Nitrotoluene

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2002

Reference 2

Reference Type:

publication

Title:

4-Nitrotoluene - CAS N°: 99-99-0 - SIDS Initial Assessment Report.

Author:

OECD

Year:

2003

Bibliographic source:

UNEP Publications

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-nitrotoluene

EC Number:

202-808-0

EC Name:

4-nitrotoluene

Cas Number:

99-99-0

Molecular formula:

C7H7NO2

IUPAC Name:

1-methyl-4-nitrobenzene

Test material form:

solid

Specific details on test material used for the study:

- Name of test material (as cited in study report): p-nitrotoluene solified-melted
- Analytical purity: 99.8%
-Appearence: greyish congealed mass
- Lot/batch No.: CHN 0462
- Storage condition of test material: room temperature
- Manufacturer: Bayer AG, Germany

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany,
- Age at study initiation: (P) 12 weeks;
- Weight at study initiation: (P) Males: 320-344 g; Females: 196-220 g;
- Fasting period before study:
- Housing: during adaptation period in groups; at study start: individually; during pairing: 1 male and 1 female/cage; during lactation: individual females with their pups
- Diet (e.g. ad libitum): standard diet ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): approximately 50%
- Air changes (per hr): at least 10 times per hour
- Photoperiod (hrs dark / hrs light): artificial illumination, 12 hour light/dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

Polyethylene glycol 400

Details on mating procedure:

During the two-week mating period each female was paired daily. Females with positive sperm detection were not mated again. Females in which insemination had not been detected by the end of this two-week mating period, were mated for another week with the first male of the dose group which had successfully inseminated a female paired with it.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A UV-VIS (ultraviolet-visible) spectrometric method for quantifying the test item in the liquid formulation was developed. For analytical investigations, representative samples from these formulations covering the concentration range used in the study were taken. These samples were diluted with acetonitril into the working range and subsequently quantified with a spectrometric method (UV-VIS). The detection wave length was 277 nm. Standard solutions of the authentic test item were used for calibration. Linearity, Precision, Specificity and Accuracy of the analytical method fulfil the predefined acceptance criteria. Additionally system suitability tests in terms of specificity, precision and linearity indicated, that qualified analytical procedures were followed during the study.
During the conduct of the study a computerized system was used for analytical investigations, which was formally not released at that time. However, this fact does not limit, the validity of the analytical results, since, prior to each analysis, a calibration was performed. In addition, this system was meanwhile also formally released for use.

Duration of treatment / exposure:

Exposure period: males: 35 days, females: up to 46 days
Premating exposure period (males): 2 weeks
Premating exposure period (females): 2 weeks
Duration of test: 47 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

400 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

0 mg/kg bw = 12 animals
25 mg/kg bw = 12 animals
100 mg/kg bw = 12 animals
400 mg/kg bw = 7 animals

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

Appearance, behaviour, excretory products, mortality were observed twice daily; body weight were recorded twice a week or weekly of inseminated females up to (and on day of delivery), on day 4 pp and on day of necropsy; feed consumption was recorded weekly, water consumption daily; gross pathological examination of all male and female rats was performed at necropsy; weight of liver, spleen and kidneys of all rats were recorded at necropsy.
Following female specific data were recorded and evaluated: duration of gestation, gestation indexcourse of birth, lactation behavior, number of corpora lutea in the right and the left ovary, number of implantation sites

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight and epididymis weight (left and rightt estis; epididymis individually)

Litter observations:

Investigations in the F1 pups at birth and up to day 5 post partum: twice daily: appearance, general behavior, mortality Clinical findings, sex ratio of pups at birth, individual pup weights at birth and on d 4 after birth, viabilty index Pups were sacrificed on day 4 to 5 post partum.

