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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 May 2007 - 11 July 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Benzenamine, reaction products with aniline hydrochloride and nitrobenzene
EC Number:
309-912-6
EC Name:
Benzenamine, reaction products with aniline hydrochloride and nitrobenzene
Cas Number:
101357-15-7
Molecular formula:
This is a UVCB substance. See section 1.2 for individual components.
IUPAC Name:
Benzenamine, reaction products with aniline hydrochloride and nitrobenzene
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: approximately 10 - 12 weeks
- Weight at study initiation: 214 - 311 g
- Housing: 5 animals per cage (pre-mating); 1 male and 1 female per cage (mating); individually (post-coitum)
- Diet: pelleted rodent diet (SM R/M-Z, SSNIFF® Spezialdiäten GmbH, Soest, Germany) ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days prior to pairing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4 - 24.0 ºC
- Humidity (%): 42 - 85 %
- Air changes (per hr): approximately 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From 9 May 2007 - 11 July 2007

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: formulations of test material in vehicle were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. Dosing solutions were stored at ambient temperature prior to use.
- Dose volume: 5 mL/kg bw. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Samples: duplicate samples (approximately 250 or 500 mg) were accurately weighed into volumetric flasks of 25, 100 or 250 mL. For determination of accuracy, samples were taken at 50 % height or at 90 %, 50 % and 10 % height. The latter set of samples was also used for the determination of the homogeneity of the formulations. For determination of stability, additional samples were taken at 50 % height. The flasks were filled up to the mark with methanol. The solutions were ultrasonicated for 10 minutes. The solutions were further diluted with methanol to obtain concentrations within the calibration range.

- Analytical conditions:
- Instrument: Alliance Separation Module 2695 (Waters, Milford, MA, USA)
- Detector: Dual λ Absorbance Detector 2487 (Waters)
- Column: 100 mm x 4.6 mm i.d. Symmetry Shield RP-18, dp = 3.5 µm (Waters)
- Injection volume: 100 µL
- Mobile phase: 0.1 % H3PO4 in 70/30 (v/v) methanol/Milli-Q water
- Flow: 0.6 mL/min
- UV detection: 290 nm

- Sample Injection: calibration solutions were injected in duplicate. Test samples and procedural recovery samples were analysed by single injection

- Calibration curves: a calibration curve was constructed using four concentrations. For each concentration, two responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration² weighting factor. The coefficient of correlation was > 0.99.
Details on mating procedure:
Females were caged together with males on a one-to-one basis during the mating period.
Duration of treatment / exposure:
From day 6 to day 19 post-coitum, inclusive.
Frequency of treatment:
Once daily 7 days per week, approximately the same time each day with a maximum of 5 hours and 12 minutes difference between the earliest and latest dose.
Duration of test:
4 weeks.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 15, 150 and 1000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
24 females (0, 15, 150 mg/kg/day); 25 females (1000 mg/kg/day).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In order to set the dose levels for the main study, a dose range finding study was performed. Four groups of 6 females were exposed to 0, 15, 150 and 1000 mg/kg/day for days 6 to 19 post-coitum inclusive by oral gavage. These dose levels were based on a 28-day toxicity study in which rats were exposed to the same dose levels. Under the conditions of the study, no mortality occurred and there were no treatment related findings in clinical signs, body weights and food consumption. There were no toxicologically relevant changes noted during macroscopic and microscopic examination and so 0, 15, 150 and 1000 mg/kg/day were selected as dose levels for the main developmental study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for viability/mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily from day 0 post-coitum onwards. The time of onset, degree and duration was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 3 and 6 - 20 (daily) post-coitum

FOOD CONSUMPTION: Yes
- Time schedule: days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 20 post-coitum
- Organs examined: Blood was collected for haematological examination. External thoracic and abdominal examinations were performed for the detection of macroscopic abnormalities. Liver and spleen were collected and fixed in 10 % buffered formalin. Each ovary and uterine horn was dissected and examined. Furthermore, the female genital tract including placentas was preserved in 10 % buffered formalin for possible histological examination.

HAEMATOLOGICAL EXAMINATION: Blood samples were collected under isoflurane anaesthesia. Blood samples were taken from the aorta and examined for the following parameters: white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils, red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets.

