2-(3-oxobenzo[b]thien-2(3H)-ylidene)benzo[b]thiophene-3(2H)-one

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive toxicity study

Based on the data available from different studies, NOAEL for 3H,3'H-2,2'-bi-1-benzothiophene-3,3'-dione; Thioindigo (CAS no. 522-75-8)was considered to be in range of 250-500mg/kg bw/day .When male and female rats were treated with 3H,3'H-2,2'-bi-1-benzothiophene-3,3'-dione; Thioindigo orally. Thus, comparing this value with the criteria ofCLP regulation3H,3'H-2,2'-bi-1-benzothiophene-3,3'-dione; Thioindigo (CAS no. 522-75-8)isnot likely to classify as reproductive toxicant.

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:

Experimental data of read across substances

Justification for type of information:

Weight of evidence approach based on structurally similar chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

WoE report is based on two reproductive toxicity studies on rats
1.Three-generation reproduction toxicity study of test material in Rats.
2.Reproductive and developmental toxicity study of test material was performed on wistar rats.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: 1.CD-1 2.Harlan-Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

1.TEST ANIMALS
- Source: Charles River Breeding Laboratories (Wilmington, MA)
- Age at study initiation: (P) x wks; (F1) x wks: P- 80-85 days
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g: No data available
- Fasting period before study: No data available
- Housing: Animals were housed individually in stainless-steel wire-mesh cages, except during the mating, lactation and post-weaning periods when the females were placed in plastic shoe-box cages containing bedding. Each rat was identified with a metal ear tag. If the tag was lost, the animal was re-tagged and/or toe-clipped.
- Diet (e.g. ad libitum): Basal diet (Purina Rodent Chow from Ralston Purina Co., Inc., St Louis, MO), ad libitum
- Water (e.g. ad libitum): Water, ad libitum
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21°C
- Humidity (%):40-60%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycle

Route of administration:

oral: feed

Type of inhalation exposure (if applicable):

not specified

Vehicle:

other: Basal diet

Details on exposure:

1.PREPARATION OF DOSING SOLUTIONS: Test material was mixed with the powdered chow in a twin shell blender to provide theoretical dose levels of 2.5, 25, 75 and 250 mg/kg/day.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Basal diet (Purina Rodent Chow from Ralston Purina Co., Inc., St Louis, MO)
- Storage temperature of food: Stored in environmentally controlled room with limited access.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Basal diet
- Concentration in vehicle: 0, 2.5, 25, 75 and 250 mg/kg/day.
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

Details on mating procedure:

Details on study schedule
- F1 parental animals not mated until [...] weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were [...] days of age.
- Age at mating of the mated animals in the study: [...] weeks
(Explain how study was performed on perents and offspring separately whatever information we have)
F0 geneartion:
F0 animals were meated twice to give F1a and F1b
F1a - on 21-day examined for external abnormalities and killed.
F1b - random selections were made of
ten male and 20 female pups from the control and treated groups of the F1b litters to serve as the second generation (F0 parental rats. The remaining F1b pups were examined for external abnormalities, killed and discarded.

F1 geneartion:
F0 animals were meated twice to give F2a and F2b
F2a - examined for external abnormalities
and killed at the end of the 21-day lactation
period.
F2b – selections were made of ten male and 20 female pups from the control and treated groups to serve as the third generation (F2) parental rats.

F2 geneartion:
F0 animals were meated twice to give F3a , F3b and F3c
F3a – On 21 days old and necropsied and tissues were collected for histopathology.
F3b - F2 animals were remated to give F3a, On 21 days necropsied and tissues were collected for histopathology.
F3c - F2 animals were remated to give F3c. On day 19 of gestation, half of the dams from the control and treated groups were killed.

