Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In the study performed according to the OECD 422 (selected based on systemic effects on parents) no effects attributed to the Solvant Black 46 was observed at the maximum tested dose of 100 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, USA and bred at IIBAT animal house facility
- Age at study initiation: Between 12 and 14 weeks. Females were virgin
- Weight at study initiation: Males: 328 g to 363 g; Females: 222 g to 251 g.
- Fasting period before study: none
- Housing: Males were housed in groups in cages, each cage containing 5 animals during pre-mating and post-mating period. Females were housed in groups of 5 animals per cage during pre-mating. 1 male and 1 female was kept together in a cage until pregnancy occurs. Pregnant females were caged individually. Animals from reversal group were housed group wise and sex wise with 5 animals per cage.
- Diet: Ad libitum - Standard gamma irradiated pellet food.
- Water: Ad libitum - Reverse osmosis water
- Acclimation period: five days in the test room
- Sanitation: Bedding material, cages, grills were changes once every 3 day and water bottles were changed daily. Cages, grills and water bottles were autoclaved.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 °C to 22.8 °C. Recorded once daily
- Humidity (%): 56% - 64%. Recorded once daily
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light


IN-LIFE DATES:
IN-LIFE DATES:
- (P) Males groups 1 to 5: From July 31, 2012 to August 28, 2012
- (P) Females groups 1 to 5: From July 31, 2012 to September 09 - September 14, 2012
- Males and females groups 6 and 7: From July 31, 2012 to September 22, 2012
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Solvent Black 46 was prepared freshly daily by mixing with corn oil and magnetically stirred to obtain a violet homogenous suspension just before the administration. The intensity of the black colour of the suspension was found to be increasing from low dose to high dose.

VEHICLE
- Justification for use and choice of vehicle : non toxic vehicle which allows an homogenous solution/suspension.
- Amount of vehicle (if gavage): dose volume maintained at 10 mL/kg b.w.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until proof of pregnancy (maximum 5 days).
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- No unsuccessful attemps.
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
(P) Males groups 1 to 5: 15 days before mating, continously during mating (1 to 5 days) and after mating until the dosing period of 28 days.
(P) Females groups 1 to 5: 15 days before mating, continously during mating (1 to 5 days), during pregnancy (21 to 23 days) and 3 days post partum (Total: from 41 to 44 days)

Males and Females from reversal groups: 41 days (until first scheduled kill of dams)
Frequency of treatment:
daily
Details on study schedule:
(P) Males groups 1 to 5: sacrifice at the end of the exposure period of 28 days.
(P) Females groups 1 to 5: sacrifice at the end of the exposure period of 41 to 44 days

Males and Females of the reversal groups: Maintained for 14 days after dosing period prior sacrifice (sacrifice at day 54). No mating.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 and Group 6 (reversal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
75 mg/kg bw/day (nominal)
Remarks:
Group 4
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 5 adn Group 7 (reversal)
No. of animals per sex per dose:
(P) Males from groups 1 to 5: 10/group
(P) Females from groups 1 to 5: 10/group

Males and Females from the reversal groups: 5/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: doses were selected based on the results of the range finding study carried out.
- Rationale for animal assignment (if not random): Randomization

- Range finding study:
Carried out using one control (G1) and three dose groups with 3 males and 3 females of each 100 (G2), 300 (G3) and 1000 (G4) mg/kg b.w. of the test substance.
Daily oral administration for 7 days. Control group animals were treated similarly, but with corn oil alone.
Observed for morbidity /mortality and signs of toxicity daily.

Death of one male, two females from G4 and one female from G3.

Bluish perianal staining observed in all animals of all the test substance treated group from day 1 to 7.
Dullness observed in one male and two females from G3 and all animals of G4.
Piloerection noted in one male and one female of G3 and two male and female of G4.
Abdominal distension observed in one female of G3 as well as one male and all females of G4.
Respiratory distress recorded in one male of G4.
Enlarged payers’s patches observed in all animals of test substance treated groups at macroscopic examination.
Gross lesions observed in G3 and G4 animals: distended stomach with bluish discoloration of mucosal surface, small intestine and large intestine with bluish discoloration of mucosal surface and seminal vesicles small in size.
Adrenals enlarged in all animals
Thymus small in size in two male and one female of G4 group.

Selected dose for the main study: 25, 50, 75 and 100 mg/kg b.w
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for all animals, to record any sign of toxicity
- Parameters eximaned: changes in skin, fur, eyes, and mucous membranes, occurrence of secretions, excretions (coloration and intensity of faeces and/or urine) and autonomic activity (e.g. lacrimation, piloerection, palpebral closure, palpebral reflex, pupil light response, and unusual respiratory pattern). Changes in gait, mobility, arousal, rearing, posture, vocalizations, activity levels and response to handling, approach response, touch response, click response, tail pinch response, toe pinch as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling), bizarre behavior (e.g. self-mutilation, walking backwards), were also recorded. Pertinent behavioral changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality, were also recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
* (P) Males groups 1 to 5: On pre-mating days 0, 7, mating days 0, 7 and 13.
* (P) Females groups 1 to 5: On pre-mating days 0, 7, mating day 0, pregancy days 0, 7, 14, 20 and on post partym day 4.
* Males and females of the reversal groups: On days 0, 7, 14, 21, 28, 35, 42, 49 and on day 53 (at termination).

BODY WEIGHT: Yes
- Time schedule for examinations:
* (P) Males groups 1 to 5: Prior administration, on pre-mating days 0, 7, mating days 0, 7 and at termination
* (P) Females groups 1 to 5: Prior administration, on pre-mating days 0, 7, mating day 0, Pregnancy days 0, 7, 14, 20, within 24 hours of parturition and on post partum day 4
* Males and Females of the reversal groups: Prior administration, on days 7, 14, 21, 28, 35, 42, 49 and on day 53 (at termination).

GROSS PATHOLOGY
- Macroscopical examination for any abnormalities with special attention to reproductive system's organs.
- Record of the number of implantation sites and corpora lutea.
- Tissues preserved in 10 % neutral buffer formalin for histopathological examination: all gross lesions, brain (representative regions including cerebrum, cerebellum and pons), spinal cord, stomach, small and large intestines (including Peyer’s patches), liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea and lungs, uterus, urinary bladder, mesenteric lymph nodes, mandibular lymph nodes, peripheral nerve (sciatic), and a section of bone marrow (sternum).

ORGAN WEIGHT
- Weights of following organs of all male animals were recorded: 1. Testes 2. Epididymis
- Weights of following organs for randomly selected five males and five females of group G1, G2, G3, G4, G5 and all animals from groups G6 and G7: Liver, Kidneys, Adrenals, Thymus, Spleen, Brain and Heart.

