Nitrogen trifluoride

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP, but comparable with the requirements of the TG with exposure limited to the period of organogenesis Read-across approach based on similarities in the mode of toxicological action - through the formation of methaemoglobin

Data source

Reference

Reference Type:

publication

Title:

Development toxicity evaluation of inhaled nitrobenzene in CD rats

Author:

Tyl, R. W., France, K. A., Fisher, L. C., Dodd, D. E., Pritts, I. M., Lyon, J. P., O'Neal, F. O. & Kimmerle, G.

Year:

1987

Bibliographic source:

Fundamentals of Applied Toxicology,8, pp 482-92.

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

exposure limited to the period of organogenesis

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Nitrobenzene

EC Number:

202-716-0

EC Name:

Nitrobenzene

Cas Number:

98-95-3

Molecular formula:

C6H5NO2

IUPAC Name:

Nitrobenzene

Test material form:

gas

Test animals

Species:

rat

Strain:

Sprague-Dawley

Administration / exposure

Route of administration:

inhalation: gas

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

Generation of test atmosphere:
Exposure apparatus was a glass and stainless steel chamber with a vol. ~4320 L. Animals were housed in their cages during exposure.
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 and humidity in the chamber were 22.8 - 23.3 °C and 46 - 50%, respectively. Air flow rate was 1000-1500 L/min. Air change rate: 14/h
The test atmosphere was sampled once every hour (samples taken from the breathing zone). A Perkin-Elmer 3920B GC equipped with a flame ionization detector was used to monitor the nitrobenzene vapor concentrations in the chamber. Calibration of the GC was done with dynamically generated gas standards of nitrobenzene. 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.

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

M & F cohoused 1:1/cage. Vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

exposure from GD6 - 15

Frequency of treatment:

once daily, 6h/d

Duration of test:

15 d with sacrifice on GD 21

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

10 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

40 ppm (nominal)

Doses / concentrations 4

Dose / conc.:

1.06 ppm (analytical)

Doses / concentrations 5

Dose / conc.:

9.8 ppm (analytical)

Doses / concentrations 6

Dose / conc.:

39.4 ppm (analytical)

No. of animals per sex per dose:

25-26/gp

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

Clinical observations: performed daily
Body weight: performed on GD 0, 6, 9, 12, 15, 18 and 21.
Post-mortem examinations: yes. Dams sacrificed on GD 21. Organs examined included: uterus, ovaries, cervix, vagina and abdominal and thoracic cavities. Liver, spleen and kidney weights were determined.

Ovaries and uterine content:

Ovaries and uterine content: examined after termination. Examinations included: gravid uterus weight, number of corpora lutea, number of implantations, number of early resorptions, number of late resorptions.

Fetal examinations:

Fetal examinations: external examinations. Number of live fetuses/litter, soft tissue examinations (half/litter), skeletal examinations (half/litter), head examinations (half/litter)

Statistics:

Appropriate statistics undertaken with p<0.05 (two-tailed) was used as the criterion for significance.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs of toxicity observed were thoses typical of inhalation studies: occasional localized alopecia, slight erosion of the tail tip, and, rarely, periocular wetness; there were no exposure- related or concentration-related signs of toxicity

Mortality:

no mortality observed

Description (incidence):

All animals survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

unaffected by treatment. However, maternal weight gain (a more sensitive measure) was significantly depressed relative to controls at 40 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 post-exposure period (days 15-18 and 15- 21) was significantly elevated at 40 ppm relative to controls by 20% and 13%, respectively, so that maternal body weight at sacrifice (day 21) was equivalent across all groups.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

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.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

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

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Effect levels (maternal animals)

Dose descriptor:

NOAEC

Effect level:

ca. 1 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Results (fetuses)

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

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

External malformations:

effects observed, treatment-related

Description (incidence and severity):

There was a significant increase of +33% in the incidence of total malformations (but not in the incidence of any individual malformations or of malformations by category) at 10 ppm but not at 10.0 or 40.0 ppm relative to that of controls.

Skeletal malformations:

no effects observed

Description (incidence and severity):

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

Visceral malformations:

no effects observed

Description (incidence and severity):

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

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Dose descriptor:

NOAEC

Effect level:

> 40 ppm

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

FETAL EFFECTS:

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 cervical and 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%.

 

Table 7.8.2-01: Maternal body weight gain:

	0 ppm	1 ppm	10 ppm	40 ppm
% pregnant females at sacrifice	96.2	96.2	96.2	100
DAYS	Body weight gain (g)
Pre-exposure period (GD 0-6)	28.5 ± 6.6	27.9 ± 7.9	29.6 ± 6.6	27.2 ± 5.8
GD 6-9	11.8 ± 8.4	10.0 ± 3.9	9.5 ± 6.0	4.6 ± 7.2***
GD 9-12	11.7 ± 9.0	14.0 ± 5.2	13.2 ± 4.8	12.2 ± 7.4
GD12-15	15.0 ± 6.4	13.5 ± 3.9	16.9 ± 4.0	14.7 ± 6.8
Post exposure period (GD 6-15)	38.6 ± 7.0	36.8 ± 6.9	39.6 ± 6.3	31.4 ± 7.6**
Post exposure period (GD 15-18)	29.3 ± 935	32.5 ± 6.7	33.1 ± 4.6	35.2 ± 5.9**
Post exposure period (GD 18-21)	44.8 ± 10.2	47.1 ± 8.6	48.5 ± 7.0	48.5 ± 6.6
Overall entire post exposure period (GD15-21)	74.2 ± 13.7	79.7 ± 12.1	81.6 ± 8.9	83.8 ± 8.4**

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

 

Table 7.8.2-02: Maternal organ weights at 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 (%)	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

 

Table 7.8.2-03: Malformations observed in fetuses exposedin utero

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

*p<0.05

Values in parenthesis refer to litter data

 

Table 7.8.2-04: Significant variations in fetuses

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

*p<0.05

Values in parenthesis refer to litter data

Applicant's summary and conclusion

Executive summary:

Pregnant CD (Sprague-Dawley) rats were exposed to nitrobenzene vapor (CAS Registry No. 98-95-3) at 0, 1, 10, and 40 ppm (mean analytical values of 0.0, 1.06, 9.8, and 39.4 ppm, respectively) on gestational days (gd) 6 through 15 for 6 hr/day. At sacrifice on gd 21, fetuses were evaluated for external, visceral, and skeletal malformations and variations. Maternal toxicity was observed: weight gain was reduced during exposure (gd 6-9 and 6-15) to 40 ppm, with full recovery by gd 21, and absolute and relative spleen weights were increased at 10 and 40 ppm. There was no effect of treatment on maternal liver, kidney, or gravid uterine weights, on pre- or post-implantation loss including resorptions or dead fetuses, on sex ratio of live fetuses, or on fetal body weights (male, female, or total) per litter. There were also no treatment-related effects on the incidence of fetal malformations or variations. In summary, during organogenesis in CD rats, there was no developmental toxicity (including teratogenicity) associated with exposure to nitrobenzene concentrations that produced some maternal toxicity (10 and 40 ppm) or that produced no observable maternal toxicity (1 ppm).