- M/F ratio per cage: 1:2 ratio
- Length of cohabitation: 15 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy Vaginal smears were performed daily until sperm or a copulatory plug was found.
- 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): Female was placed on corn-cob bedding in an individual plastic shoe-box cage and remained there until it was remated.
- Any other deviations from standard protocol: Each female was rested for a minimum of 10 days after lactation before being mated again.
Each mating was with a different male from the same dosage group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to initiation of the study, assays were performed to determine the homogeneity and stability of test material in the prepared diets. The concentrations of the test compound in the diets were determined weekly during the first 13 wk of the study and monthly thereafter. All lots of basic feed used in the study were analysed for heavy metals, chlorinated hydrocarbons and aflatoxin. These analyses demonstrated that the basic feed contained acceptably low levels of contaminants, that the diets were prepared properly and that the dietary content of the test material was stable

Duration of treatment / exposure:

Study 1.
224 days
Study 2.Upto 3 generation

Frequency of treatment:

Daily

Details on study schedule:

No data available

Remarks:

Doses / Concentrations:
Study 1.
0, 2.5, 25, 75 and 250 mg/kg/day
Study 2.
0, 5, 50, 150 or 500 mg/kg

No. of animals per sex per dose:

Total: 1410
F0 geneartion:
0 mg/kg bw/day: 20 male, 40 female
2.5 mg/kg bw/day: 10 male, 20 female
25 mg/kg bw/day: 10 male, 20 female
75 mg/kg bw/day: 10 male, 20 female
250 mg/kg bw/day: 10 male, 20 female

F1a geneartion:
0 mg/kg bw/day: 20 male, 40 female
2.5 mg/kg bw/day: 10 male, 20 female
25 mg/kg bw/day: 10 male, 20 female
75 mg/kg bw/day: 10 male, 20 female
250 mg/kg bw/day: 10 male, 20 female

F1b geneartion:
0 mg/kg bw/day: 20 male, 40 female
2.5 mg/kg bw/day: 10 male, 20 female
25 mg/kg bw/day: 10 male, 20 female
75 mg/kg bw/day: 10 male, 20 female
250 mg/kg bw/day: 10 male, 20 female

F2a geneartion:
0 mg/kg bw/day: 20 male, 40 female
2.5 mg/kg bw/day: 10 male, 20 female
25 mg/kg bw/day: 10 male, 20 female
75 mg/kg bw/day: 10 male, 20 female
250 mg/kg bw/day: 10 male, 20 female

F2b geneartion:
0 mg/kg bw/day: 20 male, 40 female
2.5 mg/kg bw/day: 10 male, 20 female
25 mg/kg bw/day: 10 male, 20 female
75 mg/kg bw/day: 10 male, 20 female
250 mg/kg bw/day: 10 male, 20 female

F2c geneartion:
0 mg/kg bw/day: 20 male, 40 female
2.5 mg/kg bw/day: 10 male, 20 female
25 mg/kg bw/day: 10 male, 20 female
75 mg/kg bw/day: 10 male, 20 female
250 mg/kg bw/day: 10 male, 20 female

F3a geneartion:
0 mg/kg bw/day: 20 male, 40 female
2.5 mg/kg bw/day: 10 male, 20 female
25 mg/kg bw/day: 10 male, 20 female
75 mg/kg bw/day: 10 male, 20 female
250 mg/kg bw/day: 10 male, 20 female

F3b geneartion:
0 mg/kg bw/day: 10 male, 20 female
2.5 mg/kg bw/day: 10 male, 20 female
25 mg/kg bw/day: 10 male, 20 female
75 mg/kg bw/day: 10 male, 20 female
250 mg/kg bw/day: 10 male, 20 female

Control animals:

yes, concurrent vehicle

Details on study design:

No data available

Positive control:

No data available

Parental animals: Observations and examinations:

1.Morbidity and mortality, body weights, food consumption, food efficiency, Foetal development and Histopathology were examined.

Oestrous cyclicity (parental animals):

1.F2 genearion - abnormal conditions, live and dead foetuses, resorptions and corpora lutea were examined.

Sperm parameters (parental animals):

No data available

Litter observations:

1.Morbidity and mortality, body weights, food consumption and food efficiency of F1 and F2 geneation were examined.

Postmortem examinations (parental animals):

1.Gross pathology and Histopathology were examined.

Postmortem examinations (offspring):

1.Foetal development and Histopathology were examined.

