Nitrobenzene

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 13 Jun 1983 to 21 May 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable publication which meets basic scientific principles

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

1987

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: internal Protocol of Bushy Run Research Center (BRRC) and approved by the Sponsor (BRAC Project 83-73-30501)

Principles of method if other than guideline:

30 animals were used per sex and dose. Except for a two week postnatal period without exposure, the F1 generation was treated identically to the F0 generation. F2 pups were sacrificed 21 days after birth. The concentration of nitrobenzene was monitored hourly by GC.

GLP compliance:

yes

Remarks:

Bushy Run Research Center

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

Details on test material:

- Name of test material (as cited in study report): nitrobenzene
- Supplier: E.I. Dupont de Nemours and Company
- Analytical purity: > 99.95% as analysed before, during and after the study, blanketed with nitrogen
- Lot/batch No.: 1-B

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan
- Age at study initiation: (P) 5-6 wks; (F1) 3 wks
- Weight at study initiation: (P) Males: 302 - 307 g; Females: 179 - 181 g; (F1) Males: 200 - 211 g; Females: 146 - 155 g
- Fasting period before study: yes, but no time schedule given
- Housing: during quarantine 3/cage (separated by sex) in 23.5x20x18 cm stainless steel wire mesh cages; during pre-mating exposure period 2/cage (separated by sex) in 35 cm x 17 cm x 18 cm high stainless steel wire-mesh cages; during mating one male and one female in 35 cm x 17 cm x 18 cm high stainless steel wire-mesh cages; after mating females were housed individually in 35 cm x 17 cm x 18 cm high stainless steel wire-mesh cages; Prior to parturition, the mated females individually in 24x30x14.5 cm high polycarbonate shoebox cages with stainless steel wire-frame tops
- Diet (e.g. ad libitum): Certified Agway Prolab RMH 3000 pellet feed, Agway Inc ., Syracuse, NY; ad libitum
- Water (e.g. ad libitum): drinking water; ad libitum
- Acclimation period: at least 2 weeks


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel chamber with glass door; approx. 4320 L
- Method of holding animals in test chamber: animals were in their cages
- Source and rate of air: room air
- System of generating vapor: Liquid nitrobenzene was metered from either a piston pump or a syringe pump into a heated glass evaporator. The temperature in the evaporator was maintained at the lowest level sufficient to vaporize the liquid. The resulting vapor was carried into the chamber by a contercurrent air stream that entered the bottom of the evaporator.
- Temperature, humidity, pressure in air chamber: 23 - 25 °C; 40 - 55%; slight negative pressure
- Air flow rate: 1000 - 1500 L/min


TEST ATMOSPHERE
- Brief description of analytical method used: a gas chromatograph equipped with a flame ionization detector was used
- Samples taken from breathing zone: yes; once every hour

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: one week
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Perkin- Elmer 3920B gas chromatograph equipped with a flame ionization detector was sed to monitor the vapor concentration. Daily nominal concentration (an estimated concentration calculated from the amount of teat material delivered and the chamber airflow during the exposure period) were also calculated for each chamber

Duration of treatment / exposure:

Duration of test: 56 weeks:
- Exposure period: Premating: 10 w, mating: 2 w, gestation: 19 d (females only), post partum: d 5-21 (dams only)
- Premating exposure period (males): 10 weeks
- Premating exposure period (females): 10 weeks

Frequency of treatment:

6 h/d, 7 d/w (pre-mating: 5 d/w) for 10 weeks

Details on study schedule:

After parturation, the dam and the offspring were not exposed for four consecutive days. F1 rats were allowed a two-week growth period without exposure. After the first mating, F1 males of the high dose and control groups were caged individually and allowed a 9-week (one spermatogenesis cycle) nonexposure recovery period before they were mated again. F2 pups were never exposed.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1, 10 and 40 ppm
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
5.1, 51.2 and 204.8 µg/L
Basis:
nominal conc.

No. of animals per sex per dose:

30

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily prior, during and after exposure period


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly


BODY WEIGHT: Yes
- Time schedule for examinations:
F0: at day 0 and weekly.
F1: At birtht or soon thereafter on a per litter basis (separated by sex), then individually
on postnatal days 4, 7, 14 and 21. Animals selected to continue on the study were weighed on the first day of exposure and weekly thereafter. Recovery group F1 males were weighed every two weeks during the 9-week recovery period rats were also weighed the morning prior to sacrifice.
F2: at birth, or soon thereafter, on a per litter basis separated by sex, then weighed individually on postnatal days 4, 7, 14 and 21 .