Other: see the study in chapter 7.5.1 "Repeated dose toxicity: oral - 28 days"
Oestrous cyclicity (parental animals):
not performed
Sperm parameters (parental animals):
not performed
Litter observations:
Performed for litter produced by females of groups 1 to 5.

Examined as earliest after delivery.
Observation made:
* Numbers and sex
* still and live births
* runts and presence of gross abnormalities
* weighed within 24 hours of parturition and on post partum day 4.

Postmortem examinations (parental animals):
SACRIFICE
- (P) Male animals: All surviving animals were sacrificed at the end of the dosing period, i.e. on day 28.
- (P) Maternal animals: All surviving animals were sacrificed at the end of the dosing period, i.e. on post partum day 4.
- Male and female animals of the reversal groups: All surviving animals were sacrified at the end of the 14 days observations period, following the end of the dosing period, i.e. on day 53.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [2] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
External examinations for gross abnormalities.
Statistics:
Examined parameters were checked for normality:
- Normal data was subjected to one-way ANOVA
- Non-normal data was subjected to Kruskal-Wallis One-Way ANOVA on Ranks

Student's Newman-Keul's Test was employed for post ANOVA comparison.
Reproductive indices:
sex ratio: (Number of male pups/ Number of female pups) x 100
Offspring viability indices:
not applicable
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Animals of G4, G5 & G7 exhibited dullness and piloerection at various intervals. Mortability: one dead male in G5, one dead male in G7 and one dead female in G5. Abdominal distension and respiratory distress were observed in those dead animals.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Death of one animal in each groups of high (G5) and high reversal (G7) males and one female in high (G5) dose groups could be considered as treatment related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistical significant decrease in the body weight in males (in G7 from day 14 till the sacrifice and in G4 and G5 on day 28) and in female (in G5 on premating day 14 and gestation day 0) as compare to their respective control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistical significant decrease in the body weight in males (in G7 from day 14 till the sacrifice and in G4 and G5 on day 28) and in female (in G5 on premating day 14 and gestation day 0) as compare to their respective control.
Food efficiency:
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Observed weekly individual clinical signs like home cage, handling and standard arena (open field) were similar to daily observations and did not showed any significance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment related histopathological findings were not observed. The histopathological findings observed were either related to, agonal to spontaneous, or were incidental and of the type routinely observed in Wistar rats of this age.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment related histopathological findings were not observed. The histopathological findings observed were either related to, agonal to spontaneous, or were incidental and of the type routinely observed in Wistar rats of this age.
Other effects:
no effects observed
Description (incidence and severity):
Test substance intake: statistically significant decrease in G2, G3 & G5 females on few days during gestation and postpartum was observed. These changes were not considered as treatment related, since there was no dose dependency and consistency.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
G1: control
G2: low dose - 25 mg/kg b.w
G3: low intermediate dose - 50 mg/kg b.w
G4: high intermediate dose - 75 mg/kg b.w
G5: high dose - 100 mg/kg b.w
G6: control reversal
G7: high reversal - 100 mg/kg b.w

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
Mortality: No morbidity/mortality was observed in G1, G2, G3, G4 and G6 animals.
Death of two males: one from G5 on day 21st and one from G7 on day 16th.
Death of one female from G5 on day 27th.

No clinical signs of toxicity were observed in males and females of G1, G2 and G6 group animals.

The bluish perianal staining in animals of G3, G4, G5 & G7 groups was considered due to the colour of test substance and this effect was disappeared in G7 group animals on 46th day of observation period in all males or at 49th day of observation in all females after withdrawal of treatment.
Further there were no other relevant changes observed to relate this finding as significant. Hence this effect was not considered as adverse effect.

However, animals belong to G4, G5 & G7 groups exhibited dullness and piloerection at various intervals. These signs were recovered after withdrawal of treatment. In addition, abdominal distension and respiratory distress were observed in dead animals of G5 and G7 (attached Table no. 13 in background material).

Observed weekly individual clinical signs like home cage, handling and standard arena (open field) were similar to daily observations and did not showed any significance (attached Tables no. 6 and 14 in background material).


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
Observed body weight in G2 and G3 was comparable with respective control group (G1) throughout the observation period.
However, G4, G5 and G7 dose groups showed a decreasing trend from day 14 with respect to their controls (G1 & G6) and continued up to sacrifice in males or until postpartum day4 in females. This decrease was statistically different in high dose reversal from day 14 to till the end, and on day 28 in high intermediate and high dose groups.

Statistical significant decrease was observed in G3 females on day 0 of the gestation with respect to the control (G1) group, this effect was not consistent and was observed only on that day, hence not considered as treatment related. (attached tables no. 1, 2, 3, 15, 16, 17 in background material).

Food consumption recorded in treated groups was comparable with respective control groups and there were no significant difference. However, the statistically significant decrease in G2, G3 & G5 females on few days during gestation and postpartum was observed. These changes were not considered as treatment related, since there was no dose dependency and consistency (attached tables 4, 5, 18, 19 in background material).


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): G1 to G5
Mating was observed within 4 days, which was normal, in all the groups including control. There were no significant changes attributed to the test substance on the mating period (attached tables 11 and 22 in background material).

No treatment related effect was observed on sex ratio of the pups in any of the treated groups when compared with control group (G1) of animals (attached tables 10, 11, 24 in background material).

Normal gestation length between 21-23 days was observed in females of all the treated groups and was comparable with gestation length of the control group females (attached table 23 in background material).

No statistical significant changes were observed on mean corpora lutea and Implantations in G2, G3, G4 females when compared to the G1. Whereas, in high dose (G5) group a decrease was observed in total corpora lutea and subsequent Implantations with respect to control (G1) group. However these changes were not statistically significant and the mean corpora lutea count was also comparable with respective control group. This decrease is due to the death of one female in this dose group. Hence these changes could not be considered as treatment related (attached tables 9, 10 and 24 in background material).

Mean litter size of the G2, G3 and G4 was comparable with G1. Although not statistically significant, a decrease was observed in the G5 group when compared to G1 due to the death of one animal in G5 group. Hence this change could not be attributed to treatment related (attached table 24 in background material).

Mean litter weight of the G2, G3 and G4 was comparable with G1. The observed decrease in G5 group was not statistically significant when compared with G1 due to the death of one animal in G5 group. Hence this change could not be attributed to treatment related (attached table 10, 24 in background material).

No test substance related effect was observed on the number of dams delivered with live pups in any of the treated group of animals and is comparable with control group (attached tables 10, 11, 24 in background material).

Loss of offspring (pre implantation, post implantation and post natal) in the treated groups (G2, G3 & G4) was comparable with control (G1) group. A statistically insignificant pre implantation loss was observed in G5 group when compared to G1 (attached tables 9, 10, 11, 24 in background material).