Statistics:

1.Fertility indices were compared using the chisquare test criterion with Yates' correction for 2 × 2 contingency tables (Steel & Torrie, 1960) to judge the significance of differences. Gestation, 4-day, 14-day and 21-day survival indices were compared by the rank sum tests described by Snedecor & Cochran (1967) and Weil (1970) to judge the significance of differences. The numbers of pups born alive were compared by analysis of variance and the t test, as described by Steel & Torrie (1960), using the multiple comparison tables of Dunnett (1964) to judge the significance of differences.

Reproductive indices:

1.Fertility indices, gestation and lactation indices were examined.

Offspring viability indices:

Yes, viability on day 4, 14 and 21 were examined.

Clinical signs:

no effects observed

Description (incidence and severity):

When treated with 250 mg/kg body weight/day, Slightly bluish-coloured fur and Bluishgreen- coloured faeces were observed in treated rats as compared to control. When treated with 75 mg/kg body weight/day, Bluishgreen- coloured faeces were observed in treated rats as compared to control. Occasional ocular or nasal porphyrin discharge, soft tools, respiratory congestion and slight alopecia were observed in treated rats which were in similar frequency with control.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

No effect on survival of treated rats were observed as compared to control.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effect on body weight of treated rats was observed as compared to control.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Although variations in food consumption were recorded for all groups, no marked or consistent differences were observed between control and treated groups.

During the mating period, the diets fed were prepared on the basis of the females' body weights and food consumption. Therefore, the amount of colouring consumed by male rats during this period was slightly lower than the required dosage level.

Food efficiency:

no effects observed

Description (incidence and severity):

No effect on food efficiency of treated rast were observed as compared to control.

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

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

1&2.no indications of any influence on reproductive parameters

Mortality: No effect on survival of treated rats were observed as compared to control.

Clinical signs: When treated with 250 mg/kg body weight/day, Slightly bluish-coloured fur and Bluishgreen- coloured faeces were observed in treated rats as compared to control.

When treated with 75 mg/kg body weight/day, Bluishgreen-
coloured faeces were observed in treated rats as compared to control.

Occasional ocular or nasal porphyrin discharge, soft tools, respiratory congestion and slight alopecia were observed in treated rats which were in similar frequency with control.

Body weight: No effect on body weight of treated rats was observed as compared to control.

Food consumption: Although variations in food consumption were recorded for all groups, no marked or consistent differences were observed between control and treated groups.

During the mating period, the diets fed were prepared on the basis of the females' body weights and food consumption. Therefore, the amount of colouring consumed by male rats during this period was slightly lower than the required dosage level.

Test substance intake: During the weaning period, the pups were not prevented from eating the food provided for the dams; the consumption of colouring by female rats during the gestation and weaning periods therefore appeared to be slightly higher than anticipated.

Reproductive function: estrous cycle
F2 parentes: No effect on live and dead foetuses, resorptions and corpora lutea of treated rats were observed as compared to control.

Reproductive function: sperm measures: No data available

Reproductive performance
F1 parentes: When treated with 2.5 and 250 mg/kg body weight/day, significant decrase in fertility indices of treated female rats were observed as compared to control.

Because of the lower fertility indices observed at the 2.5 mg/kg bw/day dosage group in the F 2 litters, the study was extended to the F3b litter.

No effect on F0 and F3 generation fertility indices were observed as compared to control.

The changes were considered to represent variation rather than compound-related effects.

Organ weights No effect on organ weight of treated rats were oberved as compared to control.

Gross pathology: No effect on Gross pathology of treated rats were oberved as compared to control.

Histopathology: No data available

other findings
Food efficiency: No effect on food efficiency of treated rast were observed as compared to control.

Dose descriptor:

NOAEL

Effect level:

> 250 - <= 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect on survival, body weights, food consumption, food efficiency, Estrous cyclicity, Foetal development, organ weights and gross pathology

Remarks on result:

other: No reproductive toxic effects observed

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Clinical signs:

no effects observed

Description (incidence and severity):

Slightly bluishcoloured fur was noted inpups given 250mg/kg/day and bluish-greencoloured faeces were produced by pups given 75 or 250 mg/kg/day. No other changes considered to be compound related were noted in general behaviour or appearance. Changes noted occasionally in control or treated pups included alopecia, respiratory congestion and runting.

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

No effect on survival of F1 pups were observed as compared to control.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The average body weights of the pups were similar in control and treated groups. Although the average number of pups born per litter varied among the groups, no dose relationship was apparent.

