Nitrotoluene

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

There is no reproduction toxicity study available for nitrotoluene (CAS 1321-12-6), but a read-across can be made from 2-nitrotoluene (CAS 88-72-2) and 4-nitrotoluene (CAS 99-99-0) in the course of an weight-of-evidence approach.

In rats, 2-nitrotoluene administered in feed at 5000 ppm for 13 weeks causes damage to the testes and the epididymis with a simultaneous reduction in the sperm count and the motility of the sperm in males, and a prolongation of the menstrual cycle among the females with a NOAEL of <=179 mg/kg bw/d (NTP, 1992).

In a two-generation study with 4-nitrotoluene in rats (Sunao, 2005), the NOEL and NOAEL of 4-Nitrotoluene for the parental animals was less than 40 mg/kg/day. However, with regard to the reproductive capacity of the parental animals no notable effects occurred up to a dose of 160 mg/kg/day. The NOEL and NOAEL for growth and development of the offspring are concluded to be 40 mg/kg/day.

Following a precautionary principle, the testicular effects of 2-nitrotoluene (CAS 88-72-2) justify a need for fertility classification of nitrotoluene (CAS 1321-12-6).

Link to relevant study records

Reference

Reference 1

Endpoint:

fertility, other

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

NTP study (standard protocol comparable to OECD 408 with acceptable restrictions (no post exposure period, slight deviations from guideline recommended housing conditions, limited chemical chemistry, not all recommended organs were weighed). Study indicative of reproduction toxicity.

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)

Deviations:

yes

Remarks:

(no post exposure period, slight deviations from guideline recommended housing conditions, limited chemical chemistry, not all recommended organs were weighed)

Principles of method if other than guideline:

NTP standard protocol for determination of subchronic toxicity after 90 days repeated dose application. Groups of rats animals were fed diets containing o-nitrotoluene for 13 weeks.

GLP compliance:

yes

Specific details on test material used for the study:

- Source: Aldrich Chemical Co. (Milwaukee, WI),
- Analytical purity: >96%
- Impurities: < 1% (mostly m- and p-nitrotoluene)
- Storage: RT
- Stability: reanalysis performed at approx. 4 months intervals indicated that the test
substance was stable under the storage conditions chosen

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: 6 weeks
- Mean weight range at study initiation: 132 - 145g (male), 110 - 116g (female)
- Housing: 5/cage
- Diet: ad libitum, NIH-07 feed (Zeigler Brothers, Gardners, PA)
- Water ad libitum
- Acclimation period: 10-15 days
- Other: 5 viral screens performed at the study start and termination indicated no positive antibody titer

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9 - 26.1
- Humidity (%): 32-90%
- Air changes (per hr): 16-29
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Details on study schedule:

Groups of rats animals were fed diets containing o-nitrotoluene for 13 weeks. Reproductive system evaluation were then performed in male and female animals

Dose / conc.:

625 ppm

Remarks:

(males: 45 mg/kg bw, females: 44 mg/kg bw; estimated from consumed diet)

Dose / conc.:

1 250 ppm

Remarks:

(males: 89 mg/kg bw, females: 87 mg/kg bw; estimated from consumed diet)

Dose / conc.:

2 500 ppm

Remarks:

(males: 179 mg/kg bw, females: 178 mg/kg bw; estimated from consumed diet)

Dose / conc.:

5 000 ppm

Remarks:

(males: 353 mg/kg bw, females: 340 mg/kg bw; estimated from consumed diet)

Dose / conc.:

10 000 ppm

Remarks:

(males: 694 mg/kg bw, females: 675 mg/kg bw; estimated from consumed diet)

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Dose descriptor:

LOAEL

Remarks:

fertility

Effect level:

<= 179 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

340 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

reproductive function (oestrous cycle)

Remarks on result:

other: not applicable

Remarks on result:

other: not applicable

Remarks on result:

other: not applicable

Reproductive effects observed:

yes

Lowest effective dose / conc.:

179 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

GROSS PATHOLOGY AND TESTES WEIGHTS AND HISTOPATHOLOGY

In all males from the 10000 ppm exposure group, the testes was smaller than in controls; alterations in the color (pale, mottled focus) of testis were also observed. Absolute testis weights at 5000 and 10000 ppm dose groups experienced a statistically significant reduction by 71.7% and 36.6%, respectively compared to controls. Relative testis weight in the 10000 ppm dose group was reduced by 34.5% compared to controls (p <= 0.01).

Absolute epididymal weights were markedly lower than controls in animals in the 2500, 5000, and 10000 ppm groups by 20, 44 and 76%, respectively.

 

REPRODUCTIVE ORGANS

MALES

In animals of the 5000 and 10000 ppm dose group, a decrease in epididymal sperm motility (6.5% and 73.1%, respectively versus 78% in control) and epididymal sperm concentration (179 million and 14 million, respectively versus 417 million/g caudal epididymal tissue) and in testicular spermatid count/ 1E +05 ml (70.1 and 11.9, respectively versus 91.5) was seen in rats from the 5000 and 10000 ppm groups. Testicular degeneration was seen in rats from the 5000 (10/10 animals, moderate) and 10000 ppm groups (10/10 animals, marked).

FEMALES

There were no histopathologic, treatment-related effects in the uterus or ovaries in any group. Only effect seen was an increase in estrous cycle length in the females of the highest dose group (10000 ppm, ca 6.63 days versus 4.90 in controls), with only 4/10 animals having a measurable estrous cycle (see table 2 below)

Table1:Summary of Reproductive Tissue Evaluations in Male Rats in the 13-Week Feed Study of o-Nitrotoluene (n=10 animals/dose)

Study Parameter		0 ppm	2500 ppm	5000 ppm	10000 ppm
Weights (g)	Necropsy weight	353 ± 5	309 ± 6**	254 ± 4**	198 ± 3**
	Left testicle	1.48 ± 0.028	1.39 ± 0.025*	1.07 ± 0.045**	0.54 ± 0.024 **
	Left epididymis	0.50 ± 0.011	0.40 ± 0.014 **	0.28 ± 0.012	0.12 ± 0.008 **
	Left epididymal tail	0.20 ± 0.010	0.16 ± 0.008 *	0.12 ± 0.007 **	0.05 ± 0.003 **
Spermatid measurements	Spermatid heads 10^7/ g testis	12.4 ± 0.44	12.58 ± 0.48	13.17 ± 0.85	4.41 ± 1.19 **
	Spermatid heads 10^7/ testis	18.3 ± 0.65	17.39 ± 0.62	14.02 ± 0.97 **	2.35 ± 0.63 **
	Spermatid count (mean/10^4/ml suspension)	91.48 ± 3.23	86.93 ± 3.11	70.08 ± 4.83 **	11.88 ± 3.10 **
Spermatozoal Measurements	Motility (%)	78 ± 1	76 ± 2	73 ± 1*	6.5 ± 5.5 **
	Concentration (10^6/g cet)^1	417 ± 28	433 ± 38	179 ± 22**	14 ± 5 **

¹g cet = grams of caudal epididymal tissue.

