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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
Study period:
From 13 Juni 1983 to 21 May 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study without detailed documentation

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
1987
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report Date:
1984
Reference Type:
secondary source
Title:
European Union Risk Assessment Report (Draft), NITROBENZENE CAS No: 98-95-3
Author:
European Chemicals Bureau
Year:
2007
Bibliographic source:
European Commission 2007 Nitrobenzene RAR
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
only examination of organogenesis period
GLP compliance:
yes
Remarks:
Bushy Run Research Center
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): nitrobenzene, Dupont de Nemours and Company
- Analytical purity: > 99.94%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CD-CRL:COBS CD(SD)BR
- Source: Charles River Breeding Laboratorien, Inc., Kingston, NY
- Age at study initiation: 61 - 66 days on arrival
- Weight at study initiation: 230 g (females)
- Housing: 2 per sex per cage during quarantine, two (one male, one female) per cage during breeding, and study females singly in stainless steel wire-mesh cages (23.5 cm x 20 cm x 18 cm)
- Diet (e.g. ad libitum): (Certified Rodent Chov #5002, Batch No. FEB 06 84 1C and FEB 28 84 1E, Ralston Purina Co., Richmond, IN; ad libitum
- Water (e.g. ad libitum): water (Municipal Authority of Westmoreland County, Creensburg, PA); ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 65
- Photoperiod (hrs dark / hrs light): 12 / 12


IN-LIFE DATES:
- Date of birth: 10 Mar 1983 (males), 15 Mar 1983 (females)

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: room air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: glass and stainless steel chamber with a volume of approx. 4320 L
- Method of holding animals in test chamber: animals were in cages
- System of generating vapour: Liquid nitrobenzene was metered from a piston pump into a heated glass evaporator. The temperature in the evaporator was maintained at the lowest level sufficient to vaporize the liquid. The resulting vapour was carried into the chamber by passage of conditioned air through the evaporator. Chamber atmospheres containing nitrobenzene were filtered before leaving an exhaust stack.
- Temperature, humidity, pressure in air chamber: 22.8 - 23.3 °C; 46 - 50%
- Air flow rate: 1000 - 1500 L/min
- Air change rate: 14/h


TEST ATMOSPHERE
- Brief description of analytical method used: A gas chromatograph (GC) equipped with a flame ionization detector was used. Calibration of the GC was done with dynamically generated gas standards of nitrobenzene.
- Samples taken from breathing zone: yes; once every hour
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A Perkin-Elmer Model 3920B gas chromatograph (CC) equipped with a flame ionization detector was used to monitor the nitrobenzene vapor concentrations in the chambers. Each chamber atmosphere was analyzed for nitrobenzene approximately once every hour during each 6-hour exposure. Daily nominal concentrations (an estimated concentration calculated from the amount of test material delivered and the chamber airflow during the exposure period) were also calculated for each chamber.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
day 6-15 of gestation
Frequency of treatment:
daily, 6 h/d
Duration of test:
all study females were sacrificed on gestation day 21
No. of animals per sex per dose:
25 - 26
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: as determined by the sponsor

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: gestation day 0, 6, 9, 12, 15, 18 and 21


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: uterus, ovaries, cervix, vagina and abdominal and thoracic cavities


OTHER: Liver, spleen and kidney weights were determined.
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 early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all live fetuses per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
continuous data: Levene's test for equal variances, analysis of variance and t-test with Bonferroni probabilities; nonparametric data: Kruskal-Wallis test followed by the Mann-Whitney U test; incidence data were compared using Fisher's exact test. The fiducial limit of 0.05 (two-tailed) was used as the criterion for significance.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Mortality: there were no maternal deaths
Clinical signs: clinical observations taken daily indicated only those signs typical of the CD-rat strain in an inhalation study e.g., occasional localized alopecia, slight erosion of the tail tip, and, rarely, periocular wetness; there were no exposure- related or concentration-related signs of toxicity


BODY WEIGHT (PARENTAL ANIMALS)
Periodic maternal body weights were unaffected by treatment. However, maternal weight gain (a more sensitive measure) was significantly depressed relative to controls at 40.0 ppm, for days 6-9 (-61%) and 6-15 (-19%) of the treatment period. Weight gains for the latter part of the exposure period (days 9-12 and 12-15) were unaffected by treatment. Weight gain during the postexposure period (days 15-18 and 15- 21) was significantly elevated at 40.0 ppm relative to controls by 20% and 13%, respectively, so that maternal body weight at sacrifice (day 21) was equivalent across all groups.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Gestational parameters were unaffected by treatment. The control and treatment groups did not differ in number of corpora lutea per dam; in total, nonviable (resorptions and dead fetuses) and viable implantations (live fetuses) per litter; in percentage pre- or postimplantation loss; in sex ratio (% males); or in fetal body weight (males, females, all fetuses) per litter