ORGAN WEIGHTS (PARENTAL ANIMALS)
No treatment related organ weight changes were observed in any of the groups when compared to concurrent control group. The observed statistical significant changes in absolute and relative weights of adrenals in G2 group males and absolute weight of thymus in G4 group females when compared to concurrent control group (G6) was not considered as treatment related, since there was no correlation with either macro or microscopic observations (attached tables 26, 27, 31 and 32 in background material).

GROSS PATHOLOGY (PARENTAL ANIMALS)
No test substance related gross pathological findings were observed.

Test substance related bluish discoloration at perianal area was recorded in in G3, G4 and G5 group. However, this was considered due to color of test substance which was excreted through feces.
There was no correlating macroscopic or histopathology findings of alimentary tract. Hence, it was not considered to be adverse effect
The macroscopic findings observed were either related to physiological, agonal to spontaneous, or were incidental and of the type routinely observed in Wistar rats of this age (attached tables 29 and 33 in background document).

HISTOPATHOLOGY (PARENTAL ANIMALS)
No test substance related histopathological findings were observed.

The histopathological findings observed were either related to, agonal to spontaneous, or were incidental and of the type routinely observed in Wistar rats of this age (attached tables 30 and 33 in background documents).


Key result
Dose descriptor:
NOAEL
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
not specified
Food efficiency:
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
no effects observed on sex of live pups
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
GROSS PATHOLOGY (OFFSPRING)
Gross external examination of live pups sacrificed on day 4 post-partum did not reveal any abnormality that could be attributed to the treatment (attached table 28 in background material).
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

The tables are attached in the field "Attached background material", exept for the table 11 below.

Table 11 - Summary of effects on reproduction/development tabular report.

OBSERVATIONS

VALUES

Group

G1

G2

G3

G4

G5

Dose (mg/kg b.w.)

0

25

50

75

100

Pairs started (N)

10

10

10

10

10

Females showing evidence of copulation (N)

10

10

10

10

10

Females achieving pregnancy (N)

10

10

10

10

10

Conceiving days 1-5(N)

10

10

10

10

10

Conceiving days ≥6-(N)

0

0

0

0

0

Pregnancy ≤ 21 days(N)

0

3

0

2

3

Pregnancy =22 days(N)

8

7

9

7

6

Pregnancy ≥ 23 days (N)

2

0

1

1

0

Dams with live young born (N)

10

10

10

10

10

Dams with live young at day 4 pp(N)

10

10

10

10

10

Corpora lutea/dam (mean)

14,30

14,40

14,60

15,00

15,33

Implants/dam (mean)

13,60

13,90

14,10

14,70

13,78

Live pups/dam at birth (mean)

12,50

12,70

12,90

13,70

12,89

Live pups/dam at day 4 (mean)

12,30

12,70

12,70

13,70

12,78

Sex ratio (m/f) at birth (mean)

92,31

144,23

115,00

101,47

118,87

Sex ratio (m/f) at day 4 (mean)

92,19

144,23

111,67

101,47

116,98

litter weight at birth (mean)

78,04

74,81

81,96

84,24

79,20

litter weight at day 4 (mean)

125,75

117,43

129,20

133,14

116,04

ABNORMAL PUPS          

Dams with 0 (N)

10

10

10

10

9

dams with 1 ( N)

0

0

0

0

0

Dams with ≥ 2(N)

0

0

0

0

0

LOSS OF OFFSPRING

Pre - implantation (corpora lutea minus implantations)

Females with 0(N)

7

7

7

8

3

Females with 1(N)

0

1

2

1

3

Females with 2(N)

2

2

0

1

0

Females with ≥ 3(N)

1

0

1

0

3

Pre-natal/ post - implantations (implantations minus live Birth)

Females with 0(N)

4

7

3

4

4

Females with 1(N)

3

0

3

3

3

Females with 2(N)

1

1

3

2

1

Females with ≥3(N)

2

2

1

1

1

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

Females with 0(N)

8

10

8

10

8

Females with 1(N)

2

0

2

0

1

Females with 2(N)

0

0

0

0

0

Females with ≥3(N)

0

0

0

0

0

Conclusions:
On the basis of the results obtained in the present study, the No Observed Adverse Effect Level (NOAEL) of Solvent Black 46 for reproduction/developmental toxicity screening test is considered as 100 mg/kg b.w.
Executive summary:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar ratswas performed to evaluate Solvent Black 46.

One hundred and twenty rats were randomized into seven groups, five main groups containing 10 rats/group/sexviz.,G1 (Control), G2, G3, G4, G5 (treated groups) and two reversal groups containing 5 rats/group/sexviz.,G6 (control reversal) and G7 (High reversal).

The test substance Solvent Black 46 was suspended in corn oil and administered by gavage daily at the doses of 25, 50, 75 and 100 mg/kg b.w. to the rats belonging to G2 (low), G3 (Low intermediate), G4 (High intermediate), G5 (High) and G7 (High reversal) groups respectively. In males dosing was carried out up to 28 days, where as in females test substance was administered during premating, mating, gestation and up to day 3 post partum.

Animals from reversal group were dosed up to first sacrifice of dams andkept untreated for 14 days to evaluate the reversibility of effects after withdrawal of the treatment. Control and control reversal groups were treated similarly but with corn oil alone. The dose volume was maintained at 10 ml/kg b.w. in all the groups.

Males were sacrificed after completion of four weeks of dosing (Day 28th) and females were sacrificed on day 4 post partum. Rats in reversal groups were sacrificed after 14 days of additional observation period.

Observed body weight in low (G2) and low intermediate (G3) dose groups was comparable with respective control group (G1) throughout the observation period. However, high intermediate (G4), high (G5) and high reversal (G7) dose groups showed a decreasing trend from day 14 with respect to their controls (G1 & G6). This decrease was statistically different in high dose reversal (G7) from day 14 to till the end, and on day 28 in high intermediate and high dose groups.

Food consumption recorded in treated groups was comparable with respective control groups and there was no significant difference. However, the statistically significant decrease in G2, G3 & G5 females on few days was not considered as treatment related, since there was no dose dependency and consistency.

Mortality/morbidity was not observed in low (G2), low intermediate (G3), high intermediate (G4) group of animals. However, the death of one animal in each group of high (G5), high reversal (G7) males and one female in high (G5) dose groups could be considered as treatment related.

No clinical signs of toxicity were observed in males and females of G1, G2 and G6. The bluish perianal staining in animals of G3, G4, G5 & G7 groups was considered due to the colour of test substance, further there were no other relevant changes observed to relate this finding as significant. Hence this effect was not considered as adverse effect. However, animals belong to G4, G5 and G7 groups exhibited dullness and piloerection at various interval. In addition, abdominal distension and respiratory distress were observed in dead animals of G5 and G7.