* Significantly different (P<= 0.05) from the control group by Dunn's or Shirley's test.

** Significantly different (P<= 0.01) from the control group by Dunn's or Shirley's test.

Table 2:Summary of Estrous Cycle Characterization in Female Rats in the 13-Week Feed Study of o-Nitrotoluene (n= 10 animals/dose except where indicated)

		0 ppm	2500 ppm	5000 ppm	10000 ppm
Necropsy body weight	-	205 ± 3	179 ± 3 **	170 ± 2 **	158 ± 3 **
Estrous cycle length	-	4.9 ± 0.1	4.7 ± 0.17	5.3 ± 0.21	6.63± 0.8 **^2
Estrous stages as % of cycle	Diestrus	36.7	39.2	40.8	62.5
	Proestrus	7.5	15.8	15.0	10.8
	Estrus	32.5	25.0	25.0	15.8
	Metestrus	23.3	20.0	19.2	10.8

²n=4, estrous cycle length longer than 12 days or unclear in 6 of 10 animals

³Evidence by multivariate analysis of variance (MANOVA) suggests that females in all dose groups differ from controls in the relative frequency of time spent in the estrous stages; P=0.06 for the 2,500 ppm group, P=0.04 for the 5,000 ppm group, and P = 0.01 for the 10,000 ppm group

** Significantly different (P<=0.01) from the control group by Dunn's or Shirley's test

Executive summary:

The study was comparable to OECD guideline (408) Repeated Dose Toxicity (oral).10 animals/sex/dose (F344/N rats) were fed diets containing o-nitrotoluene at nominal concentrations of 0, 625, 1250, 2500, 5000, or 10000 ppm. Compound intake via feed over the entire study period of 13 weeks was estimated at 45, 89, 179, 353, 694 mg/kg bw in male rats and 44, 87, 178, 340, 675 mg/kg bw in females. Only animals in the 3 highest dose groups were subjected to reproductive system evaluations. Gross and histopathological examinations of the reproductive systems indicated that o-nitrotoluene might be a reproduction toxicant. Testicular degeneration (5000 and 10000 ppm) with concomitant decrease in testicular spermatid heads counts (5000 and 10000 ppm), decrease in sperm motility (5000 and 10000 ppm) and decrease in sperm concentrations (5000 and 10000 ppm) in the caudal epididymal tissue, were seen in male rats. Small testes (10000 ppm), reduced absolute (5000 and 10000 ppm) and relative testes weights (10000 ppm) and reduced absolute epididymis weights (2500, 5000 and 10000 ppm) were also seen. In female rats, o-nitrotoluene increased the length of the oestrus cycle at the nominal concentration of 10000 ppm in diet. LOAEL (male) is <=179 mg/kg bw/d. NOAEL (female) is 340 mg/kg bw/d.

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

4-Nitrotoluene (4-NT) (Lot number PAF0942, purity: 98.0%) was purchased from Wako Pure Chem. lnd., Ltd (Osaka, Japan).

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

Source: Male and female Cij :CD®(SD)IGS rats were purchased from Charles River Japan, lnc (Yokohama, Japan).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Source: Male and female Cij :CD®(SD)IGS rats were purchased from Charles River Japan, lnc (Yokohama, Japan).
Age at study initiation: 5 weeks
Weight at study initiation: -
Fasting period before study: No
Housing: After quarantine and acclimation, only healthy animals showing favorable body weight gain and a good general condition were used for the study. Groups of twenty-four males and females were allocated to each treatment using body weight-stratified randomization.The animals were placed individually in stainless steel cages with metal net floors (280W x 440D x 150H mm, Tokiwa Scientific Equipment Co., Tokyo, Japan) in a breeding room with a barrier system under constant conditions of temperature (21-25°C), relative humidity (40-70%), ventilation ( 10-15 times/hr), and 12-hr light and darkness cycling. During the period from gestation day 17 (GD 17) to weaning day, the animals were bred individually or with individual littermates in polycarbonate cages with flat floors (265 W x 426D x 200Hmm, Tokiwa Scientific Equipment Co.) and standard bedding (Sun-flake, Charles River Japan, lnc.)..
Diet (e.g. ad libitum): The solid feed (NIH- 07M, sterilized with 30 kGy gamma rays, CLEA Japan, Inc. , Tokyo, Japan)
Water (e.g. ad libitum): Hita-city public water (chlorine-added) using automatic water-suppliers or bottles.
Acclimation period: Yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25°C
- Humidity (%): 40-70%
- Air changes (per hr): 10-15 times/ hr
- Photoperiod (hrs dark / hrs light): 12 hours

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
Rate of preparation of diet (frequency): Each animal received 5 mL/kg for seven days per week containing doses of 0, 40, 80, and 160 mg/kg/day. At the administration, a disposable catheter (Terumo Co., Tokyo, Japan, polyvinyl chloride urethral catheter, external diameter 8/3 mm) was used to connect with an injection syringe for the females at five weeks old or older and the males at 5-9 weeks old, and a disposable catheter (Terumo Co., polyvinyl chloride urethral catheter, external diameter 10/3 mm) for the males 10 weeks old or older. For the males and females after weaning to four weeks old, a metal gastric tube was connected with an injection syringe for test substance administration.
VEHICLE
Justification for use and choice of vehicle (if other than water): olive oil
Lot/batch no. (if required): Lot nurnber 0l6OAA
Purity:98.0%

Details on mating procedure:

M/F ratio per cage: 1/1
Length of cohabitation: overnight
Proof of pregnancy: vaginal plug and/ or sperm in vaginal smear referred to as day 0 of pregnancy
After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
After successful mating each pregnant female was caged: transferred to individual cages and the day of delivery was determined.