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

Sperm parameters (parental animals):

Parameters examined in Pa and F1 male parental generations:
testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in F1/F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies


GROSS EXAMINATION OF DEAD PUPS: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed after the mating period (except the recovery group).
- Maternal animals: All surviving animals were necropsied on postnatal day 21 or soon thereafter.


GROSS NECROPSY
- Gross necropsy of vagina, uteri, ovaries, testes, epididymides, seminal vesicles, prostate, and tissues with gross lesions


HISTOPATHOLOGY / ORGAN WEIGHTS
The reproductive tissues were prepared for microscopic examination and weighed (testes and epididymides), respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 4 or 21 days of age.
- These animals were not subjected to postmortem examinations.


GROSS NECROPSY
- Gross necropsy of vagina, uteri, ovaries, testes, epididymides, seminal vesicles, prostate, and tissues with gross lesions


HISTOPATHOLOGY / ORGAN WEIGTHS
The reproductive tissues of F1 animals were prepared for microscopic examination and weighed (testes and epididymides), respectively.

Statistics:

For continuous data: Bartlett's test for homogeneity of variance, ANOVA, Duncan's multiple ranbge test, t teest, F-test, Cochran t-test, Student's t-test. For discontinous data: Kruskal-Wallis-test, Mann-Whitney-U-test. Contingency data: Fisher's exact test. Probability of 0.05 was used as the level of significance

Reproductive indices:

fertility index; incidence of parturations; duration of gestation; gestation index; number of implantations; number of resorptions; postimplantational loss

Offspring viability indices:

live birth index; sex ratio; 4-day survival index; lactation index

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- Clinical signs: no treatment-related clinical signs were noted for F0, F1 and the recovery group animals
- Mortality: no mortality of F0, F1 and the recovery group animals during all phases of the study

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
For the F0 (21 days and older) rats, there were no biologically significant alterations in absolute body weights or body weight gains due to nitrobenzene exposure. During gestation, the F0 female rats exposed to 40 ppm had significant lower body weight gains (-14%) when compared to control F0. However, this finding was attributed to the decreased number of pregnant rats. The significant differences in female body weight (-21%) during gestation in the recovery phase of the study were also attributed to the lack of pregnancies in the females mated with the F1 males formerly exposed to 40 ppm nitrobenzene.
There were no biologically important alterations in absolute body weights or body weight gains in F1 animals due to exposure. During the pre-mating 10-week exposure regimen (days 0 to 65), the 1 ppm (+11%) and 40 ppm F1 females (+14%) gained more weight compared to the control females and several statistically significant differences were observed. Following parturition, the increases in body weights of the 1 ppm and 40 ppm F1 females reflected the higher hody weights that had already been observed in these females during the pre-mating 10-week exposure.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Fertility index:
Wheras the fertility index of F1 females in the 1 ppm (27/30) and 10 ppm group (29/30) was not changed, a significant decrease (-47%) of the fertility index was observed in the 40 ppm dose group (16/30) compared to control (30/30). The fertility index of F1 animals was not significantly changed in the 1 ppm (27/30) and 10 ppm group (26/30), while a significant decrease (-90%) of the fertility index was observed in the 40 ppm dose group (3/30) compared to control (30/30).
In the recovery groups the index after expocure to 40 ppm was decreased by 52% (14/30 compared to 29/30 in control).

- Incidence of parturition: No significant change in the number of parturions per pregnancy in F0 animals of all dose groups was apparent. However, a significant decrease in the number of parturions per pregnancy was noted in the 40 ppm group (-77%) and not in the 1 or 10 ppm dose groups of F1 females.
No change was observed in the recovery animals.

- Gestation: no significant change in the gestation lenght (22 days) in all dose groups for F0, F1 and for recovery animals was noted. The number of pregnancies with live litters per number of pregnancies was 29/30, 27/27, 29/29 and 16/16 for 0, 1, 10 and 40 ppm for F0 animals, respectively. A significant decrease of 77% was observed for F1 animals of the 40 ppm group, while no change was found in the 1 and 10 ppm groups. Also, no change in the gestation index was found in the recovery group.

