Dinitrotoluene

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Feb 1980 - 15 Sep 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Reliability as cited in OECD SIDS Dinitrotoluene (isomers mixture) CAS No.: 25321-14-6, 2004. Comparable to guideline study with acceptable restrictions (insufficient number of DNT-treated pregnant animals per dose (6-13))

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Reference 2

Reference Type:

publication

Title:

Teratological and postnatal evaluation of dinitrotoluene in Fischer 344rats

Author:

Price, C.J. et al.

Year:

1985

Bibliographic source:

Fundamental and Applied Toxicolgy 5, 948-961

Reference 3

Reference Type:

publication

Title:

Teratological evaluation of technical grade dinitrotoluene in Fischer 344rat

Author:

Wolkowski-Tyl, R. et al.

Year:

1981

Bibliographic source:

Teratology 23, 70A

Reference 4

Reference Type:

secondary source

Title:

Dinitrotoluene (isomers mixture), CAS No.: 25321-14-6

Author:

OECD SIDS

Year:

2004

Bibliographic source:

OECD SIDS

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

lower number of DNT-treated pregnant animals per dose (6-13 instead of 20)

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dinitrotoluene

EC Number:

246-836-1

EC Name:

Dinitrotoluene

Cas Number:

25321-14-6

Molecular formula:

C7H6N2O4

IUPAC Name:

Reaction mass of 1-methyl-2,4-dinitrobenzene and 2-methyl-1,3-dinitrobenzene

Details on test material:

- Name of test material (as cited in study report): technical grade dinitrotoluene (DNT)
- Analytical purity: 2,4-DNT (76%), 2,6-DNT (19%), 3,4-DNT (2.4%), 2,3-DNT (1.5%), 2,5-DNT (<1%) and 3,5-DNT (<1%) .

Test animals

Species:

rat

Strain:

Fischer 344

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Inc ., Wilmington, MA, and Portage, MI
- Both males and females were approximately 56 days of age at the time of arrival at Research Triangle Institute (RTI), thus providing animals for the study which were young, but sexually mature at the time breeding was initiated. Males and females were bred between 3-7 months after arrival at RTI.
- Weight at study initiation: 145-226 g at gestational day 0 (females were matched across dose groups by body weight on gestational day 0)
- Housing: males were housed singly and females were group housed (4-per-cage)
- Diet and water: Purina Certified Rodent Chow (5002) and tap water were available ad libitum throughout the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.22 +/- 1.11
- Air changes (per hr): 12 to 14 times per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Dose volume: 2 mL/kg
- Dosage solutions of DNT were formulated at concentrations of 7.00, 17.50, 18.75, 37.50, 50.00 and 75.00 mg/mL.
- Dosing solutions were stored at 5°C, and used for dosing up to 8 days after formulation

VEHICLE
- purity: laboratory-grade

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

High performance liquid chromatography (HPLC) in the normal phase mode was used for the analysis of DNT dosage formulations.
Samples were quantified from the absolute peak height of the major DNT isomer ( i.e. 2,4-DNT) measured manually and results calculated from
an external standard calibration curve.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: presence of sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

14 days: gestational day 7 through 20

Frequency of treatment:

Daily

Duration of test:

Sacrifice on gestational day 20

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 14, 35, 37.5, 75, 100, and 150 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

test concentration [mg/kg/day]: total no. of animals (pregnant and non-pregnant)
14: 22
35: 13
37.5: 22
75: 13
100: 23
150: 13
vehicle control: 37
positive control: 36

Control animals:

yes, concurrent vehicle

Details on study design:

- Positive control: Hydroxyurea, 200 mg/kg bw/day in sterile distilled water, p.o.
- Dose selection rationale: is based upon pilot data which indicated that exposure of timed-pregnant Fischer 344 rats to DNT (75 or 150 mg/kg/day, po) on gd 9 through 12 was well tolerated

Examinations

Maternal examinations:

CLINICAL SIGNS OF TOXICITY:
- Time schedule: on gestational days (gd) 0, and 7 through 20 (prior to daily dosing)

BODY WEIGHT: Yes
- Time schedule for examinations: on gestational days (gd) 0, and 7 through 20 (prior to daily dosing)

FOOD CONSUMPTION
- Time schedule for examinations: determined twice a week, beginning on day 0 of gestation
- Calculation of average food intake (g) per animal per day

WATER CONSUMPTION
- Time schedule for examinations: determined twice a week, beginning on day 0 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- liver weight, spleen weight

OTHER
- hematology on day of sacrifice: Methemoglobin assay, reticulocyte count, complete blood count

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early, middle, late, and full resorptions: Yes

Fetal examinations:

- For each fetus and "full" resorption: body weight, crown-rump length, placental weight and sex
- External examinations: Yes; all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
- Other: hematology: Methemoglobin assay, reticulocyte count, complete blood count

Statistics:

Due to the practical considerations in processing large numbers of dams and fetuses, an experimental design with three replicates was employed. Data were pooled across replicates and nonparametric statistics were applied: Kruskal-Wallis one-way analysis of variance by ranks (Siegel, 1956) was used to test for differences among DNT-treated and vehicle control groups. Kruskal-Wallis was significant at p<0 .05, the Mann-Whitney U test was
utilized to make individual comparisons between vehicle and each DNT treated group (Siegel, 1956). Jonckbeere's test for k independent samples (Jonckheere, 1954) was employed to identify significant dose response trends. Nominal level measurements were analyzed by Fisher's Exact Test (pairwise comparisons) or Chi Square (comparisons among dose groups) (Siegel, 1956) . Statistical comparisons between vehicle control and positive control, ( i .e ., hydroxyurea-treated) groups were conducted using the Mann-Whitney U test (Siegel, 1956) or Fisher's Exact Test (Siegel, 1956). Statistical analysis of hematological data from methemoglobin analysis (RTI), differential and complete blood counts (CIIT) were performed using a t-test to compare vehicle control to hydroxyurea or DNT treatment groups. The alpha level for each statistical comparison was 0.05 and the significance level (two-tailed) for each statistical comparison was reported at p<0.05 or p<0.01 .