Observed weekly individual clinical signs like home cage, handling and standard arena (open field) were similar to daily observations and did not showed any significance.

All dams were allowed to litter naturally and the size and weight of litters, live births, runts, sex of live pups and the presence of gross abnormalities were recorded within 24 hours of parturition (day 0) and on day 4 post partum.

No test substance related effect was observed in parameters like fertility, mating period, gestation length, mean corpora lutea, mean implantation, mean litter size, mean litter/pup weight, implantation losses, and sex ratio of offspring in G2,G3 and G4 groups when compared to control. Whereas, in high dose (G5) group a significant decrease was observed in total (but not mean) corpora lutea and subsequent Implantations with respect to control (G1) group. Similarly a decreased litter weight on post-partum day 4 was observed. However these changes were not statistically significant and this decrease is due to the death of one female in this dose group. Hence these changes could not be attributed to treatment related.

Complete gross pathology was conducted on all adult animals and examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.

No treatment related gross pathological findings were observed. Test substance related bluish discoloration at perianal area was recorded in G3, G4 and G5 group. This was considered due to color of test substance which was excreted through feces and there was no correlating macroscopic or histopathology findings of alimentary tract. Hence, it was not considered as an adverse effect. The macroscopic findings observed were either related to physiological, agonal to spontaneous, or were incidental and of the type routinely observed in Wistar rats of this age. No test substance related gross abnormalities were found in pups of any of treated group.

Organ weight of testes and epididymis was recorded from all animals while weight of Liver, Kidneys, Adrenals, Thymus, Spleen, Brain and Heart was recorded in randomly selected five males and five females.

No treatment related organ weight changes were observed in any of the groups when compared to concurrent control group. The observed statistical significant changes in absolute and relative weights of adrenals in G2 group males and absolute weight of thymus in G4 group females when compared to concurrent control group (G6) was not considered as treatment related, since there was no correlation with either macro or microscopic observations.

Detailed histological examination in all animals of control and high dose group was performed on the Ovaries, Testes, and Epididymis with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell strucutre. In addition, full histopathological examination was carried out on the selected five males and five females of control and high dose group.

Treatment related histopathological findings were not observed. The histopathological findings observed were either related to, agonal to spontaneous, or were incidental and of the type routinely observed in Wistar rats of this age.

On the basis of the results obtained in the present study, the No Observed Adverse Effect Level (NOAEL) of Solvent Black 46 for Reproduction toxicity is considered as 100 mg/kg b.w.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Good quality of whole 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

At the maximum tested dose, there were no adverse effect observed

Effects on developmental toxicity

Description of key information

In the study performed according to the OECD 414 no effects attributed to the Solvant Black 46 was observed at the maximum tested dose of 20 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2016-08-04 to 2017-02-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
females and males
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Vivo Bio Tech Ltd., Telangana, India. CPCSEA Registration No. 1117/PO/RcBiBt/S/07/CPCSEA.
- Age at study initiation: 15-16 weeks
- Weight at study initiation:
* Males : 289 g - 362 g
* Females : 201g - 239 g
- Fasting period before study: no
- Housing:
CAGES: Polycarbonate cages (size 31 cm (L) X 21 cm (W) X 20 cm (H)), cleaned and disinfected before the animals were brought in and cleaned at regular interval during the study.
* Acclimatation period: 1-3 rats /sex/cage
* During mating: 2 females and 1 male / cage
* Pregnant females, post-mating: 1 female/cage.
BEDDING MATERIAL: sterilized corn cob produced from pure corn, dried and free from dust, batch SPAR-33/2016, from Sparconn Life Sciences, Bangalore. Renewed as often as necessary to keep the animals dry and clean

STUDY ROOM: before initiation, the study room was cleaned and disinfected. During study: the floor of the study room and work tops were swept and mopped with disinfectant solution every day and when required.

- Diet (e.g. ad libitum): ad libitum: conventional laboratory pellet diet (batch 040716, 040816 and 040916) from Nutrivet Life Sciences, Pune.
- Water (e.g. ad libitum): ad libitum: aqua guard filtered drinking water in bottles. Samples of drinking water were subjected periodically to bacteriological tests and to chemical contaminant analysis (latests test results available)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22.4°C
- Relative Humidity (%): 45.4 to 66.5%
- Air changes (per hr): 12 time / hr, and filtered adequately
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: To: 2016-08-05 to 2016-09-23
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared daily: the defined quantity of the test substance was transfered to the mortar and triturated using pestle. Vehicle is added in to mortar and mixed well.
The formulation was transfered to the measuring cylinder and make the volume up to desired quantity.
At the time of dosing, dose formulations were stirred continously on a magnetic stirrer for maitaining homogeneity of testing solutions.

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil was used based on the solubility testing
- Concentration in vehicle: up to 20 mg test item / bw
- Amount of vehicle (if gavage): dose volume not exceeding 0.4 ml/100g bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The testing solution samples at 0 mg/mL, 1.25 mg/mL, 2.5 mg/mL and 5 mg/L were analysed by HPLC. Two replicates of 2 ml samples from upper, middle and lower layer were sent to Analytical department for the determination of homogeneity and test substance content. The analysis was carried out 2 times during treatment at starting and end of the treatment period.

The analytical parameter has been validated with respect to the following parameters:
SPECIFICITY: evaluated by analysing the blank, standard, vehicle and sample.
LINEARITY: carried out by preparing and analysing standards solutions of 6 concentrations (covering the target analyte concentration). The coded calibration solutions were injected into the HPLC, a plot was drawn between the concentration and the peak area response. The correlation coefficient, slope and intercept of the linear regression were calculated.
ASSAY ACCURACY AND PRECISION: carried out by fortifying the standard in the vehicle at 2 levels (covering the target analyte concentration). Five preparations were carried out at each concentration level selected. One control at each concentration level was maintained. Mean, SD, % RSD were calculated.