Postmortem examinations (parental animals):

HISTOPATHOLOGY / ORGAN WEIGHTS
epididymides, testes, prostate, seminal vesicles, coagulation glands ovaries with oviduct, uterus, uterine cervix and vagina, mammary gland with mamilla, liver, kidneys, pituitary, spleen and all other organs with macroscopic findings. Following male specific data were recorded and evaluated: testicular and epididymal weight (left and right testis/epididymis) individually

Postmortem examinations (offspring):

The following parameters were investigated and assessed for the F1 pups at birth and up to day 5 post partum:
-Appearance (including externally visible malformations), general behaviour and mortality: twice daily (once daily on weekends, on public holidays and on day of sacrifice).
Clinical findings in pups which died or were found dead before first weighing on day 0 post partum are listed together with the clinical finding on their dams (pups of female no. 59 of the 25 mg/kg dose group). For better clarification of findings one dead pup of litter 59 was fixed and evaluated according to the method of WILSON for visceral findings.
-Sex ratio of the pups at birth
-Individual pup weight at birth (day 0 post partum) and on day 4 after birth. Pups of female no. 59 (25 mg/kg dose group) were not weighed since they died before 1st weighing on day 0 post partum.
The pups were sacrificed by carbon dioxide asphyxia on day 4 to 5 post partum.

Statistics:

Analysis of variance (ANOVA), in case of sinificant results Dunnett's test 2 By N Chi²test, in case of significant differences Fisher's exact test with Bonferroni correction
 

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Morbidity was characterized by piloerection, sunken flanks, and respiratory sounds in both genders and additionally hypoactivity and alterations of gait in females and resulted either in spontaneous death of one male or sacrifice in moribund condition of 2 males and 5 females up to day 8 of study. Clinical findings were related to distinctly to severely impaired feed intake and body weight loss. Thereafter recovery occurred to a certain extent in both genders but clinical signs reappeared in single females of the 400 mg/kg dose groups during gestation and lactation. One further female of the 400 mg/kg dose group was sacrificed moribund on day22 post coitum due to clinilcal symptoms. Necropsy revealed besides other findings intrauterine death of its litter and treatment relationship could not be excluded.
Further on, salivation after administration was seen in males and females of all study groups including the control group and with increased incidence in dosed groups.
Here it was assumed that salivation was primarily caused by the vehicle and more intense in dosed animals due to the smell and/or gustatory component of p-nitrotoluene.

Mortality:

mortality observed, treatment-related

Description (incidence):

Severe morbidity occurred in both genders with mortality in males after start of the treatment with p-nitrotoluene at the 400 mg/kg dose level.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Severe body weight loss was transiently seen in males of the 400 mg/kg dose group after start of treatment, followed later on by distinctly increased body weight gain in surviving males so that final body weight of these males was only affected to a marginal degree. In females severe body weight loss was as well seen after start of treatment at the 400 mg/kg level, followed by intermittent recovery with distinctly increased body weight gain during the 2nd Week of study and thereafter by reduced body weight gain during late gestation and lactation. At the lower dose levels marginally impaired body weight gain during lactation could not be completely excluded for females of the 25 g/kg and 100 mg/kg dose groups despite of lack of dose dependency.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Severely impaired feed intake was observed in both genders at the 400 mg/kg dose level during the first week of treatment. Recovery did thereafter occur in both genders during the 2nd half of the premating period. Impaired feed intake was again observed during the late week of gestation and during lactation in females of the 400 mg/kg dose groups. At the lower dose levels treatment relationship could not be completely excluded for slight reduction of feed intake during lactation in females on 25 mg/kg and 100 mg/kg dose despite the lack of dose dependency.

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

In both genders water intake and excretion of urine were increased at the 400 mg/kg dose level, while effects on fecal excretion i.e slightly increased incidence of light colored and soft feces were only assumed for females of the 400 mg/kg dose groups during premating and again at the end of gestation or during lactation.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Findings included increased turnover of erythrocytes in spleen at the dose level of 100 mg/kg and above (in males increased amount of iron pigment at 100 mg/kg and above and increased hematopoiesis and congestion at 400 mg/kg; in females increased hematopoiesis and congestion at 100 mg/kg and above and increased amount of iron pigment at 400 mg/kg) and iron pigment and variable glycogen content in the liver in both genders.
In the kidney lipofuscin pigment and tubular vacuolation (restricted to sacrificed females) were seen in females and singles cell necrosis of renal tubular epithelia in males at the 400 mg/kg dose level. In males of the 400 mg/kg dose groups debris was observed in the epididymides together with exfoliation of spermatidis in a single male.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Insemination index, fertility index and gestation index

Due to severe maternal toxicity 5 females of the 400 mg/kg dose group were sacrificed moribund on day 5 or 8 of the study before pairing. The number of pregnant female at the 400 mg/kg dose level did thus not exactly fulfil the requirements of OECD guideline no 421 (at least 8 pregnant females per group).
However, based on severe toxicity at the 400 mg/kg dose level it would have been inappropriate for animal welfare reasons to add further animals to this screening study. An effect of the lower number of females available for the evaluation of the reproductive parameters in the 400 mg/kg dose group was not assumed since all 7 females available for insemination were inseminated and these females became pregnant.