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:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no compound-related effects on organ Weights.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No compound-related gross pathological change in any of the rats that were killed and necropsied

Histopathological findings:

no effects observed

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Mortality: No effect on survival of F1 and F2 pups were observed as compared to control.

Clinical signs: When treated with 250 mg/kg body weight/day, Slightly bluish-coloured fur and Bluishgreen- coloured faeces were observed in treated pups as compared to control.

When treated with 75 mg/kg body weight/day, Bluishgreen-
coloured faeces were observed in treated pups as compared to control.

Respiratory congestion and runting were observed in treated rats which were in similar frequency with control.

Body weight:
F1 and F2 pups:
No effect on body weight of treated pups was observed as compared to control.

Food consumption:
F1 and F2 pups:
No effect on food consumption of treated pups was observed as compared to control.

Organ weights: No effect on organ weight of treated pups were oberved as compared to control.

Gross pathology: No effect on Gross pathology of treated pups were oberved as compared to control.

Histopathology: No gross anatomical abnormalities were observed in fetouse of treated rats as compared to control.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 250 - <= 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect on survival, body weights, food consumption, food efficiency, Estrous cyclicity, Foetal development, organ weights, Gross pathology and Histopathology

Remarks on result:

other: No developmental toxic effects was observed

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Clinical signs:

no effects observed

Description (incidence and severity):

Slightly bluishcoloured fur was noted inpups given 250mg/kg/day and bluish-greencoloured faeces were produced by pups given 75 or 250 mg/kg/day. No other changes considered to be compound related were noted in general behaviour or appearance. Changes noted occasionally in control or treated pups included alopecia, respiratory congestion and runting.

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

No effect on survival of F2 pups were observed as compared to control.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The average body weights of the pups were similar in control and treated groups. Although the average number of pups born per litter varied among the groups, no dose relationship was apparent.

Food consumption and compound intake (if feeding study):

no effects observed

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

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

No compound-related gross pathological change in any of the rats thatwerekilled and necropsied

Histopathological findings:

no effects observed

Description (incidence and severity):

No compound-related microscopic pathological lesions in any of the rats that were killed and necropsied

Other effects:

no effects observed

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect on survival, body weights, food consumption, food efficiency, Estrous cyclicity, Foetal development, organ weights, Gross pathology and Histopathology

Remarks on result:

other: No developmental toxic effects observed

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Reproductive effects observed:

not specified

Treatment related:

not specified

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Summary of reproduction and lactation data for F1 litters

Dosage group (mg/kg/day)	Fertility indexɫ				Gestation survival index‡		Survival index§
	Females		Males				4-day		14-day		21 –day
	F1a litters
0	14/20	70	8/10	80	184/184	100	182/184	99	137/137	100	137/137	100
0	18/20	90	10/10	100	247/249	99	243/247	98	179/179	100	168/179	94
2.5	20/20	100	10/10	100	257/264	97	256/257	100	192/193	99	192/193	99
25	18/20	90	10/10	100	245/248	99	242/245	99	180/180	100	176/180	98
75	17/20	85	10/10	100	217/218	100	217/217	100	164/164	100	164/164	100
250	17/20	85	10/10	100	212/214	99	191/212	90	152/152	100	17152/152	100
	F1b litters
0	13/20	65	7/10	70	157/159	99	157/157	100	126/126	100	126/126	100
0	19/19	100	10/10	100	242/244	99	240/242	99	190/190	100	189/190	99
2.5	18/19	95	10/10	100	228/238	96	225/228	99	178/178	100	178/178	100
25	19/20	95	10/10	100	257/263	98	252/257	98	187/188	99	187/188	99
75	18/20	90	10/10	100	220/222	99	218/220	99	172/172	100	172/172	100
250	15/20	75	8/10	80	182/187	97	181/182	99	143/143	100	143/143	100

ɫ Figures tabulated are (F) no. of pregnancies/no, of matings, followed by the index, and (M) no. of fertile males/total no. of males mated, followed by the index.

‡ No. of newborn pups alive/total no. born, followed by the index.

