Registration Dossier

Administrative data

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:
read-across source
Related information:
Composition 1
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
equivalent or similar to
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
Test material information:
Composition 1
Species:
rat
Strain:
other: 1.CD-1 2.Harlan-Wistar
Sex:
male/female
Details on test animals 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.
Estrous 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
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Reproductive function: estrous 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.