Analytical verification of doses or concentrations:

not specified

Frequency of treatment:

continuos

Details on study schedule:

F1 parental animals not mated until 13 weeks after selected from the F1 litters.
Selection of parents from F1 generation when pups were 5 days of age.
Age at mating of the mated animals in the study: 13-15 weeks

Dose / conc.:

40 mg/kg bw/day

Dose / conc.:

80 mg/kg bw/day

Dose / conc.:

160 mg/kg bw/day

No. of animals per sex per dose:

24

Control animals:

yes

Details on study design:

Dose selection rationale:
The doses of the test substance were determined based on the results of a preliminary two-generation reproductive toxicity study of 4-Nitrotoluene in rats (executed by Chemical Evaluation and Research Institute, Tokyo, Japan). In the preliminary study, 4-Nitrotoluene at doses of 0, 125 , 250, 500, or 1,000 mg/kg/day was administered orally by gavage to eight rats of each sex per group for four weeks before mating and during the mating period, and after that until weaning of the offspring in the females, and after completion of mating until one day prior to the day of autopsy in the males. The effects on reproductive capacity in the parental animals and on the growth in the offspring were investigated . As a result, the dose of 1,000 mg/kg resulted in death or a moribund condition in all males and females. With the doses of 250 and 500 mg/kg, deaths or cases in a critical condition were found in both sexes and deaths of the offspring were also observed. At 125 mg/kg, the dams showed increase in liver weight and slight effects on the spieen. From these findings, the highest dose for the present study was determined as 160 mg/kg/day, at which slight effects on the dams were anticipated, with middle and low doses were set at 80 and 40 mg/kg/day, respectively, with division by a common ratio of 2.

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: All animals were observed for their clinical signs once or more daily during the period from the start of the administration until one day prior to autopsy after weaning (postnatal day (PND) 21) of the delivered pups in the females, including nursing conditions of the F1 pups, and during the period until the one day prior to autopsy in the males.
BODY WEIGHT: Yes
The body weights of the males were measured on the start day of administration and subsequently once a week until autopsy as a rule. The body weights of the females were measured on the start day of administration and subsequently once a week, and after gestation, on GDs 0, 7, 14, and 20, and on PNDs 0, 4, 7, 14, and 21. Body weight gain in males was calculated from the values during the period from the start of the administration up to week 14. Gain in females was calculated from the values during the period from the start ofther administration up to week 10 of administration for the pre-breeding period, up to GD 20 for the gestation period, and up to PND 21 for the lactation period.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was measured at the same time as body weights, or supplied and remaining food amounts were measured for each cage whenever the food was supplied. The mean food consumption value per rat was calculated using the number of food-supply days. In the females during the lactation period, the values were calculated as the food consumption per litter. However, measurement of food consumption was not performed during the mating period. Feeding efficiency was calculated based on the body weight values on the start day of administration for the males, and on the start day of administration, GD 0 and PND 0 for the females .
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

Oestrous cyclicity (parental animals):

On the day of autopsy when the estrous cycle indicated the estrous stage after weaning (PND 21) of the F1 pups for the females, blood was collected from six animals of each sex in each group from the abdominal aorta after ether anesthesia under non-fasting conditions and sera were obtained for measurement of serum T3, LH, FSH, and testosterone concentrations by EIA methods, T4 concentrationsby a FPIA method. Estradiol concentrations were determined by an RIA method at Panapharm Laboratories Co., Ltd (Kumamoto,Japan). As no abnormalities were detected in any hormone at the dose of 160 mg/kg, measurement for the doses of 40 and 80 mg/kg was omitted except in the case of T4 in both sexes.

Sperm parameters (parental animals):

Sperm examinations were conducted for 10 males mated with the females delivering the F1 pups in each group at autopsy. The numbers of sperm in the testis and in the caudal epididymis were counted under a microscope using a hemocytometer. Epididymal sperm mortility was investigated using a Hamilton sperm mortility analyzer (HTM-IYOS; Hamilton Thorne Research, lnc.,Beverly, MA, USA). Smearstained specimens were prepared and epididymal sperm morphology was observed for 200 individual sperm under a microscope and the rate for morphologically normal sperm was determined.

Litter observations:

External surfaces and the sex ratios of the delivered live pups were examined on PND 0. The animals were observed for clinical signs everyday during the lactation period. On PND 4, the anogenital distance (AGD) was measured, and then the number of the pups in each litter was adjusted randomly to eight (four males and four females where possible) and the pups not selected were sacrificed under ether anesthesia, and stored in 10% neutral buffered formal in solution after examination of the external surfaces. Body weights were measured individually on PNDs 0, 4, 7, 14, and 21 and viability was assessed on PND 0, PND 4, and PND 21 (weaning rate).

Postmortem examinations (parental animals):

SACRIFICE
On the day when the females showed the estrous stage on observation of the estrous cycle after weaning of the F1 pups, and on the day of autopsy for the males, they were sacrificed under anesthesia, and underwent a macroscopic examination of all organs and tissues. Dead animals or animals in a moribund conditions, and the dams whose delivered pups all died were autopsied as early as they were found. As with surviving animals the tissues were collected, organs were weighed, and histopathological examinations were conducted.
HISTOPATHOLOGY/ ORGAN WEIGHTS
In the F0 and F1 parents, histopathological examinations were conducted for the liver, heart, kidneys, testes, epididymis, prostate, seminal vesicles, ovaries, uterus, vagina, brain, spleen, thymus, pituitary gland, thyroid (including the parathyroid), adrenal glands and mammary glands resected and routinely processed for paraftin-embedding and sectioning.

Postmortem examinations (offspring):

SACRIFICE
The F1 offspring not selected as parental animals on PND 25 - 27, and the F2 offspring on PND 21 were sacrificed under anesthesia, and macroscopically examined. Dead animals or animals in a moribund condition were autopsied as early as they were found. On the day when the females showed the estrous stage on observation of the estrous cycle after weaning of the F1 pups, and on the day of autopsy for the males, they were sacrificed under anesthesia, and underwent a macroscopic examination of all organs and tissues. Dead animals or animals in a moribund conditions, and the dams whose delivered pups all died were autopsied as early as they were found. As with surviving animals. the tissues were collected, organs were weighed, and histopathological examinations were conducted.
HISTOPATHOLOGY / ORGAN WEIGTHS
In the F0 and F1 parents, histopathological examinations were conducted for the liver, heart, kidneys, testes, epididymis, prostate, seminal vesicles, ovaries, uterus, vagina, brain, spleen, thymus, pituitary gland, thyroid (including the parathyroid), adrenal glands, and mammary glands resected and routinely processed for paraffin-embedding and sectioning.