Insemination index and fertility index were not affected by treatment at a dose level up to and including 400 mg/kg bw/day. All available females of all study groups were inseminated.

Gestation index was not affected by treatment with p-nitrotoluene at a dose level up to and including 100 mg/kg/bw/day.
One female of the 400 mg/kg/ dose group was sacrificed moribund on day 22 of gestation. At necropsy a dead litter was found in the uterus and together with increased prenatal loss in the remaining litters of this dose group a treatment related effect could not be excluded for marginally reduced gestation index at the 400 mg/kg dose level.

Time to insemination

Determination of the time to insemination did not reveal toxicologically relevant effects in comparison to control values at a dose level up to and including 400 mg/kg bw/day when all females were included for calculation.
Even if one remated female in the control group was excluded from calculation of the mean time to insemination, the marginally increased value of the 400 mg/kg dose group lay well within the range of historical control data of preceding reproduction/developmental toxicity screening studies and fertility studies. Thus a toxicologically relevant effect on time insemination was not assumed.

Duration of gestation

A treatment related effect on duration of gestation was not relevant at a dose level up to and including 400 mg/kg bw/day.

Course of birth
Treatment related effects on course of birth could not be completely excluded at the 400 mg/kg dose level

Lactation behaviour
Treatment related effects on lactation behaviour could not be completely excluded at the 400 mg/kg dose level

Number of copora lutea

Mean number of corpora lutea was lower than the current control value in all dosed groups and statistically significantly reduced at the 100 mg/kg dose level. However, the control value of the current study was high in comparison to previous reproduction/developmental toxicity screening studies and fertility studies with the rat strain used and dose relationship was not evident for this finding. Therefore reduced number of corpora lutea at the 100 mg/kg dose level was regarded as incidental and a treatment related effect on mean number of corpora lutes per litter was not assumed at a dose level up to and including 400 mg/kg bw/day.


Number of implantation sites

At all dose levels tested the mean number of implantation sites per litter lay well within the range of the normal scattering for the rat strain used and showed no statistical significant significance.
Thus a treatment related effect on mean number of implantation sites was not evident at a dose level up to and including 400 mg/kg bw/day.

Prenatal loss

The difference between the number of implantation sites and the total number of pups littered (living and dead) yields the prenatal loss.
In females with viable pups prenatal loss was not affected to a toxicologically relevant degree at a dose level up to and including 100 mg/kg bw / day while prenatal loss was statistically significantly increased at the 400 mg/kg dose level. Further on one female of the 400 mg/kg dose group which was sacrificed moribund on day 22 of gestation revealed a litter of dead fetuses in the uterus at necropsy. Based on these findings a treatment related effect on prenatal loos was relate to statistically significantly reduced feed intake and body weight gain during gestation at the 400 mg/kg dose level:

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

25 mg/kg bw/day

Sex:

male

Basis for effect level:

other: No adverse effects observed at this dose.

2

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

100 mg/kg bw/day

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

3

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

25 mg/kg bw/day

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

4

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

25 mg/kg bw/day

Sex:

male/female

Results: P1 (second parental generation)

Effect levels (P1)

Remarks on result:

other: not apllicable

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Hematomas at different localisation were observed in pups of the 400 mg/kg dose groups and treatment relationship could not be completely excluded since hematomas in viable pups were only observed in the highest dose groups and related to signs of lower viability of pups (no milk ingestion, slightly increased postnatal loss and reduced birth weights).
All other clinical findings in the dosed groups (hypoactivity, pale skin, discolored and later missing tip of tail) showed either no dose relation and/or were comparable to the type and incidence of clinical signs seen in control pups of the same age in the current or previous reproduction/developmental toxicity screening and pre-and postnatal developmental toxicity studies thus representing the normal type and range of clinical findings in pups of this age.
Therefore, and due to lack of dose relationship, toxicological relevance was not assumed for these clinical findings in F2 pups of dosed groups.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