The test item formulations were found to be homogeneous.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/2
- Length of cohabitation: until pregancy occurs or 2 weeks elapsed.
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: observation of sperm positive vaginal smear referred to as day 0 of gestation.
Duration of treatment / exposure:
From gestation day 5 to gestation day 19 (i.e. 15 days).
Frequency of treatment:
Once daily
Duration of test:
19 days from gestation day 0 (i.e. 20 days from gestation day 0).
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Basis: actual ingested
Dose / conc.:
5 mg/kg bw/day (nominal)
Remarks:
Basis: actual ingested
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Basis: actual ingested
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
Basis: actual ingested
No. of animals per sex per dose:
25 females per dose:
G1: 0 mg/kg bw/day
G2: 5 mg/kg bw/day
G3: 10 mg/kg bw/day
G4: 20 mg/kg bw/day
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: range finding study
- Rationale for animal assignment: based on body weight, all animals were assigned in descending order. Highest body weight animals were selected for mating. Randomization was done based on day 0 body weight. After confirmation of mating by vaginal smear examination, animals were assigned in unbaised manner to control and treated groups. The inseminated by same male were evenly distributed across the group.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule : at arrival in the laboratory, after completion of the acclimatization period and before group allocation and D0, D3, D5, D8, D11, D14, D17 and D20 (day of scheduled killed) of pregnancy

FEED CONSUMPTION: Yes
- Time schedule : D0, D3, D5, D8, D11, D14, D17 and D20 (day of scheduled killed) of pregnancy

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20: Organs examined: gross observation of all maternal organes (for any structural abnormalities or pathological changes) with emphasis on uterus, number of live/viable foetuses, number of dead fetuses, number of corpora lutea, number of implantations sites, degree of resorption and weight of placenta recoded for the terminally sacrified females. The gravid uteri (including cervix) were weighed at necropsy

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 resorptions: Yes




Fetal examinations:
Each fetuses were marked, sexed, weighted and crown rump length was measured
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]

Statistics:
Raw data were processed using statistical software Sigma Plot 11.0: the mean and standard deviation were calculated using the excel and all data were summarized in tabular form.

Homogeneity checked for all continuous data (body weight, feed consumption, weight of uterus, relative weight of uterus, weight of ovaries, number of live fetuses, pre-implantation loss, number of implantation site, number of resorption, post-implantation loss, CL count, mean weight of fetus, mean weight of placenta, mean foetal CRL measurement) using Shapiro Wilk Test, followed by ANOVA and Kruskal Wallis one Way ANOVA
Data showing singificance in their variances were subjected to Holm-Sidak and Dunn's methods.
Indices:
Pre-implantation loss, post-implantation loss, mean numbers of resorption, embryonic deaths and total foetuses
Historical control data:
No
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were observed in any of the animals throughout the study period (see table 1).
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality and morbidity were oberved among any of the groups of animals throughout the study period (see Table 2)
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in the body weight of dams of treated groups when compared to control group during study period.
There was no statistically significant difference in the percent body weight change of dams of treated groups when compared to control group during study period.
(see Tables 3 and 4).
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
Even if not a feeding study, food consumption was examined: there was no statistically significant diffrence in feed consumption of dams of trested groups when compared to control group during the study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
External and internal gross examination of all dams in control and all treated groupds did not reveal any abnormality of pathological significance (see Table 5).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
weight of uterus, weight of ovaries
Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in pre- and post-implantation loss in any of the treated groups as compared to control group (see Table 7).
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
no total litter loss by resorption was observed.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in number resorptions in any of the treated groups as compared to control group (see Table 7).
Dead fetuses:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in number of lives fetuses in any of the treated groups as compared to control group (see Table 7).
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Intrauterine growth was unaffected by test item administration in G2, G3 and G4 compared to control group. All females were determined to be gravid, with the exception of 6 females in G1, 2 females in G2 and 4 females in G4.
(see Table 6).
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pregnancy rate of female in the group of control, and treated animals in G2, G3 and G4 was 76%, 92%, 100% and 84% respectively.
Other effects:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in absolute and relative weights of uterus, weight of ovaries, corpus luteum, number of implemantation site in any of the treated groups as compared to control group (see Table 7).
Key result
Dose descriptor:
NOAEL
Effect level:
>= 20 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no test item related changes at the highest tested dose
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in mean weight of fetus of both sexes of any of the treated group fetuses as compared to control group (see Table 8).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in number of lives fetuses in any of the treated groups as compared to control group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Male/female sex ratio of fetuses in the group of control, and treated animals at in G2, G3 and G4 was 101/100, 135/139, 141/157 and 107/125 respectively (see Table 12).
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The external gross examination of fetuses showed small fetuses:
- G1 : 2 fetuses
- G2 : 5 fetuses
- G3: 1 fetus
- G4: 2 fetuses

(see Table 9)

These observations were not dose dependent and inconsistent hence, not considered as treatment related, but considered as spontaneous in origin.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related fetal skeletal malformations noted at any of the dose levels tested.
(see Table 11)
Visceral malformations:
no effects observed
Description (incidence and severity):
Visceral examination did not show any malformation in fetuses of treatment groups and control group (see Table 10).
Other effects:
no effects observed
Description (incidence and severity):
Head razor examination did not show any abnormality in treated group and control group (see Table 10)
Key result
Dose descriptor:
NOAEL
Effect level:
>= 20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There was no test item induced adverse effects on structural development or growth in fetuses under the experimental conditions
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 1: Summary of General Clinical Signs Observation

Sex: Female

Group

Dose (mg/kg b. wt.)

No. of Animals

Animal Number

Gestation Day

Clinical Signs

G1

0

25

01-25

0-20

Normal

G2

5

25

26-50

0-20

Normal

G3

10

25

51-75

0-20

Normal

G4

20

25

76-100

0-20

Normal

 Keys:No. = Number, mg/kg b. wt. = milligram/ kilogram body weight

Table 2: Summary of Mortality and Morbidity

Sex: Female

Group

Dose

(mg/kg b. wt.)

No. of Animals

Gestation Day

Observation

Morning 

Evening

G1

0

25

0-20

No mortality/ morbidity

No mortality/ morbidity

G2

5

25

0-20

No mortality/ morbidity

No mortality/ morbidity

G3

10

25

0-20

No mortality/ morbidity

No mortality/ morbidity

G4

20

25

0-20

No mortality/ morbidity

No mortality/ morbidity

 Keys:No. = Number, mg/kg b. wt. = milligram/ kilogram body weight

Table 3: Summary of Body Weight (gram)

Sex: Female

Group (N)

G1 (19)

G2 (23)

G3 (25)

G4 (21)

Dose (mg/kg b. wt.)

0

5

10

20

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

0

235.53

10.48

233.87

9.69

233.76

11.41

233.81

12.03

3

243.21

11.83

241.22

9.15

240.60

12.14

240.00

12.05

5

245.89

12.04

245.87

9.45

245.96

12.27

244.76

11.43

8

250.89

12.56

250.65

10.42

251.36

12.90

248.38

11.25

11

261.11

12.58

261.61

11.21

262.44

12.83

257.52

11.68

14

273.68

13.60

273.57

12.04

274.24

14.41

268.52

12.27

17

296.26

17.43

297.22

14.84

298.04

15.46

290.52

14.00

20

329.74

23.17

330.83

18.44

331.44

18.08

321.05

16.37

 Keys:N= Number of Animals, SD= Standard Deviation, mg/kg b. wt. = milligram/ kilogram body weight

Table 4: Summary of Body Weight Change (%)

Sex: Female

Group (N)

G1 (19)

G2 (23)

G3 (25)

G4 (21)

Dose (mg/kg b. wt.)