Statistics:

The main tests applied were:
The Bartlett's analysis for homogeneity of variance (Bartlett, 1937) was conducted for the bodyweights, body weight gain , food consumption, feeding efficiency, organ weights, the number of days required for mating, gestation length, the number of implantations (scars), sperm examination items (the number of sperms), and hormone measurements in the F0 and F1 generations. the body weights in the F2 generation, and the number of pups delivered, the number of the delivered live pups, AGD, and the organ weights of the Fand F2 pups. When the variance was not uniform , the Kruskal-Wallis's rank sum test (Kruskal and Wallis, (1952) was conducted.
Then if a significant difference was detected. the non-parametric Dunnett method was applied for analysis of significant differences. When a significant difference was detected, testing by he 11011-parametric Dunnett method was conducted.
Chi2 test, incl. correction of Yates, for comparing e.g. death ratios of pups and male-to-female sex ratios of progeny.
The Kruskal-Wallis test was conducted for analyses of sperm examination items (sperm motility and morphology), and feeding efficiency in the F0 and F1 generations, and rates for abnonnalities of external surfaces viability on PND 0 and PND 4, and weaning rates in the F1 and F2 pups.
Fisher's exact test to compare mating and/or fertility rates.
One-way ANOVA was conducted. If a significant difference was detected , the Dunnett's multiple comparison test (Dunnett, 1964) was applied for detection of differences between control and treatment groups.

Reproductive indices:

Based on the observations during the reproductive period in the parental animals, the following indices were calculated. As criteria for sexual maturation, the females were examined for vaginal opening on PND 36 and the males for preputial separation of the penis on PND 43. The females were examined for their estrous cyclicity using vaginal smears for 2 weeks before mating. After mating was performed, the mating index, fertility index, gestation index, number of days required for copulation, gestation length, number of implantations , delivery index and number of the pups delivered were assessed.

Offspring viability indices:

External surfaces and the sex ratios of the delivered live pups were examined on PND 0. The animals were observed for clinical signs everyday during the lactation period. On PND 4, the anogenital distance (AGD) was measured , and then the number of the pups in each litter was adjusted randomly to eight (four males and four females where possible) and the pups not selected were sacrificed under ether anesthesia, and stored in 10% neutral buffered formal in solution after examination of the external surfaces. Body weights were measured individually on PNDs 0, 4, 7, 14, and 21 and viability was assessedl on PND 0, PND 4, and PND 21 (weaning rate).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One female in the 160 mg/kg group showed staining around the nose and mouth, reduced locomotor activity, crouching, and cyanosis during the gestation period and another exhibited reduced locomotoractivity, reduced respiratory rate, and eyelid closure during the lactation period, and both eventually died. lncreased salivation was observed throughout the administration period in both sexes of the 0, 40, 80, and 160 mg/kg groups, and this was significant for the frequency in the groups receiving 40 mg/kg or more.
The authors attributed salvination to the presumably irritant effects of the test substance, because no other related changes were observed, and stated, that therefore, its toxicological significance must be limited.

Dermal irritation (if dermal study):

not examined

Mortality:

not specified

Description (incidence):

No precise information on the number of deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight changes in the males of all dose groups showed a similar growth trend to that of the controls. In the females, body weight changes in all dose groups all showed a similar trend to that in the controls during the pre-mating period. However, body weights significantly increased on GDs 7 and 14 in the 80 mg/kg group during gestation period and on PND 21 in the 160 mg/kg group during the lactation period. However, this effect was not regarded as adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the males, no significant variation in food consumption was found among the treatment groups and the controls. However, a significant decrease in the feeding efficiency was observed at weeks 0-3 and weeks 0-10 of administration in the groups treated with doses of 80 mg/kg or more. In the females this was similarly the case on GDs 0-20. Significantly elevated values for food consumption were observed at weeks 4-10 in the 80 mg/kg group, at week 5 and weeks 7-10 in the 160 mg/kg group, on GD 7 in the 80 and 160 mg/kg groups, and on PND 4 in the 40 and 80 mg/kg groups. However, these effects was not regarded as adverse.
The elevated food consumption throughout the administration period in the P0 females was considered to be caused by nibbling after administration.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

ln the 160 mg/kg group, significant increase in the frequency of blackened spleens in the females was found at autopsy. Histopathological findings in the same group included increases in renal eosinophilic microbodies and hyaline droplets in the males, hemosiderin deposition of the spleen in both sexes, and increase in extramedullary hematopoiesis of the spleen in the females. In the 80 mg/kg group, increased renal hyaline droplets in the males and increased splenic hemosiderin deposition in the females were also observed.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

- Among indices related to the reproductive capability of the parents, no abnormalities in the following were found in any dose group: oestrous cyclicity, the mating indices for both sexes, the delivery index, gestation index, the number of sperm in the testis and epididymis, epididymal sperm motility and morphology (morphologically abnormal sperm rate).
- No change in the number of days required for copulation was evident.
- A significantly prolonged gestation length was found in the groups given 80 or 160 mg/kg.
- The number of implantations were significantly increased in the groups receiving 40 mg/kg or more.
- Significant increase in the number of pubs delivered was observed in the 160 mg/KG group..
- A change in vaginal opening was observed.
- No significant differences were found between the controls and the 160 mg/kg groups with regard to serum T3, LH, FSH, testosterone, and estradiol concentrations.

Dose descriptor:

LOAEL

Remarks:

parental general toxicity

Effect level:

40 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Remarks:

reproductive capacity

Effect level:

160 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related adverse effects on reproductive capacity observed up to the highest tested dose.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the gestation period, one 160 mg/kg female presented with reduced locomotor activity, a reduced respiratory rate, eyelid closure, and subnormal temperature and she paled and eventually died. Other findings in the same group were staining around the nose and mouth, hypothermia and emaciation in one animal and reddish tears and staining lower abdomen in another female. Another female in this dose group died on the day of delivery. Salivation was evident throughout the administration period in both sexes of the 0, 40, 80, 160 mg/kg groups, and the frequency was increased significantly in the groups receiving 40 mg/kg or more.
The authors attributed salvination to the presumably irritant effects of the test substance, because no other related changes were observed, and stated, that therefore, its toxicological significance must be limited.