All pups of litter no. 59 of the 25 mg/kg dose group were found dead in the cage before first weighing. One of the pups showed a huge hematoma under the skin and bones skull.
These findings of the litter no. 59 were regarded as incidental and without relation to treatment due to the lack of dose relationship and since only a single litter was affected.
The overall number of dead and missing pup after birth (day 0 to 4 post partum) in the 25 mg/kg, 100 mg/kg and 400 mg/kg dose groups showed no dose relationship but was percentage was slightly increased at the 400 mg/kg dose level. Together with lower birth weights and marginally reduced viability index, reduced viability of F1 pups at the 400 mg/kg dose level as a result of treatment could not be completely excluded.
Further on treatment relationship was assumed for lack of milk ingestion (no visible milk spots) in few pups of the 400 mg/kg dose group.

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Description (incidence and severity):

The mean number of male F1 pups per litter showed neither statistically significant nor toxicologically meaningful differences towards the current or historical control values at all dose levels tested. The lowest number of male F1 pups occurred in the 100 mg/kg dose group without dose relationship. Thus, a treatment related effect on sex ratio of F1 pups was not evident at a dose level up to and includin 400 mg/kg bw /day.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Effect levels (F1)

1

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

2

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

25 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose.

Results: F2 generation

Effect levels (F2)

Remarks on result:

other: not applicable

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

F0-GENERATION
all rats including controls showed severe salivation

(most probably due to vehicle)

MORTALITY, 400 mg-dose group:
3 males and 5 females in the premating period and
1 female sacrificed moribund on day 22 p.c.: ventral posture,

 hypoactivity, piloerection, intrauterine death of its litter)

FOOD INTAKE
----MALE, 400 mg-dose group, significantly different from control:
week1: 23.9g/d(control males) versus 14.32g/d (p<0.01)
week2: 23 g/d(control males) versus 26.7g/d (p<0.01)  

 ---FEMALE, control, low, mid, high dose [g/day]:
week1: 14.6, 14.8, 14.9, 4.7 (p<0.01)
week2: 15.7, 15.7, 16.2, 18.7 (p<0.05)

 

day 0-4p.p.: 38.9, 32.4, 34.6, 27.2 (p<0.01)

DEVELOPMENT OF BODY WEIGHT GAIN(d1-d15); significant changes:
----male/female, 400 mg-group:
d1-4: -20.8g/-17.6g (control:8.1g/1.3g);
d4-8: 9.5g/-10.4g (control: 8.4g/2.8g);
d8-15: 19.4g/17.0g (control 9.6g/5.5g);
-----females body weight gain reduced when compared to controls
------------------------during gestation:control, low, mid, high dose: 111.3g, 109.3g, 105.5g, 72.2g (p<0.01))
--------------------------during lactation:
control, low, mid, high dose: 27.2g, 16.1g, 16.6g, 4.0g(p<0.01)

CLINICAL SIGNS OF INTOXICATION,
--400 mg-group:
m/f: piloerection, respiratory sound, sunken flanks, increased water intake and urination, reduced amount of feces
females: hypoactivity, alteration of gait and increased incidence of soft and light colored feces
-- 100 mg-group: no findings
--25 mg-group:
1 female: ventral posture, hypoactivity, high stepping gait, piloerection

EFFECTS ON REPRODUCTION (control, low to high dose):
--INSEMINATION INDEX
(no of females inseminated/no of females paired X 100):
100%, 100%, 100%, 100%.
Determination of the time to insemination did not reveal toxicologically relvant effects in comparison to control
values
--FERTILITY INDEX
(no of females with implantation sites/ no of females inseminated X 100):
75%, 75%, 100%, 100%
--GESTATION INDEX
(no of females with viable pups/no of females with implantation sites X 100): 100%, 100%, 100%, 85.7%
------No of corpora lutea: 21.56, 18.44, 16.42(stat. sign.), 17.67
------No of implantation sites/litter: 13, 12.89, 12.75, 12.33
------Mean number of pups delivered (living and dead): 12.1, 11.6. 12.1, 9.2
---PRENATAL LOSS
(Difference between no of implantation sites and the total no of pups littered (living and dead)):
0.89, 1.33, 0.67, 3.17 (stat. sign.)
-------Duration of gestation(days): 22.78, 22.44, 22.33, 22.67
--COURSE OF BIRTH:
------400 mg-group: 1 female was sacrificed of d22 p.c.: all fetuses of its litter were dead
-------25 mg-group: delivering of only 1 pup with a huge hematoma, loss of all other pups on day of birth