0

5

10

20

Day

Mean

SD

Mean

SD

Mean

SD

Mean

SD

0-3

3.27

2.19

3.16

1.09

2.93

1.22

2.66

1.49

0-5

4.41

2.33

5.16

1.65

5.22

1.33

4.72

1.81

0-8

6.52

2.38

7.19

1.99

7.53

1.63

6.28

2.07

0-11

10.88

2.91

11.89

3.15

12.29

2.04

10.20

2.50

0-14

16.21

3.11

17.00

2.98

17.32

2.59

14.92

3.26

0-17

25.79

5.12

27.07

3.09

27.53

3.55

24.34

3.98

0-20

40.02

8.12

41.41

4.11

41.85

5.53

37.42

5.29

Table 5: Summary of Individual Gross observation

Sex: Female

Group

Dose (mg/kg b. wt.)

No. of Animals

Animal Number

Macroscopic Observation

External

Internal

G1

0

25

01-25

NAD

NAD

G2

5

25

26-50

NAD

NAD

G3

10

25

51-75

NAD

NAD

G4

20

25

76-100

NAD

NAD

Keys:NAD = No Abnormality Detected, mg/kg b. wt. = milligram/ kilogram body weight

Table 6: Summary of Number of gravide uterus and pregnancy rate

Group (N)

G1 (25)

G2 (25)

G3 (25)

G4 (25)

Dose (mg/kg b. wt.)

0

5

10

20

No. of Gravid Uterus

19

23

25

21

Pregnancy Rate (%)

76

92

100

84

Keys:N= Number of animals,mg/kg b. wt. = milligram/ kilogram body weight, % = Percent

Table 7: Summary of Maternal Evaluation Data

Group (N)

G1 (19)

G2 (23)

G3 (25)

G4 (21)

Dose (mg/kg b. wt.)

0

5

10

20

Parameters

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Weight of Dam (g)

329.737

23.1729

330.826

18.4407

331.44

18.081

321.048

16.3661

Weight of Uterus (g)

55.77

13.24

62.66

8.79

64.12

9.39

58.19

9.83

Relative Uterus Weight (%)

16.76

3.28

18.90

2.11

19.09

2.60

18.1017

2.8028

Weight of Ovaries (g)

0.16

0.04

0.16

0.02

0.15

0.02

0.14

0.03

CL Count

12.16

1.74

12.91

1.41

12.80

1.47

13.14

1.59

Number of Live Fetus

10.47

2.63

11.91

1.56

11.88

1.67

11.05

2.09

Number of Dead Fetus

0.05

0.23

0.00

0.00

0.04

0.20

0.00

0.00

Number of Implantation Site

11.79

2.02

12.61

1.75

12.72

1.40

12.00

2.24

Number of Resorption

1.21

1.32

0.65

0.78

0.76

0.97

0.95

0.97

Pre-Implantation Loss (%)

3.17

7.43

2.53

6.88

0.19

9.73

8.32

14.69

Post-Implantation Loss (%)

12.05

13.15

5.21

6.17

6.69

7.45

7.53

7.97

Keys:N = Number of Dams, n = Number of Dams including Dams with Complete Resorption, g = Gram, CL = Corpus Luteum, SD = Standard Deviation,% = Percent,mg/ kg b. wt. = milligram/ kilogram body weight

Table 8: Summary of Fetal weight (g), Placenta weight (g) and CRL Measurment (cm)

Group (N)

G1 (19)

G2 (23)

G3 (25)

G4 (21)

Dose (mg/kg b. wt.)

0

5

10

20

Sex

Parameters

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Males

Fetus Weight (g)

3.4058

0.3107

3.4789

0.2757

3.5694

0.2939

3.5248

0.2209

Placenta Weight (g)

0.4700

0.0536

0.4494

0.0456

0.4412

0.0385

0.4359

0.0356

CRL (cm)

3.4288

0.1919

3.4309

0.1224

3.4588

0.1384

3.4644

0.1780

Females

Fetus Weight (g)

3.2420

0.2542

3.3270

0.2318

3.4079

0.2326

3.3480

0.2943

Placenta Weight (g)

0.4688

0.0948

0.4405

0.0464

0.4331

0.0460

0.4271

0.0493

CRL (cm)

3.3418

0.1759

3.3712

0.0975

3.4447

0.1538

3.3383

0.1725

Male and Female

Fetus Weight (g)

3.3323

0.2504

3.4010

0.2269

3.4747

0.2487

3.4331

0.2525

Placenta Weight (g)

0.4734

0.0802

0.4437

0.0439

0.4371

0.0390

0.4329

0.0409

CRL (cm)

3.3829

0.1762

3.4049

0.1032

3.4518

0.1443

3.3951

0.1690

Keys:N= Number of animals, SD= Standard Deviation, g = Gram, cm = Centimeter, CRL = Crown to Rump Length,mg/kg b. wt. = milligram/ kilogram body weight.

 Note:Fused placenta was observed in fetus number 09 and 10 of animal number 08. Hence placenta weight was not considered in mean weight of Placenta in male and female separately but considered in combined male and female.

Table 9: Summary of External, Visceral and Head Razor Evaluation Data

Group

Fetus

Litters

G1

G2

G3

G4

G1

G2

G3

G4

Dose (mg/kg b. wt.)

0

5

10

20

0

5

10

20

Number Examined Externally

201

274

298

232

19

23

25

21

Dead Fetus

1

0

1

0

1

0

1

0

Small Fetus

2

5

1

2

1

3

1

2

NAD

198

269

296

230

19

23

25

21

Number Examined Viscerally

104

142

156

123

19

23

25

21

NAD

104

142

156

123

19

23

25

21

Number Examined by

Head Razor

104

142

156

123

19

23

25

21

NAD

104

142

156

123

19

23

25

21

Keys:NAD = No Abnormality Detected, mg/kg b. wt. = milligram/ kilogram body weight

Table 10: Summary of Percent (%) External, Visceral and Head Razor Evaluation Data

Group

% Fetus

% Litters

G1

G2

G3

G4

G1

G2

G3

G4

Dose (mg/kg b. wt.)