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

No precise information on the number of deaths.
One female of the 160 mg/kg bw group died.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the males, body weights were decreased significantly at weeks 5 and 6 of the administration in the 80 mg/kg group and during the entire administration period in the 160 mg/kg group, and body weight gain was also decreased significantly in the 160 mg/kg group. In the females, body weights changed simlarly in all dose groups and the controls during the gestation period. However significantly decreased body weights were observed throughout the increased the pre-mating period and on PND 0 in the 160 mg/kg group.
These changes were considered to reflect toxicity.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the males, significantly lowered values for food consumption were observed at weeks 1 and 2 of the administration in the 160 mg/kg group and significant decrease in the feeding efficiency was also found at weeks 0-3 and 0-10 of administration in the groups receiving 80 mg/kg and 160 mg/kg, respectively.
In the females, significantly low values for food consumption were evident at week 1 in the 80 mg/kg group and at weeks 1 and 2 in the 160 mg/kg group, along with low values for feeding efficiency at weeks 0-7 and 0-10 and on GDs 0-20 in the 160 mg/kg group. During the lactation period, similar changes were observed in all dose groups and the controls. Significantly elevated food consumption was noted at weeks 9 and 10 of the administration and on GDs 7 and 14 in the 160 mg/kg group. The elevated food consumption throughout the administration period in the P1 females was considered to be caused by nibbling after administration.

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Significantly elevated relative hepatic weights were evident in both sexes of the treated groups, a long with high values for the relative renal and splenic weights in the 160 mg/kg males and the absolute and relative splenic weights in the females receiving this dose. Other findings included significantly higher relative renal weights in the 40 mg/kg females than in controls, and lower absolute brain, pituitary gland, heart, testis, and epididymis weights in the 160 mg/kg males and elevated relative kidney and adrenal gland weights in the counterpart females.

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the 160 mg/kg group, the frequency of clearly visible dotted patterns on the renal surfaces in the males was increased significantly. The spleens is black colored were also observed in the males and females at this dose and histopathological examination revealed significantly increased eosinophilic microbodies in the males, and hemosiderin deposition in the females. In the groups 80 mg/kg or more significant increased renal hyaline droplets and increased of splenic hemosiderin deposition was noted in the males.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

- Among indices related to the reproductive capability of the parents, no abnormalities in the following were found in any dose group: oestrous cyclicity, the mating indices for both sexes, the delivery index, gestation index, the number of sperm in the testis and epididymis, epididymal sperm motility and morphology (morphologically abnormal sperm rate) and preputial separation of the penis.
- A lower fertiIity index was found in all groups, including the controls as compared with the values for the F0 parents.
- A significant reduction in the number of days required for copulation was observed in the 160 mg/kg group.
- No abnormality was found in the parameter of gestation length.
- No abnormalities were observed in the number of implantations.
- No abnormalities observed in the number of pubs delivered.
- A significant decrease vaginal opening was observed in the rate was found in the 160 mg/kg group. The authors attributed this effect as secondary effect to delayed development due to the lowerting of body weight.
- No significant differences were found between the controls and the 160 mg/kg groups with regard to serum T3, LH, FSH, testosterone, and estradiol concentrations. However, the T4 concentration in the females of the 160 mg/kg group was significantly low, but a similar change was absent in the F0 generation and no other related change was found, so that it was not considered a treatment-related effect.

Dose descriptor:

LOAEL

Remarks:

parental general toxicity

Effect level:

40 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Remarks:

reproductive capacity

Effect level:

160 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related adverse effects on reproductive capacity observed up to the highest tested dose.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the offspring of both sexes, no abnormalities were observed in clinical signs or viability in the 40 and 80 mg/kg groups. However, the viability of PDN 4 of the puppies was decreased at the dose 160 mg/kg.

Dermal irritation (if dermal study):

not specified

Mortality / viability:

not examined

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significantly lowered body weights in the male offspring were found on PND 0 in the 80 mg/kg group and on PNDs 0-21 in the 160 mg/kg group. In the female offspring, significant decrease was noted on PNDs 0-21 in the 160 mg/kg group.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

No abnormalities were found in the sex ratio or external surfaces of the live pups delivered at any dose.

Anogenital distance (AGD):

effects observed, treatment-related

Description (incidence and severity):

In the males of the 160 mg/kg group an increase in AGD (mm/cubic root of body weight) in the offspring was observed.
In the females, no significant differences were found between any of the dose groups and the controls.

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Significantly lowered absolute brain weights were found in males receiving 80 mg/kg or 160 mg/kg and in the females treated with highest dose. Lowered values for the brain weights in the weanlings found in the male groups of 80 mg/kg or more and in the females of the 160 mg/kg group for both generations could be explained by body weight change and were considred non treatment-related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Autopsy of the pups which died during the lactation period for the F1 weanlings not selected as the F1 parents, and for F2 weanlings demonstrated no alterations due to 4-Nitrotoluene in any of the dose groups.

Histopathological findings:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Physical development:
Pinna unfolding, incisor eruption, and eye opening were normal. In the offspring of both sexes, no abnormalities were found regarding the systemic pain response, negative geotaxis, and the air righting and pinna reflexes.

Response test:
In the offspring of both sexes, no abnormalities were found regarding the systemic pain response, negative geotaxis, and the air righting and pinna reflexes.

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Dose descriptor:

NOAEL

Remarks:

growth and development

Generation:

F1

Effect level:

40 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related adverse effects observed at this dose.

Dose descriptor:

LOAEL

Remarks:

growth and development

Generation:

F1

Effect level:

80 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Clinical signs:

no effects observed

Description (incidence and severity):

In the offspring, no abnormalities were observed in clinical signs in the 40 and 80 mg/kg groups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

In the offspring, no abnormalities were observed in viability in the 40 and 80 mg/kg groups. However, the viability of PDN 4 of the puppies was decreased at the dose 160 mg/kg.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significantly lowered body weights in the both sexes were found on PND 0 in the 80 mg/kg group and on PNDs 0-21 in the 160 mg/kg group.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

No abnormalities were found in the sex ratio or external surfaces of the live pups delivered at any dose.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

In the males of the 160 mg/kg group (mm/cubic root of body weight) a tendency for increase in AGD, in the offspring was observed. However, this was considered to be due to lowering of the body weights. Although AGD in the males of F2 generations of the 160 mg/ kg group increased or tended to increase, no other related change, including serum hormone levels, was observed and the cause is unclear.
In the females, no significant differences were found between any of the dose groups and the controls.