F1-PUPS
-----LACTATION BEHAVIOUR:
1 pup of the 100 mg- group, 2 pups of the 400 mg-group: no milk ingestion

POSTNATAL DEVELOPMENT OF F1 PUPS (control, low to high dose):
-----NO of PUPS DELIVERED(mean-no):
12.11, 11.56, 12.08, 9.17
----NO of LIVE PUPS (mean-no,d0/d4):
12.11/12, 11.56/11.25, 11.92/11.33, 9.17/8.33
--LIVE BIRTH INDEX(%):
100, 100, 98.61, 100
--VIABILITY INDEX (d4 pp, %):
98.99, 88.89, 95.41, 92.06
--SEX RATIO (d0, % males/liter):
49.94, 48.66, 44.00, 46.43
--PUP CLINICAL OBSERVATIONS (frequency/pups/litters, day 0-day 5):
----Found dead: 1/1/1; 14/14/1; 5/5/1; 1/1/1
----Missing: 0/0/0; 0/0/0; 2/2/1; 4/4/2
----Hypoactivity: 0/0/0; 1/1/1; 0/0/0; 1/1/1
----Hematoma: 0/0/0; 0/0/0; 0/0/0; 5/2/2
----Pale skin: 0/0/0; 5/1/1; 1/1/1; 1/1/1
Tip of tail
dark discolored: 0/0/0; 4/1/1; 0/0/0; 0/0/0
milk spot not
detectable: 0/0/0; 0/0/0; 1/1/1; 2/2/2
tip of tail
missing: 0/0/0; 1/1/1; 0/0/0; 0/0/0
--MEAN PUP WEIGHT:
d0: m/f/total and d4: m/f/total:
-------control -group:
6.30/5.96/6.12 and 9.64/9.22/9.43;
------25 mg-group:
6.27/5.94/6.07 and 9.42/9.09/9.22;
------100 mg-group:
5.43(p<0.01)/5.28(p<0.5)/5.36(p<0.05) and 8.31(p<0.05)/8.20/8.26;
------400 mg-grpup:
4.89(p<0.01)/4.69(p<0.01)/4.80p<0.01) and 6.79(p<0.01)/6.55(p<0.01)/6.68(p<0.01)

F0-GENERATION
GROSS PATHOLOGY:
--MEAN ORGAN WEIGHTS (control, low to high dose, abs(g)/rel[%]):
-----MALE
-------------LIVER
15.23/3.88, 15.52/3.96, 17.51(p<0.05)/4.26(p<0.05), 19.53(p<0.01)/5.04(p<0.01);
--------------SPLEEN:
0.67/0.17, 0.69/0.18, 0.72/0.18, 1.06(p<0.01)/0.28(p<0.01)
kidney weights, testes weight, epididymal weights were comparable to controls

----FEMALE:
liver and kidney weights were comparable to controls
--------------SPLEEN:
0.56/0.21, 0.598/0.23, 0.59/0.22, 1.07(p<0.01)/0.45(p<0.01)

HISTOPATHOLOGY(MALES, FEMALES):
-----------400 mg group:
LIVER:
periportal pigment deposits (2/12 males, 4/12 females),
variable glycogen content (4/12 males, 3/12 females);
KIDNEY:
tubular pigment (3/12 females),
mononuclear infiltration (2/12 females),
tubular vacuolation (5/12 females),
single cell necrosis (2/12 males);
SPLEEN:
Congestion (12/12 males, 10/12 females),
increased pigment (2/12 males, 1/12 females)
TESTES:
atrophy, 2/12;
EPIDIDYMIDES:
Cellular debris 4/12
100 mg-group:
SPLEEN:
Congestion (12/12 males, 2/12 females)
25 mg-groups:
no changes attributable to treatment

Applicant's summary and conclusion

Conclusions:

4-Nitrotoluene had no adverse effects on most reproductive endpoints (insemination index, fertility index, time to insemination, gestation length, number of corpora lutea and number of implantation sites, live birth index) in a rat oral Reproductive/Developmental Toxicity Screening Test (OECD TG 421), even under conditions where overt systemic toxicity was observed. A reduction in the gestation index, increased prenatal loss and reduced litter size and pup weights were reported at parentally toxic doses. Testicular degeneration was found in subchronic studies at systemically toxic dose levels characterized by reduced body weights and toxicity to the spleen subsequent to the erythrocyte damaging effect of 4-nitrotoluene (NOAELreproductive toxicity: 25 mg/kg bw/day; NOAEL(male)general toxicity: 25 mg/kg bw/day (male), LOAEL(female)general toxicity: 25 mg/kg bw/day (female)).