0

5

10

20

0

5

10

20

Number Examined Externally

201

274

298

232

19

23

25

21

% Dead Fetus

0.50

0.00

0.34

0.00

5.26

0.00

4.00

0.00

% Small Fetus

1.00

1.82

0.34

0.86

5.26

13.04

4.00

9.52

% NAD

100

100

100

100

100

100

100

100

Number Examined Viscerally

105

142

156

123

19

23

25

21

% NAD

100

100

100

100

100

100

100

100

Number Examined by

Head Razor

105

142

156

123

19

23

25

21

% NAD

100

100

100

100

100

100

100

100

Keys:NAD = No Abnormality Detected, mg/kg b. wt. = milligram/ kilogram body weight,% = Percent

Table 11: Summary of Number and Percent (%) Skeletal Evaluation Data

Skeletal Malformations / Variations

Group: No. of Fetus (% foetus)

Group: No. of Litters (% litters)

G1

(96)

G2

(132)

G3

(137)

G4

(109)

G1

(19)

G2

(23)

G3

(25)

G4

(21)

Name of Bone

Malformation/ Variation

NAD

45

(46.88%)

61

(46.21%)

67

(48.91%)

73

(66.97%)

1

(5.26%)

2

(8.70%)

3

(12.00%)

7

(33.33%)

Hyoid

Unossified / Incomplete Ossification

5

(5.21%)

3

(2.27%)

3

(2.19%)

4

(3.67%)

4

(21.05%)

1

(4.35%)

2

(8.00%)

4

(19.05%)

Frontal/ Parietal/ Interparietal/ Occipital

Unossified/Incomplete Ossification

8

(8.33%)

15

(11.36%)

13

(9.49%)

3

(2.75%)

4

(21.05%)

5

(21.74%)

7

(28.00%)

3

(14.29%)

Zygomatic

Incomplete Ossification

1

(1.04%)

5

(3.79%)

7

(5.11%)

6

(5.50%)

1

(5.26%)

1

(4.35%)

4

(16.00%)

3

(14.29%)

Fused

0

(0.00%)

2

(1.52%)

2

(1.46%)

0

(0.00%)

0

(0.00%)

2

(8.70%)

1

(4.00%)

0

(0.00%)

Rib

Supernumerary

19

(19.79%)

20

(15.15%)

28

(20.44%)

17

(15.60%)

9

(47.37%)

10

(43.48%)

12

(48.00%)

6

(28.57%)

Wavy

0

(0.00%)

2

(1.52%)

4

(2.92%)

0

(0.00%)

0

(0.00%)

1

(4.35%)

1

(4.00%)

0

(0.00%)

Incomplete Ossification

0

(0.00%)

1

(0.76%)

2

(1.46%)

0

(0.00%)

0

(0.00%)

1

(4.35%)

1

(4.00%)

0

(0.00%)

Sternebra

Unossified/Incomplete Ossification

32

(33.33%)

41

(31.06%)

42

(30.66%)

21

(19.27%)

15

(78.95%)

18

(78.26%)

18

(72.00%)

10

(47.62

Sternoschisis

4

(4.71%)

2

(1.52%)

3

(2.19%)

3

(2.75%)

3

(15.79%)

2

(8.70%)

3

(12.00%)

2

(9.52%)

Bipartite Ossification

0

(0.00%)

2

(1.52%)

1

(0.73%)

0

(0.00%)

0

(0.00%)

2

(8.70%)

1

(4.00%)

0

(0.00%)

Misaligned

0

(0.00%)

0

(0.00%)

1

(0.73%)

0

(0.00%)

0

(0.00%)

0

(0.00%)

1

(4.00%)

0

(0.00%)

Thoracic centrum

Bipartite Ossification

0

(0.00%)

3

(2.27%)

2

(3.65%)

0

(0.00%)

0

(0.00%)

3

(13.04%)

2

(8.00%)

0

(0.00%)

Dumbell Ossification

0

(0.00%)

3

(2.27%)

5

(3.65%)

0

(0.00%)

0

(0.00%)

3

(13.04%)

3

(12.00%)

0

(0.00%)

Lumbar Vertebra

Unossified / Incomplete Ossification

0

(0.00%)

1

(0.76%)

0

(0.00%)

0

(0.00%)

0

(0.00%)

1

(4.35%)

0

(0.00%)

0

(0.00%)

Thoracic Vertebra

Incomplete Ossification

0

(0.00%)

1

(0.76%)

0

(0.00%)

0

(0.00%)

0

(0.00%)

1

(4.35%)

0

(0.00%)

0

(0.00%)

Ischium

Unossified / Incomplete Ossification

0

(0.00%)

2

(1.52%)

1

(0.73%)

0

(0.00%)

0

(0.00%)

1

(4.35%)

1

(4.00%)

0

(0.00%)

Pubis

Unossified/Incomplete Ossification

0

(0.00%)

0

(0.00%)

1

(0.73%)

0

(0.00%)

0

(0.00%)

0

(0.00%)

1

(4.00%)

0

(0.00%)

Squamosal

Incomplete Ossification

0

(0.00%)

3

(2.27%)

4

(2.92%)

0

(0.00%)

0

(0.00%)

1

(4.35%)

1

(4.00%)

0

(0.00%)

Metacarpals and Metatarsals

Incomplete Ossification

0

(0.00%)

(0.00%)

1

(0.76%)

1

(0.73%)

0

(0.00%)

0

(0.00%)

1

(4.35%)

1

(4.00%)

0

(0.00%)

 Keys:NAD = No Abnormality Detected,No. = Number, % = Percent.

Table 12: Summary of Fetal Sex Ratio

Group (N)

G1 (201)

G2 (274)

G3 (298)

G4 (232)

Dose

(mg/kg b. wt.)

0

5

10

20

Sex Ratio

(Male/Female)

101/100

135/139

141/157

107/125

Key:N= Number of Fetus,mg/kg b. wt. = milligram/ kilogram body weight

Conclusions:
Based on the findings of prenatal developmental oral toxicity study of the test item in Wistar rat at dose level 0, 5, 10, 20 mg/kgbw, no test item related changes were observed in any of the doses tested hence, the maternal No Observed Adverse Effect Level (NOAEL) of the test item is considered to be 20 mg/kg bw in Wistar rat. Since, there was no test item induced adverse effects on structural development or growth in the fetuses, the developmental No Observed Adverse Effect Level (NOAEL) of the test item in fetuses is considered to be 20 mg/kg bw under the experimental conditions.
Executive summary:

The objective of this study was to provide evaluations of prenatal developmental toxicity of the test item in Wistar rats.

The results of the study provides general information concerning the effects of prenatal exposure on the pregnant animal and fetus, also include assessment of maternal effects as well as death, structural abnormalities, or altered growth in the fetus and determination of the maternal and developmental No Observed Adverse Effect Level (NOAEL).

The animals were randomly allocated to the four groups (25 Females/group). The doses selected for groups were; 0, 5, 10 and 20 mg/kg body weight.

Control group animals received vehicle (Corn oil) aloneand treatment groups were administered with test item daily by oral route, throughout the study period (Gestation day5-19).

