2-nitrotoluene

Toxicity to reproduction

Administrative data

Endpoint:

fertility, other

Remarks:

based on test type

Type of information:

experimental study

Adequacy of study:

supporting study

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.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: 408

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

Test material

Test material information

Constituent 1

Reference substance name:

2-nitrotoluene

EC Number:

201-853-3

EC Name:

2-nitrotoluene

Cas Number:

88-72-2

Molecular formula:

C7H7NO2

IUPAC Name:

1-methyl-2-nitrobenzene

Details on test material:

- 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

Test animals

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

Administration / exposure

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

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 625, 1250, 2500, 5000, or 10000 ppm (45, 89, 179, 353, 694 mg/kg bw in male rats and 44, 87, 178, 340, 675 mg/kg bw in females; estimated from consumed diet)
Basis:
nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

1

Dose descriptor:

LOAEL

Effect level:

179 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Effect level:

340 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

Executive summary:

NTP, 1992

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.