Executive summary:

Groups of 12 male and female Wistar rats each were treated daily (by gavage) for two weeks before mating, during the 2 -week mating and one week remating period, during gestation, lactation and up to the day before necropsy with the test substance solved in Polyethylene glycol 400 in doses of 0, 25, 100 and 400 mg/kg bw/day, respectively. Males were necropsied on day 36 of the study and females were necropsied between day 4 to 5 p.p.

F1 pups were sacrified between day 4 to 5 p.p.

Investigations were performed on general tolerance of the test compound by the parenteral animals (including histopatology of testes, epididymes, accessory sexual glands, ovaries, mammae with mamillae, liver, spleen, kidneys, pituitary gland and organs with macroscopic findings) as well as on effects on reproduction including early postnatal development of F1 pups.

The study was performed in 2002 in compliance with international guidelines (OECD No. 421).

Salivation after administration occurred in all study groups and both genders and was most probably based on the vehicle used.

Increased incidence of this finding in dosed groups was most possibly related to an offensive smell and/or gustatory component of the test substance and toxicological relevance was not assumed for this finding.

After start of treatment with the test substance at the 400 mg/kg dose level signs of severe systemic toxicity were observed in both genders including piloerection, respiratory sounds, sunken flanks, increased water intake and urination and reduced amount of feces. In females hypoactivity, alterations of gait and increased incidence of soft and light colored feces were additionally seen. The clinical symptoms occured together with distinctly to severely decreased feed intake and severe body weight loss and resulted in spontaneous death of one male and sacrifice in moribund condition of 2 further males and 5 females up to day 8 of study.

Another female of the 400 mg/kg dose group was sacrificed moribund on day 22 p.c.and revealed intrauterine death of its litter at necropsy (see below).

Recovery was observed thereafter in both genders during second week of the premating period but clinical symptoms, reduced feed intake and reduced body weight gain reappeared in females of the highest dose group during gestation and lactation. Necropsy of the animals of the 400 mg/kg dose group revealed alterations of the gastrointestinal tract in sacrificed/died animals of both genders as well as single cases of alterations of spleen and prenatal litter loss in females.

Increased weight of liver, spleen and to a slight degree of kidney was observed in males of the 400 mg/kg dose group.

Increased weight of kidney and liver were as well observed to a marginal or slight degree in males of the 100 mg/kg dose group. In females only weight of spleen was increased at the 400 mg/kg dose level.

At the lower dose levels treatment relationship could not be completely excluded for reduced feed intake and body weight gain of females during lactation at a dose level of 25 mg/kg and 100 mg/kg bw/day.

Histopathology of fixed organs revealed in both genders morphological evidence of increased turnover of erythrocytes in spleen at a dose level of 100 mg/kg and above and iron pigment and variable glycogen content in the liver at the 400 mg/kg dose level. Histopatological findings of the kidney consisted of single cell necrosis of renal tubular epithelia in males of the 400 mg/kg dose group while lipofuscin pigment was seen in the renal proximal tubuled in females together with vacuolation of tubules (only in sacrificed females). Further on debris was observed in the epididymides at the 400 mg/kg dose level together with exfoliation of spermatids in a single male.

With respect to reproductive parameters including early postnatal development, treatment related effects on course of birth and lactation behaviour could not be completely excluded at the 400 mg/kg dose level. Further on impaired gestation index, increased prenatal loss and reduced litter size at birth occured at the 400 mg/kg dose level together with clinical symptoms (no milk spot, hematomas), impaired pup weight up to day 4 p.p. and impaired viability index up to day 4 p.p.

Pup weight at birth was as well slightly reduced at the 100 mg/kg dose level.

Thus the following no-observed-effect levels (NOEL) were determined:

General toxicity in Males: 25 mg/kg bw/day

General toxicity in Females: < 25 mg/kg bw/day

Salivation after administration in Males and Females: < 25 mg/kg bw/day

Reproduction/Developmental Toxicity: 25 mg/kg bw/day