§The 4-day, 14-day and 21-day survival indices are preceded, respectively, by no. of pups surviving to day 4/no. liveborn, no. of pups surviving to day 14/no. retained at day 4, and no. of pups surviving 'to day 21/no. retained at day 4.

Summary of reproduction and lactation data for F2 litters

Dosage group (mg/kg/day)	Fertility indexɫ				Gestation survival index‡		Survival index§
	Females		Males				4-day		14-day		21 –day
	F2a litters
0	16/20	80	9/10	90	184/184	100	184/184	100	156/158	99	153/158	97
0	17/20	85	9/10	90	194/199	97	190/194	98	163/164	99	158/164	96
2.5	10/20	50*	7/10	70	100/100	100	100/100	100	91/91	100	90/91	99
25	10/20	50*	6/10	60	109/109	100	109/109	100	97/98	99	94/98	96
75	17/20	85	9/10	90	177/178	99	177/177	100	155/158	98	146/158	92
250	13/20	65	8/10	80	177/123	95	117/117	100	109/110	99	106/110	96
	F2b litters
0	18/20	90	10/10	100	211/222	95	209/211	99	161/161	100	161/161	100
0	16/20	80	9/10	90	178/179	99	178/178	100	147/147	100	147/147	100
2.5	8/20	40**	5/10	50*	83/84	99	79/83	95	65/66	98	65/66	98
25	12/20	60	8/10	80	129/130	99	128/129	99	104/105	99	104/105	99
75	16/20	80	9/10	90	197/203	97	196/197	99	153/153	100	153/153	100
250	15/20	75	8/10	80	135/138	98	134/135	99	114/114	100	114/114	100
	F2c litters
0	13/20	65	9/10	90	53/53	100	52/53	98	44/45	98	44/45	98
0	16/20	80	9/9	100	81/82	99	78/81	96	61/61	100	61/61	100
2.5	8/20	40*	5/10	50*	31/31	100	29/31	94	24/27	89	24/27	89
25	8/20	40*	6/10	60	33/33	100	33/33	100	28/28	100	28/28	100
75	11/20	55	9/10	90	84/92	91	83/84	99	70/70	100	69/70	99
250	10/20	50 6	6/10	60	47/47	100	46/47	98	42/42	100	42/42	100

ɫ Figures tabulated are (F) no. of pregnancies/no, of matings, followed by the index, and (M) no. of fertile males/total no. of males mated. followed by the index.

‡ No. of newborn pups alive/total no. born, followed by the index.

§The 4-day, 14-day and 21-day survival indices are preceded, respectively, by no. of pups surviving to day 4/no. liveborn, no. of pups surviving to day 14/no. retained at day 4, and no. of pups surviving to day 21/no. retained at day 4.

Values marked with asterisks are significantly lower than those for the concurrent control groups: *P < 0.05; **P < 0.01.

Summary of reproduction and lactation data for F3 litters

Dosage group (mg/kg/day)	Fertility indexɫ				Gestation survival index‡		Survival index§
	Females		Males				4-day		14-day		21 –day
	F3a litters
0	12/20	60	9/10	90	114/114	100	112/114	98	105/106	99	102/106	96
0	9/20	45	6/10	60	90/91	99	90/90	100	79/79	100	78/79	99
2.5	14/20	70	8/10	80	144/150	96	143/144	99	114/124	92	113/124	91
25	11/20	55	7/10	70	108/110	98	106/108	98	87/97	90	86/97	89
75	11/20	55	7/10	70	95/96	99	94/95	99	88/88	100	86/88	98
250	14/20	70	8/10	80	153/153	100	153/153	100	132/132	100	131/132	99
	F3b litters
0	14/20	70	8/10	80	116/119	97	104/116	90	94/95	99	94/95	99
2.5	15/20	75	8/10	80	137/142	96	136/137	99	121/121	100	120/121	99
25	15/20	75	9/10	90	140/141	99	139/140	99	125/126	99	124/126	98
75	11/20	55	7/10	70	101/104	97	101/101	100	88/89	99	88/89	99
250	15/20	75	8/10	80	146/146	100	145/146	99	127/128	99	127/128	99

ɫ Figures tabulated are (F) no. of pregnancies/no, of matings, followed by the index, and (M) no. of fertile males/total no. of males mated, followed by the index.