Test item formulation was found to be homogeneous. The mean active ingredient content of AY36 at 1.25, 2.5 and 5 mg/ml concentrationwas 1.197,2.377, 4.701 mg/ml and 1.186, 2.356, 4.730 mg/mlat first week (initialsample)and last week (finalsample)of treatment, respectively.The mean active ingredient content of BV3 at 1.25, 2.5 and 5 mg/ml concentrationwas1.205, 2.391, 4.759 mg/ml and 1.194, 2.393, 4.747mg/mlat first week (initialsample)and last week (finalsample)of treatment, respectively.

No mortality and morbidity were observed among any of the groups of animals throughout the study period.

No clinical signs were observed in any of the animals throughout the study period.

There was no statistically significant difference in the body weight of dams of treated groups when compared to control group G1 during study period.

There was no statistically significant difference in the percent body weight change of dams of treated groups when compared to control group G1 during study period.

There was no statistically significant difference in feed consumption of dams of treated groups when compared to control group during study period.

External and internal gross examination of all dams in control and all treated groups did not reveal any abnormality of pathological significance.All females were determined to be gravid, with the exception of 6, 2, 4 females in G1 (0 mg/kg body weight), G2 (5 mg/kg body weight) and G4 (20 mg/kg body weight), respectively.

There was no statistically significant difference in absolute and relative weights of uterus, weight of ovaries, number of live fetuses, pre-implantation loss, number of implantation site, number of resorptions, post-implantation loss, CL count in any of the treated groups as compared to control group. Intrauterine growth and survival of fetuses was unaffected by test item administration at 5 (G2), 10 (G3) and 20 (G4) mg/kg body weight. Pregnancy rate of female in the group of G1, G2, G3 and G4 was 76%, 92%, 100% and 84%, respectively.

There was no statistically significant difference in mean weight of fetus, mean weight of placenta and mean foetal CRL measurement in male, female and both sexes of any of the treated group fetuses as compared to control group.

The external gross examination of fetuses showed small fetuses 2 (G1), 5 (G2), 1 (G3), 2 (G4) and dead fetuses 1(G1), 1(G3). These Observations were not dose dependent and inconsistent hence, not considered as treatment related, but considered as spontaneous in origin. Male/ Female sex ratio of fetuses in the group G1, G2, G3 and G4 was 101/100, 135/139, 141/157 and 107/125, respectively.                      

Visceral examination did not show any malformation in the fetuses of treatment groups and control group.

Head razor examination did not show any abnormality in control and treatment groups.

There were no test item related fetal skeletal malformation noted at any of the dose levels tested. Skeletal malformations like Hyoid: Unossified/ Incomplete Ossification were noted for 5 (4), 3 (1), 3 (2), 4 (4) fetus (litter) in G1, G2, G3 and G4, respectively. Frontal/ Parietal/ Interparietal/Occipital: Unossified/Incomplete Ossification were noted for 8 (4), 15 (5), 13 (7), 3 (3) fetus (litter) in G1, G2, G3 and G4, respectively. Zygomatic: incomplete ossification were noted for 1 (1), 5 (1), 7 (4), 6 (3) fetus (litter) in G1, G2, G3 and G4, respectively and Zygomatic: fused were noted for 2 (2) and 2 (1) fetus (litter) in G2 and G3, respectively. Supernumerary rib were noted for 19 (9), 20 (10), 28 (12), 17 (6) fetus (litter) in G1, G2, G3 and G4, respectively. Wavy ribs were noted for 2 (1), 4 (1), fetus (litter) in G2 and G3 respectively. Incomplete ossification of ribs were noted for 1 (1), 2 (1), fetus (litter) in G2 and G3 respectively.

Sternebra: unossified/ Incomplete ossification were noted for 32 (15), 41 (18), 42 (18), 21 (10) fetus (litter) in G1, G2, G3 and G4, respectively. Sternebra: sternoschisis were noted for 4 (3), 2 (2), 3 (3), 3 (2) fetus (litter) in G1, G2, G3 and G4, respectively. Sternebra: bipartite ossification were noted for 2 (2), 1 (1) fetus (litter) in G2 and G3, respectively. Sternebra: misaligned was noted for 1 (1) fetus (litter) in G3. Thoracic centrum: Bipartite ossification were notedfor 3 (3), 2 (2), fetus (litter) in G2 and G3, respectively.

Thoracic centrum: dumbell ossification were noted for 3 (3), 5 (3) fetus (litter) in G2 and G3, respectively. Lumbar vertebra: Unossified/ Incomplete ossification was noted for 1 (1) fetus (litter) in G2. Thoracic vertebra: Incomplete ossification was noted for 1 (1) fetus (litter) in G2. Ischium: Unossified/ Incomplete ossification were noted for 2 (1), 1 (1) fetus (litter) in G2 and G3, respectively. Pubis: Unossified/ Incomplete ossification was noted for 1 (1) fetus (litter) in G3. Squamosal: Incomplete ossification were noted for 3 (1), 4 (1) fetus (litter) in G2 and G3, respectively. Metacarpals and Metatarsals: Incomplete ossification were noted for 1 (1), 1 (1) fetus (litter) in G2 and G3, respectively.

There were no test item related fetal skeletal developmental variations noted at any of the dose levels tested. These findings occured infrequently and were noted similarly in the concurrent control group, and /or were noted in a manner thatwas not considered to be treatment related. As these observations were not dose dependent therefore, these skeletal developmental variations were not considered to be test item related.

 

Conclusion

Based on the findings ofprenatal developmentaloral toxicity study of the item in Wistar rat at dose level 0, 5, 10 and 20 mg/kg body weight, no test item related changes were observed in any of the doses tested hence, the maternal No Observed Adverse Effect Level (NOAEL) of the test item is considered to be 20 mg/ kg body weight in Wistar Rat. Since, there was no test item induced adverse effects onstructural development or growth in the foetuses, the developmental No Observed Adverse Effect Level (NOAEL) of test item in fetuses is considered to be 20 mg/ kg body weight under the experimental conditions.


Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Species:
rat
Quality of whole database:
Good quality of the database
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

During the test performed according to the OCDE 414, no test item related changes were observed in any of the dose tested (maximum tested dose of 20 mg/kg bw/day) hence, the maternal No Observed Adverse Effect Level (NOAEL) of the test item is considered to be 20 mg/kg bw in Wistar rat. Since, there was no test item induced adverse effects on structural development or growth in the fetuses, the developmental No Observed Adverse Effect Level (NOAEL) of the test item in fetuses is considered to be 20 mg/kg bw under the experimental conditions.

Justification for classification or non-classification

In the 2 studies performed, no adverse effects were observed at the maximum tested doses. Therefore, the substance is not classified.