ORGAN WEIGHTS: The weight of the liver, spleen and terminal body weight were recorded from the surviving females on the scheduled day of 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 and distribution of live and dead fetuses: Yes
- Number and distribution of embryo-fetal deaths: Yes
- Number of former implantation sites: Yes
Fetal examinations:
- External examinations: Yes (all per litter)
- Soft tissue examinations: Yes: (al per litter)
- Skeletal examinations: Yes: (all per litter)
- Head examinations: Yes: (half per litter)
Statistics:
Where variable followed a normal distribution, the Dunnett-test was applied. For non-normalised data, the Steel-test was applied. The Fisher Exact-test was applied to frequency data. In all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data.
Indices:
Pre-implantation loss = [(Number of corpora lutea - number of implantation sites) / Number of corpora lutea] x 100

Post-implantation loss = [(Number of implantation sites - number of live fetuses) / Number of implantation sites] x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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

Details on maternal toxic effects:
- Mortality; one animal treated at 1000 mg/kg was sacrificed on day 7 post-coitum, based on clinical signs and body weight loss (-14%). This death was not considered to be related to treatment, as the animal had bitten through the plastic gavage tube during dosing on day 6 post-coitum. At necropsy, blue discolouration of the gastro-intestinal tract was observed. This animal had 17 normally developing fetuses (8 in the right uterus horn, and 9 in the left horn) and 19 corpora lutea. An additional animal was added to the study to replace this animal. No other mortality occurred.

- Clinical signs: One animal treated at 1000 mg/kg showed rales from days 14 to 18 post-coitum. Black discolouration or staining of faeces and different parts of the body was noted at 150 mg/kg and 1000 mg/kg in a dose dependent manner. This discolouration was related to the colour of the test material and was not considered to be of toxicological relevance. One animal showed alopecia, which was considered to be unrelated to treatment as this was an isolated finding.

- Body weights: Body weights and body weight gain of treated animals remained in the same range as the controls over the 4-week study period.

- Food consumption: Food consumption before or after allowance for body weight was similar between treated and control animals.

- Haematology: No toxicologically relevant findings were noted. A slight increase in reticulocytes was noted in females treated at 1000 mg/kg. No other treatment related findings were noted. As the slight increase in reticulocytes was an isolated finding, and as no other erythrocyte parameters were affected the toxicological relevance of this finding was doubted.

- Pathology: Reddish discolouration in parts of the abdominal adipose tissue was noted in animals dosed at 150 and 1000 mg/kg. Black discolouration of the gastro-intestinal tract was noted in one 1000 mg/kg animal. Furthermore, two animals dosed at 1000 mg/kg showed black discolouration of the skin at necropsy. This discolouration was considered to be due to the colour of the test material and of no toxicological relevance. Other findings noted among control and treated animals (including uterus filled with fluid, alopecia, adhesion of the placenta) were considered to be of no toxicological significance, since they remained within the range of biological variation for rats of this age and strain.

- Organ weights: Liver and spleen weights of treated animals were considered to be similar to those of control animals.

- Histopathology: There were no treatment related findings.

Effect levels (maternal animals)

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

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
- Litter size: A slight reduction in the number of viable fetuses was noted in animals treated at 1000 mg/kg; however, this was not statistically significant as the value was well within the historical control range.

- Sex ratio: No treatment-related effect on the sex ration was noted.

- External malformations and variation: There were no test material related external malformations. No external developmental variations were observed in any fetuses during the study. Polydactyly was noted was noted in one fetus from the group dosed at 1000 mg/kg and also in one fetus in each the control and the 15 mg/kg dose groups. The incidence of polydactyly did not indicate a relation to treatment with the test material.

- Visceral malformations and variations: Situs inversus affecting all internal organs in one fetus of the 15 mg/kg group, and enlarged right atrium of a fetus in the 1000 mg/kg group were the only findings noted and were considered spontaneous in origin and hence not related to treatment with the test material. Soft tissue developmental variations noted in the test material treated groups were right subclavian originating from the aortic arch, accessory spleen, thymus extended into neck region, and accessory liver lobule. These findings were not considered test material related because the mean litter proportions of these variations were not dose related.

