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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD Guideline 422. GLP study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Benzenamine, reaction products with aniline hydrochloride and nitrobenzene, hydrochlorides
EC Number:
309-913-1
EC Name:
Benzenamine, reaction products with aniline hydrochloride and nitrobenzene, hydrochlorides
Cas Number:
101357-16-8
Molecular formula:
Not applicable. Multiconstituent substance.
IUPAC Name:
Benzenamine, reaction products with aniline hydrochloride and nitrobenzene, hydrochlorides
Details on test material:
- Name of test material (as cited in study report): C.I. Solvent Black 5 (analogue CAS 11099-03-9).
- Lot/batch No.: 10137087
- Physical state: Blackish brown powder
- Storage condition of test material: Room temperature.
- Stability and uniformity were property has been confirmed.

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on test animals or test system and environmental conditions:
- Age at study initiation: 10 weeks old

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Methylcellulose aqueous solution
Details on exposure:
VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg
- 0.5%w/v Methylcellulose aqueous solution (suspended)
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Males: 42 days (from 14 days before mating, and through the mating period until a day before necropsy).
Females: 41-48 days (from 14 days before mating, and through the mating period until day 4 of lactation).
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (control), 40, 200 and 1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
Males: 7 (control), 12 (40 mg/kg bw/day), 12 (200 mg/Kg bw/day), 7 (1000 mg/kg bw/day), 5 (control with recovery), 5 (1000 mg/kg bw/day with recovery).
Females: 12 (control), 11 (40 mg/kg bw/day), 11 (200 mg/Kg bw/day), 12 (1000 mg/kg bw/day), 5 (control with recovery), 5 (1000 mg/kg bw/day with recovery).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In the 14-day range finding study with 5 males and 5 females at dose levels of 0, 100, 300, 1000 mg/kg/day, there were no changes attributed to the treatment at any groups. (Inspection items: clinical signs, body weight, food consumption, necropsy, organ weight, hematology and blood chemistry).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of the dosing and recovery period.
- How many animals: All animals
- Parameters checked: RBC, WBC, Hb, Plat., MCV, MCH, MCHC, Ret., Eosino., Baso., Mono., Lymph., Neutro., LUC, PT, APTT, Fibrin.,

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of the dosing and recovery period.
- How many animals: All animals
- Parameters checked: ASAT, ALTA, ALP, CPK, T. Bil., T. Prot., Albumin, T. Chol., TGL, PL, Glucosa, BUN, Creat., IP, Ca, Na, K, Cl, α1-glob., α2-glob., β-glob., γ-glob., A/G.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: sensory activity / grip strength / motor activity

POST-MORTEM EXAMINATIONS:
GROSS PATHOLOGY: Yes, all animals at the end of the dosing and recovery periods.
Maternal examinations: Absolute and relative organ weight: Adrenals, ovaries, thymus, spleen, brain, heart, lung, liver, kidneys.

HISTOPATHOLOGY: Yes, animals within control and highest dose tested (1000 mg/kg bw/day), at the end of dosing and recovery period.
Maternal examinations: Adrenal, femoral bone marrow, heart, kidney, liver, lung, spleen, stomach, thymus, thyroid, trachea, uterus, adipose tissue.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
Fetal examinations:
- External examinations: Yes: all per litter
Indices:
Gestation index, implantation index, delivery index, live birth index (Day 0), sex ratio (Days 0 and 4), viability index (Days 0 and 4).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Fetal abnormalities

Key result
Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
not specified

Any other information on results incl. tables

Delivery and litter data:

Dose (mg/kg bw/day)

0

40

200

1000

Number of females examined

12

11

11

12

Number of females with live pups

12

11

11

12

Gestation index (%)

100.0

100.0

100.0

100.0

Gestation period (days)

21.96 ± 0.40

21.95 ± 0.42

22.18 ± 0.40

22.13 ± 0.43

Number of corpora lutea

17.6 ± 1.7

16.9 ± 0.42

16.3 ± 2.1

16.1 ± 1.9

Number of implantation sites

15.4 ± 1.9

15.5 ± 2.8

15.5 ± 1.8

14.8 ± 2.1

Implantation index (%)