ORGAN WEIGHTS (PARENTAL ANIMALS)
No effects of exposure on body weight (absolute or corrected for gravid uterine weight), on gravid uterine weight, on kidney weights (absolute or relative). Absolute and relative liver weights were increased at 40.0 ppm (103.6 and 104.6% of controls, respectively) but the differences were not statistically significant. Spleen weights (absolute and relative) were significantly elevated at 10.0 ppm (+15% and +14%) and at 40.0 ppm (+40% and +41%) with a clear exposure level response.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEC
Effect level:
0.005 mg/L air
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
0.051 mg/L air
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
0.205 mg/L air
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
GROSS PATHOLOGY (OFFSPRING)
There was no significant increase in the number of litter with one or more affected fetuses at any exposure concentration relative to controls for individual and total external, visceral (including craniofacial), or skeletal malformations. There was a significant increase of +33% in the incidence of total malfomations (but not in the incidence of any individual malformations or of malfomations by category) at 10 ppm but not at 10.0 or 40.0 ppm relative to that of controls.
There were 9 external, 52 visceral (including craniofacial), and 139 skeletal findings that were classified as variations. Examples of external variations observed included ecchymosis of head and extremities and bruises on trunk; examples of visceral variations were shortened innominate artery, irregular rugae on palate, fetal atelectasis, red foci on thymus, and dilated ureters; examples of skeletal variations included poorly ossified cerviavland thoracic centra, sternebrae 2, 5, and 6, and skull plates. The incidence of five of these variations (one external, four skeletal) was significantly different for one of the nitrobenzene-exposed groups from that of controls. The incidence of litters with one or more fetuses with external variations was significantly elevated by 64% at 40.0 ppm for ecchymoses on the trunk (but not on the head or extremities). There were no effects of treatment on the incidence of visceral variations. The incidence of four skeletal variations in nitrobenzene-exposed groups differed significantly from that of controls for the following: The incidence of split (bipartite) anterior arch of the atlas was significantly elevated by +600% at 1.0 ppm but not at 10.0 or 40.0 ppm), the incidence of bilobed thoracic centrum 9 was significantly reduced at 40.0 ppm by -83%, the incidence of parietal skull plate with a "hole" in bone (defined as a precisely delineated area of nonossification surrounded by ossification) was elevated at 40.0 ppm by +138%, and the incidence of poorly ossified premaxillary bone was significantly elevated at 1.0 ppm by +267%.

Effect levels (fetuses)

Dose descriptor:
NOAEC
Effect level:
0.205 mg/L air
Basis for effect level:
other: Highest test concentration.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Maternal body weight gain:

0 ppm 1 ppm 10 ppm 40 ppm
% pregnant females at sacrifice 96.2 96.2 96.2 100
Days 0 ppm 1 ppm 10 ppm 40 ppm
Body weight change (g) 0-6 (preexposure period) 28.5 ± 6.6 27.9 ± 7.9 29.6 ± 6.6 27.2 ± 5.8
6-9 11.8 ± 8.4 10.0 ± 3.9 9.5 ± 6.0 4.6 ± 7.2***
9-12 11.7 ± 9.0 14.0 ± 5.2 13.2 ± 4.8 12.2 ± 7.4
12-15 15.0 ± 6.4 13.5 ± 3.9 16.9 ± 4.0 14.7 ± 6.8
6-15 (exposure period) 38.6 ± 7.0 36.8 ± 6.9 39.6 ± 6.3 31.4 ± 7.6**
15-18 29.3 ± 935 32.5 ± 6.7 33.1 ± 4.6 35.2 ± 5.9**
18-21 44.8 ± 10.2 47.1 ± 8.6 48.5 ± 7.0 48.5 ± 6.6
15-21 (postexposure period) 74.2 ± 13.7 79.7 ± 12.1 81.6 ± 8.9 83.8 ± 8.4**

**p<0.01; ***p<0.001

Maternal organ weightsat sacrifice:

0 ppm 1 ppm 10 ppm 40 ppm
Body weight at sacrifice (g) 371.8 ± 23.2 374.9 ± 22.6 381.3 ± 21.1 373.3 18.6
Corrected body weight (= body weight at sacrifice - gravid uterine weight; g) 273.53 ± 16.35 274.25 ± 15.51 280.42 ± 14.29 271.07 13.03
Gravid uterine weight (g) 98.25 ± 16.34 100.65 ± 12.73 100.86 ± 12.51 102.24 10.80
Liver weight (g) 13.55 ± 1.31 13.35 ± 1.79 13.78 ± 1.66 14.04 1.24
Relative liver weight (%) 4.95 ± 0.36 4.86 ± 0.49 4.90 ± 0.45 5.18 0.35
Kidney weight (g) 1.91 ± 0.17 1.84 ± 0.14 1.88 ± 0.17 1.87 0.10
Relative kidney weight (%9 0.70 ± 0.06 0.67 ± 0.04 0.67 ± 0.06 0.69 0.04
Spleen weight (g) 0.60 ± 0.10 0.60 ± 0.11 0.69 ± 0.12* 0.84 0.12***
Relative spleen weight (%) 0.22 ± 0.03 0.22 ± 0.04 0.25 0.04* 0.31 0.04***

*p<0.05;***p<0.001

Malformations observed in fetuses exposed in utero:

Fetuses Litters
0 ppm 1 ppm 10 ppm 40 ppm 0 ppm 1 ppm 10 ppm 40 ppm
External malformations 1 1 0 0 1 1 0 0
Visceral malformations 8 14 3 17 5 7 3 7
Skeletal malformations 41 58 46 46 16 21 15 15
Total malformations 49 72 49 63 18 24* 17 20

*p<0.05

Significant variations in fetuses:

Fetuses Litters
0 ppm 1 ppm 10 ppm 40 ppm 0 ppm 1 ppm 10 ppm 40 ppm
Number examined externally  347 353 361 377 25 25 25 26
Ecchymosis on trunk 21 27 34 38 14 17 17 23*
Number examined viscerally 180 181 187 196 25 25 25 26
Number examined skeletally 167 172 174 181 25 25 25 26
Anterior arch of atlas, split 1 7 5 6 1 7* 5 5
Thracic centrum 9, bilobed 6 3 3 1 6 3 3 1*
Parietal, hole in bone 9 15 21 29 8 9 11 19*
Premaxiliary, poorly ossified 3 19 13 12 3 11* 7 6

*p<0.05

Applicant's summary and conclusion