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The F2 weanlings, significantly lowered absolute brain weights were found in males receiving 80 mg/kg or 160 mg/kg and in the females treated with highest dose.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Autopsy of the pups which died during the lactation period for the F1 weanlings not selected as the F1 parents, and for F2 weanlings demonstrated no alterations due to 4-Nitrotoluene in any of the dose groups.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Physical development:
Pinna unfolding, incisor eruption, and eye opening were normal.In the offspring of both sexes, no abnormalities were found regarding the systemic pain response, negative geotaxis, and the air righting and pinna reflexes.

Response test:
In the offspring of both sexes, no abnormalities were found regarding the systemic pain response, negative geotaxis, and the air righting and pinna reflexes.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

LOAEL

Remarks:

growth and development

Generation:

F2

Effect level:

80 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Remarks:

growth and development

Generation:

F2

Effect level:

40 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related adverse effects observed at this dose.

Reproductive effects observed:

yes

Lowest effective dose / conc.:

80 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Conclusions:

In the present investigation of the effects of 4-Nitrotoluene, individual P0 and P1 female parents demonstrated worsening of clinical sighs such as reduced locomotor activity or eyelid closure and eventually death during the perinatal period at dose of 160 mg/kg. lt has been reported that formation of metohemoglobin can be induced by oral adrninistration of 4-Nitrotoluene in rats (NTP, 1992, Dunnick et a!., 1994), and may be inferred that anemiaca used by this condition , accompanied by effects on the liver and kidney, might enhance stress in dams during the perinatal period. With regard to salivation found during the adrninistration period, this was presumably due to the irritant effects of 4-Nitrotoluene because no other related changes were observed, and its toxicological significance must be limited. While no abnormalities in body weighs or body weight gain due to 4-Nitrotoluene were found in either sex of P0 parents, significant lowering of the body weights was evident in F1 males receiving 80 mg/kg during the premating period and 160 mg/kg throughout the administration period, as weil as in F1 females at the dose of 160 rng/kg during the pre-rnating period and on PND 0. These changes were considered to reflect toxicity. Decreased food consumption and feeding efficiency were found in the groups receiving 80 mg/kg or more in both sexes of P0 and P1 parents. The elevated food consumption throughout the administration period in the P0 and P1 females was considered to be caused by nibbling after administration. The significant decrease in the vaginal opening rate observed in 160 mg/kg P1 female parents might have been secondary to delayed development due to lowering of the body weight. In the P1 parents, a low fertility index was found in all groups, including the controls as compared with the P0 parents. However, no treatment related changes that could have been the cause of lowered pregnant rate was found in the autopsy or histological findings for the pituitary gland, reproductive organs, and accessory reproductive organs in either sex, including non-copulating and nonpregnant pairs in all groups. Other changes such as significant prolongation of the gestation length at 80 mg/kg and 160 mg/kg in the P0 parents and the significant reduction of the number of days required for copulation in the P1 parents and increased number of pups delivered in the P0 parents of the 160 mg/kg group, not observed in the other generation and were within the normal range for this strain , and therefore were considered tobe incidental. Similarly, there were no changesre lated to the significant increase in the number of implantations with the P0 parents, and thus its significance is unclear. As far as investigated in the present study, there were no effects apparent on pathologic exam inations of the reproductive organs or in sperm parameters and horrnone levels in the parental anirnals. lt has been reported that neither estrogenic nor androgenic effects are exerted by 4-Nitrotolulene. Thus, although an effect of 4-Nitrotoluene on reproduction could not be fully precluded in this study, no positive evidence was obtained for any influence on reproductive capacity. Furthermore horrnones demonstrated no significant variation between the controls and the 160 mg/kg group for serum T3, Ll-1, FSH, testosterone, and estradiol concentrations in either sex ofthe P0 and P1 parents. While the T4 concentration in the P1 female parents of the 160 mg/kg group was significantly low, sirnlar change was absent in the P0 generation and no other related change was found , so that it was not considered an effect of 4-Nitrotoluene. In the P0 and P1 parents, changes in absolute and /or relative weights ifthe kidney and liver, pituitary and heart, and brain, testes and epididymes lacked dosedependence and not consistently observed and therefore were not concluded to be of rnajor signiticance. In the pathological findings signiticant increase in the frequency of hernosiderin depos ition in the spleens ofmales in the 160 mg/kg P0 group and 80 mg/kg and 160 mg/kg F1 groups suggested induction of anemia through formation of metohemoglobin (Ciss et al., 1980; NTP, 1992; Dunnick et al., 1994). In the males of the same dose groups, increase in renal eosinophilic microbodies and hyaline droplets were also observed. Although testicular atrophy, in some cases associated with seminal duct necropsy, has been reported (Ciss et al., 1980), no such changes were noted in this study. Since they used Wistar rats and administered 400 mg/kg/day of 4-Nitrotoluene in 1 % methylcellulose by gavage, 5 days a week for about 6 months and we used SO rats and administered 4-Nitrotoluene in olive oil by gavage, 7 days a week, these differences in strain and methods of administration may have caused the anomalous results. A significantly reduced viability on PND 4 at 160 mg/kg and an inhibited body weight gain in either sex at 80 mg/kg or more in the F1 and F2 offspring were observed. Although AGD in the males of both generations of the 160 mg/ kg group increased or tended to increase, no other related change, including serum hormone levels, was observed and the cause is unclear. Lowered values for the brain weights in the F1 and F2 weanlings found in the male groups of 80 mg/kg or more and in the females of the 160 mg/kg group for both generations could be explained by body weight change. No abnormal changes up to a dose of 160 mg/kg in the indices of physical development, the indices of retlex response and autopsy tindings in both generations were observed. In the present study, no abnormalities were observed in the endocrine and reproductive organs and the serum hormones, and the reduced vaginal opening in the F1 generation was considered to be secondary to lower body weights for parental animals. In the offspring , inhibited increase of the body weights, decreased brain weights considered to be secondary to lower body weights, and reduced viability, a longer or a tendency for a langer male 's anogenital distance with on other related changes were observed. Taken overall, the results indicate that the possibility that 4-Nitrotoluene has an endocrine disrupting effect is very low. When 4-Nitrotoluene was administered over two generations to rats in the present study, the NOEL and NOAEL of 4-Nitrotoluene for the parental animals was less than 40 mg/kg/day. However, with regard to the reproductive capacity of the parental animals. no notable effects occurred up to a dose of 160 mg/kg/day. The NOEL and NOAEL for growth and development of the offspring are concluded to be 40 mg/kg/day.