ɫ No. of newborn pups alive/total no. born, followed by the index.

§The 4-day, 14-day and 21-day survival indices are preceded, respectively, by no. of pups surviving to day 4/no. liveborn, no. of pups surviving to day 14/no. retained at day 4, and no. of pups surviving to day 21/no. retained at day 4.

Conclusions:

NOAEL was considered to be in range of 250 - 500 mg/kg body weight/day for F0, F1 and F2 generation, when CD male and female rats were treated with 3H,3'H-2,2'-bi-1-benzothiophene-3,3'-dione; Thioindigo (CAS no. 522-75-8) orally.

Executive summary:

Data available from different studies were reviewed to determine the reproductive toxicity of 3H,3'H-2,2'-bi-1-benzothiophene-3,3'-dione; Thioindigo (CAS no. 522-75-8).The studies are as mentioned below:

Study 1

In a three generation reproduction study, groups of ten males and twenty female Charles River CD rats were fed with test material  at dietary levels providing intakes of 0, 2.5, 25, 75 and 250 mg/kg bw/day. Two females were placed in a male's cage for the entire mating period (15 days). Vaginal smears were performed daily until sperm or a copulatory plug was found; this was designated as day 0 of pregnancy. At the end of the mating period, each

female was placed on corn-cob bedding in an individual plastic shoe-box cage and remained there until it was remated. Each female was rested for a minimum of 10 days after lactation before being mated again. Each mating was with a different male from the same dosage group.

The control and treated F0 rats were maintained on their respective diets for 2 wk prior to the first mating period. They were then mated, when approximately 100 days old. The rats (F0) were mated twice. The pups (F1a) from the first mating were examined for external abnormalities and killed at the end of the 21-day lactation period. After the second mating, random selections were made of ten male and 20 female pups from the control and treated groups of the F1b litters to serve as the second generation (F1) parental rats. The remaining F1b pups were examined for external abnormalities, killed and discarded. The F0 parent rats were also killed at this time.

After weaning, the F1b pups selected for the second generation continued to be fed on their control or appropriately treated diet for 80 days and were then mated. The pups from the first mating (F2a) were examined for external abnormalities and killed at the end of the 21-day lactation period. From the second mating (F2b), selections were made of ten male and 20 female pups from the control and treated groups to serve as the third generation (F2) parental rats. The remaining pups (F2b) were examined for external abnormalities, killed and discarded. Following the third mating, one-half of the mated females were killed on day 19 of gestation and the uteri and ovaries were examined. The remaining females were allowed to deliver. After weaning, the F2c pups were examined for external abnormalities, killed and discarded. The F1 parent rats were then killed. Five male and five female rats from the control and each of the treated groups were necropsied and their tissues were collected for histopathology.

After weaning, the F2b pups selected for the third generation continued to be fed on their control or appropriately treated diet for 80 days and were then mated. Five male and five female pups (F3a) from the control and each of the treated groups were killed when 21 days old and necropsied, and tissues were collected for histopathology. The parent rats (F2) were then re-mated and the pups from the second mating (F3b) were examined for external abnormalities and killed at the end of the 21-day lactation period. The F 2 parent rats were then re-mated for the third mating (F3c). On day 19 of gestation, half of the dams from the control and

treated groups were killed by chloroform anesthesia. Uteri were examined for any abnormal conditions and the presence of live and dead foetuses and of resorptions were recorded. All ovaries were inspected and the corpora lutea were counted. Remaining F2 parent rats were then killed and discarded.

The rats were observed twice daily for changes in behaviour or appearance and for morbidity and mortality. Individual body weights and food consumption were recorded weekly. Specific parameters for the reproductive phase of this study included observations of fertility, litter size, numbers of male and female pups, viability of the newborn, survival of pups to weaning and growth of the pups.All stillborn offspring and any progeny that died during the study were examined either by skeletal clearing or by necropsy. Body weights of pups aged 4 days (before and after reduction of litters to ten pups) and aged 14 days were obtained by weighing as litters. The pups were weighed individually at 21 days of age. The thyroid, adrenals, lung. heart, spleen, stomach, jejunum, ileum, colon, liver, kidneys, urinary bladder, testes or ovaries and uterus from five male and five female rats from the F1 and F3, generations of the control and 250-mg/kg/day groups were embedded in paraffin, sectioned, stained with haematoxylin and eosin and examined microscopically.