88.08 ± 11.30

91.63 ± 9.47

95.42 ± 7.66

91.68 ± 7.22

Delivery index (%)

94.27 ± 5.71

90.07 ± 8.41

94.73 ± 3.79

96.18 ± 6.10

Number of pups delivered

14.5 ± 1.8

14.0 ± 3.0

14.6 ± 1.7

14.2 ± 2.1

Number of live pups on Day 0

14.4 ± 1.7

13.5 ± 2.7

14.5 ± 1.8

14.0 ± 1.9

Live birth index (%)

93.78 ± 5.97

87.46 ± 8.50

94.13 ± 4.49

95.18 ± 5.59

Sex ratio on Day 0 (male/total)

0.50 ± 0.11

0.45 ± 0.08

0.49 ± 0.08

0.56 ± 0.14

Viability index on Day 0 (%)

99.48 ± 1.79

97.23 ± 5.55

99.35 ± 2.14

99.02 ± 2.30

Number of live pups on Day 4

14.3 ± 1.6

13.3 ± 2.6

14.2 ± 2.0

13.8 ± 1.9

Sex ratio on Day 4 (male/total)

0.51 ± 0.12

0.47 ± 0.08

0.49 ± 0.08

0.56 ± 0.14

Viability index on Day 4 (%)

98.97 ± 2.41

98.14 ± 4.52

97.26 ± 3.85

98.85 ± 2.69

Body weight of pups Day 0 – Male

6.68 ± 0.33

6.69 ± 0.45

6.68 ± 0.50

6.58 ± 0.37

Body weight of pups Day 0 – Female

6.28 ± 0.44

6.42 ± 0.61

6.35 ± 0.48

6.14 ± 0.43

Body weight of pups Day 4 – Male

10.38 ± 0.78

10.47 ± 1.16

10.60 ± 1.18

9.85 ± 1.14

Body weight of pups Day 4 – Female

9.78 ± 0.86

10.06 ± 1.20

10.12 ± 1.15

9.33 ± 1.10

Not significantly different from control.

Applicant's summary and conclusion

Conclusions:
The NOEL/NOAEL for reproductive and developmental toxicity in rats were determined to be 1000 mg/kg bw/day since no effects were observed in the highest dose tested.
Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental screening test was performed with the test item according to OECD Guideline 422 (GLP study). Based on a preliminary 14 -days range-finding study, test item was administered orally to 12 male and female rats at 0 (control), 40, 200 and 1000 mg/kg bw/day for 42 days (from 14 days before mating, and through the mating period until a day before necropsy) and for 41-48 days (from 14 days before mating, and through the mating period until day 4 of lactation) respectively, to evaluate reproductive ability and viability and development of pups. Parental animals were subjected to necropsy at the end of the dosing. Pups were necropsied at Day 5 of lactation. No animals died in any test article group, and no test article related changes were observed in clinical signs, detailed observations, grip strength, locomotor activity, body weight, food consumption, hematology, blood chemistry, urinalysis (only performed in males), organ weight, necropsy, or histopathology. Related to the reproductive and developmental toxicity parameters, no test article-related changes were observed in any group in estrous cycle examination, copulation rate, fertility rate, gestation period, number of corporea lutea, number of implantations, implantation index, gestation index, delivery index, or nursing behaviour in dams. With respect to examination of pups, no test article related changes were observed in the number of pups delivered, number of live pups, live birth index, viability index or sex ration on Day 0 after birth, number of live pups, viability index or sex ration on Day 4 after birth, clinical signs, external examinations, body weight or necropsy. Accordingly, it was considered that the test article did not affect the developmental or growth of next generation. Based on these results, the NOEL/NOAEL for reproductive and developmental toxicity in rats was determined to be 1000 mg/kg bw/day since no effects were observed at the highest dose tested.

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