Executive summary:

In this study, no abnormalities were observed in the endocrine and reproductive organs and the serum hormones, and the reduced vaginal opening in the F1 generation was considered to be secondary to lower body weights for parental animals. In the offspring, inhibited increase of the body weights, decreased brain weights considered to be secondary to lower body weights, and reduced viability, a longer or a tendency for a langer male 's anogenital distance with on other related changes were observed. Taken overall, the results indicate that the possibility that 4-Nitrotoluene has an endocrine disrupting effect is very low. When 4-Nitrotoluene was administered over two generations to rats in this study, the NOEL and NOAEL of 4-Nitrotoluene for the parental animals was less than 40 mg/kg/day. However, with regard to the reproductive capacity of the parental animals no notable effects occurred up to a dose of 160 mg/kg/day. The NOEL and NOAEL for growth and development of the offspring are concluded to be 40 mg/kg/day.

Reference 3

Endpoint:

two-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

There is no experimental test data available for nitrotoluene (CAS 1321-12-6), but a read-across can be made from its components. Nitrotoluene (CAS 1321-12-6) is a mixture of mainly 4-nitrotoluene (CAS 99-99-0) and/or 2-nitrotoluene (CAS 88-72-2). In addition, the mixture is containing small amounts of 3-nitrotoluene (CAS 99-08-1). A wealth of data is existing for the hazard assessment of 2- and 4-nitrotoluene, which can be used for the classification of nitrotoluene (CAS 1321-12-6). Key data and classification are derived from the isomer with the most critical hazard identified for each specific end point. The available experimental test data are considered reliable and suitable for the classification of nitrotoluene (CAS 1321-12-6) under Regulation 1272/2008.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

LOAEL

Remarks:

fertility

Effect level:

<= 179 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: not applicable

Remarks on result:

other: not applicable

Remarks on result:

other: not applicable

Reproductive effects observed:

yes

Lowest effective dose / conc.:

179 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Mononitrotoluene is a mixture of mainly 4-nitrotoluene (CAS 99-99-0) and/or 2-nitrotoluene (CAS 88-72-2). In addition the mixture is containing small amounts of 3-nitrotoluene (CAS 99-08-1). A wealth of data is existing for the hazard assessment of 2- and 4-nitrotoluene. Key data and classification is derived from the isomer with the most critical hazard identified for each specific end point. For completeness key data of the other isomer is added as supporting information.

Regarding toxicity to reproduction (fertility) classification is driven by 2-nitrotoluene.

The EU RISK ASSESSMENT (2008) is summarizing the information on toxicity for fertility of 2 -nitrotoluene:

The only information available on fertility is from non-standard studies in experimental animals. In rats, 2-nitrotoluene administered in feed at 5000 ppm for 13 weeks causes damage to the testes and the epididymis with a simultaneous reduction in the sperm count and the motility of the sperm in males, and a prolongation of the menstrual cycle among the females. Reduced sperm motility was also observed at 10000 ppm (the highest dose level tested) for the mouse. The testicular effects indicate a need for fertility classification.

Information on 4 -nitrotoluene:

In a two-generation study with 4-nitrotoluene in rats (Sunao, 2005), no abnormalities were observed in the endocrine and reproductive organs and the serum hormones, and the reduced vaginal opening in the F1 generation was considered to be secondary to lower body weights for parental animals. In the offspring, inhibited increase of the body weights, decreased brain weights considered to be secondary to lower body weights, and reduced viability, a longer or a tendency for a langer male 's anogenital distance with on other related changes were observed. Taken overall, the results indicate that the possibility that 4-Nitrotoluene has an endocrine disrupting effect is very low. When 4-Nitrotoluene was administered over two generations to rats in this study, the NOEL and NOAEL of 4-Nitrotoluene for the parental animals was less than 40 mg/kg/day. However, with regard to the reproductive capacity of the parental animals no notable effects occurred up to a dose of 160 mg/kg/day. The NOEL and NOAEL for growth and development of the offspring are concluded to be 40 mg/kg/day.

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 (Bayer 2002). 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)).

Effects on developmental toxicity

Description of key information

There is no developmental toxicity study available for nitrotoluene (CAS 1321-12-6), but a read-across can be made from 4-nitrotoluene (CAS 99-99-0-2).

In a Reproductive/Developmental Toxicity Screening Test (OECD TG 421), 4-nitrotoluene caused significantly reduced pup body weights at dose levels at which maternal toxicity was observed (NOAEL developmental toxicity: 25 mg/kg bw/day; LOAEL developmental toxicity: 100 mg/kg bw/day; LOAEL maternal toxicity: 25 mg/kg bw/day).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Principles of method if other than guideline:

Additional evaluation: liver, spleen, kidney, pituitary gland, uterus, uterine cervix and vagina, mammary gland, seminal vesicle and prostate

GLP compliance:

yes

Limit test:

no

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

Species:

rat

Strain:

Wistar

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

Polyethylene glycol 400

Analytical verification of doses or concentrations:

yes

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.

Duration of treatment / exposure:

males: 35 days, females: up to 46 days

Frequency of treatment:

daily

Duration of test:

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

Dose / conc.:

25 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

400 mg/kg bw/day

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

Details on study design:

Sex: male/female
Duration of test: 46 days

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

Description (incidence and severity):

A treatment related increase of relative kidney and absolute and relative liver weights were marginally to slightly evident in males at the 100 mg/kg dose level and more pronounced at the 400 mg/kg dose level. Absolute and relative increase of weight of spleen was observed at the 400 mg/kg dose level in both genders.

Gross pathological findings:

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.

Other effects:

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.

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.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

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.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

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.

Dose descriptor:

LOAEL

Remarks:

maternal toxicity

Effect level:

<= 25 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Fetal body weight changes:

effects observed, treatment-related

Reduction in number of live offspring:

effects 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.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs of F1:
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.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: yes

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

25 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this tested dose.