 

Fur and faeces were bluish-coloured in 75 mg/kg bw/day and 250 mg/kg bw/day groups. Gestation, viability and lactation indices of all litters from exposed animals did not differ from controls. Fertility indices were statistically significantly lower for F2female rats in the 2.5 and 25 mg/kg bw/day groups only and consequently considered as not being dose-related. Fertility indices were also reduced in F2b and F2cgroup of male rats. No statistically significant changes in the fertility index were observed in the F3generation. As effects on fertility indices in the F2generation were not dose-related and effects were not identified in the F1and F3generation this effect was not considered to be compound-related. Examination of the ovaries and uteri of F1dams killed on gestation day (GD) 19 revealed no gross anatomical abnormalities. No unusual changes were observed in the stillborn pups or in pups that died during the study. No compound-related gross or microscopic pathological lesions were noted in any of the F1or F3arats that were sacrificed and necropsied. Finally, no compound-related organ-weight variations were recorded in the F1rats. ThereforeNOAEL was considered to be250 mg /kg bw/day for test material  inCharles RiverCD male and female rats by oral administration (feed) in 3 generation study.(F0,F1 andF2 generation)

 

 

Study 2.

Reproductive and development toxicity study oftestmaterial was performed on male and femaleHarlan-Wistar rats. Test material administered in diet for up to 3 generation. 10 male and 20 females were used. Asno indications of any influence on

maternal weight gain,reproductive parameters andNo developmental toxic effects were seen up to the highest dose of 500 mg/kg bw, Hence No Observed Adverse Effect Level (NOAEL) for maternal toxicity was considered to be 500 mg/kg/day.When male and femalewistar rats were treated withtest materialorally up to 3 generation.

Based on the data available from different studies,3H,3'H-2,2'-bi-1-benzothiophene-3,3'-dione; Thioindigo (CAS no. 522-75-8)did not showedreproductive toxicityat dose concentration 500mg/kg bw/day by oral route.Hence the test chemical is not likely to classify as a reproductive toxicant as per the criteria mentioned in CLP regulation.

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Data is Klimicsh 2 and from authoritative database

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive toxicity study

Data available from different studies were reviewed to determine the reproductive toxicity of 3H,3'H-2,2'-bi-1-benzothiophene-3,3'-dione; Thioindigo (CAS no. 522-75-8).The studies are as mentioned below:

Study 1

In a three generation reproduction study, groups of ten males and twenty female Charles River CD rats were fed with test material  at dietary levels providing intakes of 0, 2.5, 25, 75 and 250 mg/kg bw/day. Two females were placed in a male's cage for the entire mating period (15 days). Vaginal smears were performed daily until sperm or a copulatory plug was found; this was designated as day 0 of pregnancy. At the end of the mating period, each

female was placed on corn-cob bedding in an individual plastic shoe-box cage and remained there until it was remated. Each female was rested for a minimum of 10 days after lactation before being mated again. Each mating was with a different male from the same dosage group.

The control and treated F0 rats were maintained on their respective diets for 2 wk prior to the first mating period. They were then mated, when approximately 100 days old. The rats (F0) were mated twice. The pups (F1a) from the first mating were examined for external abnormalities and killed at the end of the 21-day lactation period. After the second mating, random selections were made of ten male and 20 female pups from the control and treated groups of the F1b litters to serve as the second generation (F1) parental rats. The remaining F1b pups were examined for external abnormalities, killed and discarded. The F0 parent rats were also killed at this time.

After weaning, the F1b pups selected for the second generation continued to be fed on their control or appropriately treated diet for 80 days and were then mated. The pups from the first mating (F2a) were examined for external abnormalities and killed at the end of the 21-day lactation period. From the second mating (F2b), selections were made of ten male and 20 female pups from the control and treated groups to serve as the third generation (F2) parental rats. The remaining pups (F2b) were examined for external abnormalities, killed and discarded. Following the third mating, one-half of the mated females were killed on day 19 of gestation and the uteri and ovaries were examined. The remaining females were allowed to deliver. After weaning, the F2c pups were examined for external abnormalities, killed and discarded. The F1 parent rats were then killed. Five male and five female rats from the control and each of the treated groups were necropsied and their tissues were collected for histopathology.