- Skeletal malformations and variations: Malformations that were noted during the study consisted primarily of bent limb bones that were noted in one fetus of the 1000 mg/kg group and five fetuses of the control group. One fetus in the 15 mg/kg group showed sternoschisis. These findings were not attributed to the test material due to occurrence in single fetuses and/or the absence of a dose relationship. In the control group one fetus had a vertebral centra anomaly in which the half of a thoracic centrum was absent.
There was a statistically significant decrease in the mean litter proportion of fetuses with reduced ossification of the skull, resulting in mean litter proportions of skeletal and total variations in the 1000 mg/kg group that were statistically significantly decreased. The values of reduced ossification of the skull were 19.9 %, 22.4 %, 18.8 % and 10.3 % in the control, 15, 150 and 1000 mg/kg groups respectively. These values indicate an advanced ossification of skull bones in the 1000 mg/kg group compared to the other groups. No patterns of advanced ossification were observed for the ossification of cervical centrum no. 1, vertebral arches, ribs and sternebra in the 1000 mg/kg group. Although a relationship to treatment cannot be dismissed, a decrease in the number of variations in fetuses that indicate a possible advancement in ossification of the skull is not considered an adverse developmental effect.
Other skeletal developmental variations observed in the test material groups were 14th rudimentary ribs, unossified hyoid, 7th cervical ribs, bent ribs, unossified sternebra nos. 5 and/or 6, slightly to moderately malaligned sternebra(e), 14th full ribs, unossified pubis, tarsals ossified, 25 presacral vertebrae and unossified vertebral centra. All these variations occurred at similar frequencies in the control, and/or occurred infrequently, and/or in a manner that was not dose related.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse test material related effects were observed.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Formulation Analysis Results

The concentrations analysed in the formulations of the groups dosed at 15 and 1000 mg/kg were between 87 % and 100 % of target, which was as expected based on the recoveries of the procedural recovery samples (86 - 100 %). The accuracies of the 150 mg/kg formulations were slightly higher (108 - 116 %) but still considered acceptable. The 15 and 1000 mg/kg formulations were homogenous and stable for at least 5 hours when stored at room temperature. No test material was detected in the control group formulations.

Table 1: Summary of Fetuses and Litters with Malformations

Dose group

Fetuses

Litters

0

15

150

1000

0

15

150

1000

No. examined externally

337

358

305

316

23

24

21

23

Polydactyly

0

0

0

1

0

0

0

1

No. examined viscerally

337

358

305

316

23

24

21

23

Heart - atrium enlarged

0

0

0

1

0

0

0

1

Microphthalmia

1

0

0

0

1

0

0

0

Situs Inversus

0

1

0

0

0

1

0

0

No. examined skeletally

337

358

305

316

23

24

21

23

Bent limb bone(s)

5

0

1

0

2

0

1

0

Vertebral centra anomaly

1

0

0

0

1

0

0

0

Polydactyly

1

1

0

0

1

1

0

0

Sternoschisis

0

1

0

0

0

1

0

0

Total no. with malformations

External

0

0

0

1

0

0

0

1

Soft tissue

1

1

0

1

1

1

0

1

Skeletal

7

2

1

0

4

2

1

0

Combined

8

3

1

2

5

3

1

2


Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, the maternal, reproductive and developmental No Observed Adverse Effect Level (NOAEL) for the test material was determined to be 1000 mg/kg bw/day.
Executive summary:

The prenatal developmental toxicity of the test material was determined in accordance with standardised guidelines OECD 414, EU Method B.31 and EPA OPPTS 870.3700. During the study, mated female Wistar rats were assigned to four dose groups, containing 24 animals (in groups dosed at 0, 15 and 150 mg/kg) and twenty five animals (in the group dosed at 1000 mg/kg), respectively. The test material was administered once daily by gavage from day 6 to 19 post-coitum. The rats of the control group received the vehicle, propylene glycol, alone. Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

Under the conditions of the study, no maternal mortality was observed in any of the dose groups. A slight increase in reticulocytes was noted in females treated at 1000 mg/kg. No other treatment related findings were noted. As the slight increase in reticulocytes was an isolated finding, and as no other erythrocyte parameters were affected, the toxicological relevance of this finding was doubted. No reproductive toxicity was observed during the study and no developmental toxicity was observed.

Based on the results in this prenatal developmental toxicity study the maternal, reproductive and developmental No Observed Adverse Effect Level (NOAEL) for the test material was determined to be 1000 mg/kg bw/day.

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