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Abnormalities:

not specified

Developmental effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

F0-GENERATION
all dosed rats including controls showed severe salivation (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, 400mg-group
m/f: severely decreased,
DEVELOPMENT OF BODY WEIGHT GAIN
 25 and 100 mg-groups: body weight gain comparable to controls
400 mg-group, m/f:
 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/5.5);
400 mg, females body weight gain reduced when compared to controls during
----------gestation (72.2 g versus 111.3g) and
----------lactation (4.0 g versus 27.2 g)
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
--      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
----Duration of gestation(days): 22.78, 22.44, 22.33, 22.67
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.)
-
COURSE OF BIRTH:
------400 mg-group: 1 femle 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 DEVELOPMENTof F1 PUPS (control, low to high dose):
-----No pups delivered(mean-no):
12.11, 11.56, 12.08, 9.17
-----No 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-group:
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(MALE; FEMALE):
---------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- and 25 mg-groups:
no changes attributable to treatment

 

Conclusions:

In a Reproductive/Developmental Toxicity Screening Test (OECD TG 421), 4-nitrotoluene caused significantly reduced pup body weights at dose levels at which maternal toxicity was observed (NOAEL developmental toxicity: 25 mg/kg bw/day; LOAEL developmental toxicity: 100 mg/kg bw/day; LOAEL maternal toxicity: 25 mg/kg bw/day).

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 LOAEL and NOAEL were determined:

LOAEL maternal toxicity : 25 mg/kg bw/day

NOAEL developmental toxicity : 25 mg/kg bw/day

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

There is no experimental test data available for nitrotoluene (CAS 1321-12-6), but a read-across can be made from its components. Nitrotoluene (CAS 1321-12-6) is a mixture of mainly 4-nitrotoluene (CAS 99-99-0) and/or 2-nitrotoluene (CAS 88-72-2). In addition, the mixture is containing small amounts of 3-nitrotoluene (CAS 99-08-1). A wealth of data is existing for the hazard assessment of 2- and 4-nitrotoluene, which can be used for the classification of nitrotoluene (CAS 1321-12-6). Key data and classification are derived from the isomer with the most critical hazard identified for each specific end point. The available experimental test data are considered reliable and suitable for the classification of nitrotoluene (CAS 1321-12-6) under Regulation 1272/2008.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

LOAEL

Effect level:

<= 25 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

25 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose.

Dose descriptor:

NOAEL

Remarks:

developmental toxictity

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Abnormalities:

not specified

Developmental effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

Mononitrotoluene is a mixture of mainly 4-nitrotoluene (CAS 99-99-0) and/or 2 -nitrotoluene (CAS 88-72-2). In addition the mixture is containing small amounts of 3-nitrotoluene(CAS 99-08-1). A wealth of data is existing for the hazard assessment of 2- and 4-nitrotoluene. Key data and classification is derived from the isomer with the most critical hazard identified for each specific end point. For completeness key data of the other isomer is added as supporting information, if available.

Information on 4 -nitrotoluene:

Regarding developmantal toxicity / teratogenicity one OECD Guideline 421 Reproduction / Developmental Toxicity Screening Test is available for 4-nitrotoluene.

In a Reproductive/Developmental Toxicity Screening Test (OECD TG 421), 4-nitrotoluene caused significantly reduced pup body weights at dose levels at which maternal toxicity was observed (NOAEL developmental toxicity: 25 mg/kg bw/day; LOAEL developmental toxicity: 100 mg/kg bw/day; LOAEL maternal toxicity: 25 mg/kg bw/day) (Bayer 2002).

The EU RISK ASSESSMENT 2008 is summarizing the information on developmental toxicity of 2 -nitrotoluene:

The only information available on developmental toxicity is derived from two non-standard reproduction studies in experimental animals. In one of them carried out in CD rats, the only effect considered as indicative of developmental toxicity was the retardation in pup growth; however, because of the absence of further details on its severity we are not sure if this effect could be used for classification; in addition, if this effect was considered as toxic, it cannot be ruled out that some toxicity was due to the transfer of the substance through the milk. In the other study carried out in Wistar rats, mortality, vitality and behaviour of pups from both treated and untreated animals were the same and no histopathological changes in organs occurred among the young animals regardeless on treatment; therefore, 2-nitrotoluene did not induce developmental toxicity; furthermore, there was no toxicity derived from transfer of the substance through the milk. Differences on results between studies could be due to differences on sensitivity between strains. The classification for developmental toxicity based on available data and the EU criteria is not justified. However, assuming the worst case, it is necessary a risk characterization for developmental toxicity using the LOAEL of 50 mg/kg.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008.

There is no reproduction toxicity study available for nitrotoluene (CAS 1321-12-6), but a read-across can be made from 2-nitrotoluene (CAS 88-72-2) and 4-nitrotoluene (CAS 99-99-0) in the course of an weight-of-evidence approach.

There was evidence of effects on fertility of 2-nitrotoluene in a study comparable to OECD TG 408 in rats based on testicular effects. The testicular effects justify a classification for fertility classification. However, in a two-generation study with 4-nitrotoluene in rats (Sunao, 2005), the NOEL and NOAEL of 4-Nitrotoluene for the parental animals was less than 40 mg/kg/day, but with regard to the reproductive capacity of the parental animals, no notable effects occurred up to a dose of 160 mg/kg/day. The NOEL and NOAEL for growth and development of the offspring are concluded to be 40 mg/kg/day. Following a precautionary principle, the testicular effects of 2-nitrotoluene (CAS 88-72-2) justify a classification of nitrotoluene (CAS 1321-12-6) for effects on fertility.

There is no developmental toxicity / teratogenicity study available for nitrotoluene (CAS 1321-12-6), but a read-across can be made from 4-nitrotoluene (CAS 99 -99 -0).

In a OECD TG 421 study, developmental toxicity of 4-nitrotoluene was only observed at dose levels, which were maternally toxic. Therefore, classification for developmental toxicity is not justified.

 

Furthermore, 2-nitrotoluene (CAS 88-72-2) is included in Annex VI of Regulation (EC) No. 1272/2008 with the following classification:

• Repr. Cat. 2 (H361)

Therefore, nitrotoluene (CAS 1321-12-6) is also classified for Repr. Cat. 2 (H361f: Suspected of damaging fertility.) under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480.