- Frequency of vaginal plug appeareance: a greater number of dropped vaginal plugs was observed in females of the 40 ppm group of the F0 (36x), F1 (64x), and recovery generation (33x) compared to females in other exposure or control groups (F0: 29x; F1: 34x; Recovery: 27x).

- Number of implantations, resorptions, postimplantation loss: Although there were less pregnancies in the 40 ppm F0 group compared to the control F0 group, there were no biologically significant differences in the number of implantations, the number of resorptions or postimplantation loss. For the 3 uteri examined in the 40 ppm F1 group, a decrease in the number of implantations was observed in 2 of the 3 uteri. However, the mean number of resorptions in these 3 uteri was similar to the control group mean. The postimplantation loss in the 40 ppm F1 generation appears high due to the low number of implantations and not to an increase in the number of resorptions. The increase in the mean number of implantations for the 10 ppm F1 generation (+21%) was considered spurious.

ORGAN WEIGHTS (PARENTAL ANIMALS)
A significant reduction in both absolute and relative testes and epididymides weights was observed in F0 males of the 40 ppm group, with values of -33% and -31% for the absolute and relative testis weight and -24% and -21% for the absolute and relative epididymides weights. A similar observation occurred in the F1 males of the 40 ppm group after several weeks of recovery with reduction by -46% and -45% for the absolute and relative testis weight and -34% and -35% for the absolute and relative epididymides weights, respectively. Mean testes and epididymides weights for males of the 1 and 10 ppm NB exposure groups of the F0 and F1 generations were similar to control values (although the F1 controls were sacrificed several weeks later than the 1 and 10 ppm F1 males).

GROSS PATHOLOGY (PARENTAL ANIMALS)
The only biological important gross lesion observed at sacrifice was a reduction in the size of the testes of the 40 ppm F0 and F1 males, as described before.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The biologically significant hictopathologic findings in this study can be narrowed to only two organs, the testes and the epididymides . Therefore, there were no microscopic changes in the reproductive organs of the female rats that could be attributed to exposure. A further simplifcation of the description of histopathologic findings can be made due to the observation that lesions related to exposure occurred only in the 40 ppm groups of the F0 and F1 generations. Specifically, the testes of the F0 generation males exposed to 40 ppm had seminiferous tubule atrophy and spermatocyte degeneration. The degree and distribution of the atrophy was marked to severe and multifocal or diffuse, respectively, in 14 of 30 rats. In addition to these testIcular lesions was the presence of giant syncytial spermatocytes in the seminiferous tubules of 22 of 30 rats. The epididymides of these males had degenerated spermatocytes in the tubular lumina and decreased numbers of spermatids.
The microscopic findings of the testes of the F1 generation males exposed to 40 ppm for 12 weeks but allowed an 9-week recovery period prior to sacrifice were similar to those lesions of the 40 ppm F0 males. Howover, there were some notable differences. For example, the plant syncytial spermatocytes were nearly absent (occurring in only 1 of 30 rats) and the active stages of spermatocyte degeneration In the seminiferous tubules were much less frequent. As with the F0 males, the epididymides of the F1 males had degenerate spermatocytes and reduced numbers of spermatids.

Effect levels (P0)

1

Dose descriptor:

NOAEC

Effect level:

0.051 mg/L air (nominal)

Sex:

male

Basis for effect level:

other: fertility, male reproductive organ toxicity and spermatogenesis

2

Dose descriptor:

NOEC

Effect level:

>= 0.205 mg/L air (nominal)

Sex:

male/female

Basis for effect level:

other: systemic toxcicity

Remarks on result:

other: Generation: all generations (migrated information)

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
There was no differences in survival index between control and treated groups for postnatal days 1, 4, or 21 of any generation. The mean survival index values for all groups, all generations, ranged between 93 and 100 percent. The listed group sizes may be smaller than the number of parturitions because 3 females from the F0 generation (one each from the 0, 1, and 10 ppm groups) and 3 females from the F1 generation (one each from the 0, 1, and 10 ppm groups) unexpectedly delivered their litters during the 6-hour (or air) exposure. Due to pup exposure to nitrobenzene by inhalation, the litter was documented, but the dam and her pups were euthanized and removed from the study.

