; n: Number of animals; #: Weighed post fixation

 

(Contd..,). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		#Thyroid along with parathyroid
G1, F & 0	Mean	0.0273
	±SD	0.0049
	n	10
G2, F & 250	Mean	0.0256
	±SD	0.0038
	n	11
G3, F & 500	Mean	0.0310
	±SD	0.0057
	n	10
G4, F & 1000	Mean	0.0288
	±SD	0.0050
	n	10

F: Female;SD: Standard Deviation; n: Number of animals;

#: Weighed post fixation (excluded non-pregnant animals for mean calculations)

SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT (%) RELATIVE TO TERMINALBODY WEIGHT RECORD

Group, Sex & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Epididymes	Testes	Prostate+Seminal vesicles with coagulating glands (PSC)	Thyroid along with parathyroid
G1, M & 0	Mean	405.22	0.4021	0.8324	0.9208	0.0066
	±SD	36.53	0.0380	0.0606	0.1453	0.0016
	n	12	12	12	12	12
G2, M &250	Mean	430.16	0.3965	0.8301	0.9676	0.0072
	±SD	41.05	0.0430	0.0732	0.1915	0.0027
	n	12	12	12	12	12
G3, M & 500	Mean	418.65	0.3797	0.7868	0.8367	0.0066
	±SD	39.47	0.0746	0.1799	0.0821	0.0012
	n	12	12	12	12	12
G4, M & 1000	Mean	419.16	0.3500	0.7534	0.8289	0.0067
	±SD	31.90	0.0639	0.1581	0.1142	0.0011
	n	12	12	12	12	12

M: Male;SD: Standard Deviation; n: Number of animals

 

 

TABLE 6 (Contd..,). SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT (%) RELATIVE TO TERMINALBODY WEIGHT RECORD

Group, Sex & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Thyroid along with parathyroid#
G1, F & 0	Mean	315.65	0.0086
	±SD	24.99	0.0013
	n	10	10
G2, F & 250	Mean	306.54	0.0084
	±SD	33.48	0.0011
	n	11	11
G3, F & 500	Mean	306.82	0.0101
	±SD	21.80	0.0019
	n	10	10
G4, F & 1000	Mean	295.65	0.0098
	±SD	16.24	0.0020
	n	10	10

F: Female;SD: Standard Deviation; n: Number of animals

Note: excluded non-pregnant animals for mean calculations

 

SUMMARY RECORD OF VAGINAL SMEAR EXAMINATION FOR DETERMINATION OF OESTRUS CYCLICITY

Group & Dose (mg/kg body weight/day)	Total No. of Females	No. of Females with Regular Oestrus Cyclicity during Pre-mating and Mating	No. of Females confirmed with pregnancy	No. of Females with Regular Oestrus Cyclicity on Lactation Day 14	No. of Females with Irregular Oestrus Cyclicity during Pre-mating, Mating and on Lactation day 14
G1 & 0	12	12	10	10	0
G2 & 250	12	12	11	11	0
G3 & 500	12	12	10	10	0
G4 & 1000	12	12	10	10	0

Applicant's summary and conclusion

Conclusions:

NOAEL was considered to be 1000 mg /kg bw/day for reproductive and developmental toxicity in P0 and F1 Sprague Dawley rats , when they were exposed with test chemical orally.

Executive summary:

Reproduction/Developmental toxicity screening of test chemical was performed according to OECD guideline 421 on male and femaleSprague Dawley. A total of 96 (48 males + 48 females) Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3and G4) consisted of 12 males and 12 females. The animals in G1 group were administered with vehicle [0.5% w/v carboxy methyl cellulose],animals in G2, G3 and G4groups were administered withtest chemical dose levels of250, 500 and 1000 mg/kg body weight for low dose, mid dose and high dose groups respectively.The vehicleand test chemical formulationswere administered orally by gavage at the dose volume of 10 mL/kg body weight.Males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 33 days of treatment). The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed).  

All animals were observed for clinical signs once daily, mortality and morbidity twice daily, detailed clinical examination weekly once, body weight and feed consumption weekly once. The serum collected from adult males and lactation day 13 pups representing each per litter was screened for T4 levels. Females were observed for oestrus cyclicity during pre-mating treatment and mating treatment period and the dams on lactation day 14 prior to sacrifice. The females were observed for copulatory interval and all the adult animals were observed for mating and fertility index. Each litter was examined after delivery (lactation day 1) and the number and sex of pups (litter size), stillbirths (dead pups born on day 1) and live births were recorded. The dams were observed for body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index throughout the lactation period. The pups were observed for clinical signs and external examinations once daily from lactation day 1 to 13. The both male and female pup weights were recorded separately on lactation days 1, 4, 7 and 13. The anogenital distance of each pup was measured on lactation day 4. The male pups were observed for retention of nipples/areolae on lactation day 13. Gross pathology and organ weighing were performed on day 34 for males and on lactation day 14 for dams. Gross pathology was performed on lactation day 4/13 for pups. The number of corpora lutea and implantation sites for dams were recorded during necropsy.

 All the tested dose group animals of either sex did not reveal any clinical signs of toxicity and no mortality/morbidity observed. There were no changes observed in mean body weight, percent change in body weight with respect to day 1 and feed consumption at all the tested dose group animals of either sex during the experimental period. No changes in organ weights (both absolute and relative) were observed at all the tested dose group animals. There were no treatment related changes observed in serum T4 levels of adult males and in serum T4 levels of lactation day 13 pups at any of the tested dose groups.Dams did not reveal any treatment related changes in oestrus cyclicity, copulatory interval, body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index at all the tested dose groups throughout the lactation period.All pups did not reveal any clinical signs or external anomalies throughout the lactation period. No treatment related changes in pup weights, ano-genital distance ratio were noted. No occurrences of nipples in male pups at any of the tested dose groups and vehicle control group.There were no treatment related gross pathological changes (both external and internal) observed at all the tested dose group adult animals and pups. There were no treatment related histopathological findings noticed during the microscopic examination. Testes were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all germ layers (cells). In addition this, ovaries did not show any pathological findings/lesions. HenceNOAEL was considered to be  1000 mg /kg bw/day for reproductive and developmental toxicity in P0 and F1 Sprague Dawley rats , when they were exposed with test chemical orally