After weaning, the F2b pups selected for the third generation continued to be fed on their control or appropriately treated diet for 80 days and were then mated. Five male and five female pups (F3a) from the control and each of the treated groups were killed when 21 days old and necropsied, and tissues were collected for histopathology. The parent rats (F2) were then re-mated and the pups from the second mating (F3b) were examined for external abnormalities and killed at the end of the 21-day lactation period. The F 2 parent rats were then re-mated for the third mating (F3c). On day 19 of gestation, half of the dams from the control and

treated groups were killed by chloroform anesthesia. Uteri were examined for any abnormal conditions and the presence of live and dead foetuses and of resorptions were recorded. All ovaries were inspected and the corpora lutea were counted. Remaining F2 parent rats were then killed and discarded.

The rats were observed twice daily for changes in behaviour or appearance and for morbidity and mortality. Individual body weights and food consumption were recorded weekly. Specific parameters for the reproductive phase of this study included observations of fertility, litter size, numbers of male and female pups, viability of the newborn, survival of pups to weaning and growth of the pups.All stillborn offspring and any progeny that died during the study were examined either by skeletal clearing or by necropsy. Body weights of pups aged 4 days (before and after reduction of litters to ten pups) and aged 14 days were obtained by weighing as litters. The pups were weighed individually at 21 days of age. The thyroid, adrenals, lung. heart, spleen, stomach, jejunum, ileum, colon, liver, kidneys, urinary bladder, testes or ovaries and uterus from five male and five female rats from the F1 and F3, generations of the control and 250-mg/kg/day groups were embedded in paraffin, sectioned, stained with haematoxylin and eosin and examined microscopically.

 

Fur and faeces were bluish-coloured in 75 mg/kg bw/day and 250 mg/kg bw/day groups. Gestation, viability and lactation indices of all litters from exposed animals did not differ from controls. Fertility indices were statistically significantly lower for F2female rats in the 2.5 and 25 mg/kg bw/day groups only and consequently considered as not being dose-related. Fertility indices were also reduced in F2b and F2cgroup of male rats. No statistically significant changes in the fertility index were observed in the F3generation. As effects on fertility indices in the F2generation were not dose-related and effects were not identified in the F1and F3generation this effect was not considered to be compound-related. Examination of the ovaries and uteri of F1dams killed on gestation day (GD) 19 revealed no gross anatomical abnormalities. No unusual changes were observed in the stillborn pups or in pups that died during the study. No compound-related gross or microscopic pathological lesions were noted in any of the F1or F3arats that were sacrificed and necropsied. Finally, no compound-related organ-weight variations were recorded in the F1rats. ThereforeNOAEL was considered to be250 mg /kg bw/day for test material  inCharles RiverCD male and female rats by oral administration (feed) in 3 generation study.(F0,F1 andF2 generation)

 

 

Study 2.

Reproductive and development toxicity study oftestmaterial was performed on male and femaleHarlan-Wistar rats. Test material administered in diet for up to 3 generation. 10 male and 20 females were used. Asno indications of any influence on

maternal weight gain,reproductive parameters andNo developmental toxic effects were seen up to the highest dose of 500 mg/kg bw, Hence No Observed Adverse Effect Level (NOAEL) for maternal toxicity was considered to be 500 mg/kg/day.When male and femalewistar rats were treated withtest materialorally up to 3 generation.

Based on the data available from different studies,3H,3'H-2,2'-bi-1-benzothiophene-3,3'-dione; Thioindigo (CAS no. 522-75-8)did not showedreproductive toxicityat dose concentration 500mg/kg bw/day by oral route.Hence the test chemical is not likely to classify as a reproductive toxicant as per the criteria mentioned in CLP regulation.

 

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Thus, comparing this value with the criteria of CLP regulation 3H,3'H-2,2'-bi-1-benzothiophene-3,3'-dione; Thioindigo (CAS no. 522-75-8)isnot likely to classify as reproductive toxicant.