CLINICAL SIGNS (OFFSPRING)
Instances of ecchymosis, hypoactivity, hypothermia, and partial cannibalism of pups were evenly distributed among control and treated groups for all generations. The only noteworthy observations occurred in litters of the control (0 ppm) group. One F1 litter of the control group had 11 dead at birth wit multiple signs of ecchymosts and cannibalism of pups . Also, an F2 litter of the control group had complete mortality occurring between postnatal days 9 and 12.

BODY WEIGHT (OFFSPRING)
For the F1 generation, body weights of the male and female pups of the 40 ppm group were lower by -11% for males and -14% for females than respective control mean values on postnatal day 21. The increase in the 1 ppm F1 male body weights on postnatal days 14 (+7%) and 21 (+8%) was considered spurious. For the F2 generation, male body weights of both the 1 and 10 ppm exposure groups were 5% lower than the male control value on postnatal day 0 only. There were no body weight differences between control and 40 ppm pups of the recovery generation.

Effect levels (F1)

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

0.051 mg/L air (nominal)

Sex:

male

Basis for effect level:

other: fertility, male reproductive organ toxicity and spermatogenesis

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Fertility index (number of pregnancies/number of females mated in %):

Generation	0 ppm	1 ppm	10 ppm	40 ppm
F0	100	90	96.7	53.3*
F1	100	90	86.7	10.0*
Recovery	96.7			46.7*

* p<0.001

Incidence of Parturition (number of parturitions/number of pregnancies in %):

Generation	0 ppm	1 ppm	10 ppm	40 ppm
F0	100	100	100	100
F1	100	92.6	100	33.3*
Recovery	100			100

*p<0.001

Gestation index (number of pregnancies with live litters /number of pregnancies in %):

Generation	0 ppm	1 ppm	10 ppm	40 ppm
F0	96.7	100	100	100
F1	100	92.6	100	33.3*
Recovery	100			100

*p<0.001

Uterine assessment:

	Generation	0 ppm	1 ppm	10 ppm	40 ppm
Number of Implantations	F0	14.1 ±2.11	14.8 ± 1.73	14.1 ± 2.48	12.9 ± 3.99
	F1	11.1 ± 3.26	12.1 ± 4.04	14.4 ± 2.79*	6.7 ± 8.96
Number of Resorptions	F0	1.2 ± 2.14	1.0 ± 1.62	0.9 ± 1.09	1.1 ± 1.00
	F1	1.1 ± 1.52	1.5 ± 1.67	1.5 ± 2.63	1.0 ± 1.00
Postimplantation Loss (%)	F0	8.8 ± 16.22	6.5 ± 8.13	6.1 ± 7.35	8.2 ± 7.42
	F1	11.7 ± 17.81	18.9 ± 27.9	11.9 ± 19.54	66.7 ± 57.74

*p<0.01

Mean organ weight:

		0 ppm	1 ppm	10 ppm	40 ppm
F0	Body weight (g)	520.4 ± 33.98	523.5 ± 38.19	511.6 ± 50.88	513.3 ± 40.8
	Absolute testis weight (g)	3.46 ± 0.362	3.4 ± 0.291	3.32 ± 0.249	2.32** ± 0.813
	Relative testis weight (% body weight)	0.67 ± 0.08	0.65 ± 0.077	0.66 ± 0.088	0.46** ± 0.166
	Absolute epididymides weight (g)	1.45 ± 0.148	1.44 ± 0.12	1.38 ± 0.121	1.10** ± 0.201
	Relative epididymides weight (% body weight)	0.28 ± 0.032	0.28 ± 0.035	0.27 ± 0.035	0.22* ± 0.04
F1 following recovery	Body weight (g)	585.7 ± 43.03			574
	Absolute testis weight (g)	3.51 ± 0.348			1.88** ± 0.507
	Relative testis weight (% body weight)	0.6 ± 0.071			0.33** ± 0.082
	Absolute epididymides weight (g)	1.49 ± 0.145			0.98** ± 0.185
	Relative epididymides weight (% body weight)	0.26 ± 0.03			0.17** ± 0.036

*p<0.01

**p<0.001