Historical control data:

No data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

14 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Maternal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: relative spleen weights; secondary effects on hematology were observed at higher doses

Description (incidence and severity):

relative spleen weights; secondary effects on hematology were observed at higher doses (See "Remarks on results including tables and figures").

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: fetotoxicity

2

Key result

Dose descriptor:

NOAEL

Effect level:

>= 150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: embryotoxicity

Fetal abnormalities

1

Abnormalities:

not specified

2

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

Treatment related:

yes

Any other information on results incl. tables

MATERNAL TOXICITY

Clinical signs and maternal mortality

The clinical signs related to DNT treatment were rough coat, lethargy and hind limb weakness observed in 7/13 high-dose females.

 

Treatment	Vehicle	Hydroxyurea	Dinitrotoluene
Dose (mg/kg/d)	-	200	14	35	37.5	75	100	150
Females treated	37	36	22	13	22	13	23	13
% Pregnant (gd 20)	91	91	77	100	92	86	92	83
% Deaths (gd 0-20)	0	0	4.5	7.7	0	0	4.3	46.2

Maternal weight gain

A significant treatment effect was observed for maternal weight gain during gestation, but only the highest dose group (150 mg/kg/day) showed a significant decrease in weight when pairwise comparisons were made with the vehicle control group. Absolute maternal weight gain (i.e., maternal weight gain during treatment minus gravid uterine weight) exhibited a significant dose-related decrease across DNT treatment groups; significant reduction of absolute maternal weight gain was observed at 14, 100 and 150 mg/kg/day DNT. Significant dose-related increases in relative maternal liver and spleen weights were observed across DNT treatment groups.

 

Water consumption did not appear to be affected by DNT treatment, but statistical analysis was not performed since average consumption was based on group housed animals which were not equally distributed across cages.

Maternal weight gain on gestational days 7 through 20

Treatment	Vehicle	Hydroxyurea	Dinitrotoluene
Dose (mg/kg/d)	-	200	14	35	37.5	75	100	150
Number sacrificed	20	20	10	7	12	6	12	5
Total wt gain (g)	61.8 ± 3.6^*	54.0 ± 4.6	64.9 ± 4.6	66.1 ± 6.0	55.8 ± 5.8	64.8 ± 6.8	52.7 ± 4.5	8.1 ± 20.1^**
Absolute wt. gain (g)	24.1 ± 2.1	21.1 ± 1.7	17.2 ± 3.9^*	26.7 ± 4.3	22.2 ± 2.1	23.1 ± 2.1	15.2 ± 1.9^**	-14.0 ± 13.4^**
Liver wt (% body wt)	4.09 ± 0.08^**	3,95 ± 0.05	3.91 ± 0.1^*	4.12 ± 0.09	3,96 ± 0.09	4.55 ± 0.1^**	4.58 ± 0.08^**	4.79 ± 0.36
Spleen wt (% body wt)	0,197 +/- 0,003**	0,209 +/- 0,009	0,185 +/- 0,007	0,223 +/- 0,011*	0,215 +/- 0,006*	0,246 +/- 0,010**	0,320 +/- 0,027**	0,284 +/- 0,059*

^*p<0.05

^**p<0.01

Maternal hematological examination 

No significant difference was observed in hematological parameters for dams sacrificed at 20 minutes or 120 minutes after final treatment on gd 20; therefore, data were pooled for analysis. At sacrifice on gd 20, dams treated with 100 mg/kg/day DNT exhibited statistically significant increases in methemoglobin, reticulocyte count, red blood cell size, red blood cell distribution width and platelet count. Statistically significant decreases in red blood cell count and hematocrit were also observed.

FETAL TOXICITY

- no significant effects on % resorptions, % dead or live fetuses;

- no significant effects on live litter size, fetal body weight, crown-rump length, sex distribution, fetal grouwth and morphological development;

- changes of fetal liver and spleen weights without dose dependence and also occurring in control groups

- 100 mg: changes of hematological parameters (as in dams), decreased reticulocyte count, decreased RBC count, and increased RBC size

- no statistically significant increased malformations at any dose: % malformed fetuses per litter (external, visceral, sceletal) in dose groups 14/35/37.5/75/100/150 mg/kg and vehicle/pos. control: 4.1/3.2/0.8/0.0/5.8/0.0 and 3.8/30.6

Total number of animals and pregnants evaluated in the study and total number of live litter and live fetuses examined:

mg/kg bw/day	total number of animals	total number of pregnants	number of live litters examined	number of live fetuses examined
14	22	13	10	9,2 +/- 0,7
35	13	7	7	9,0 +/- 0,9
37.5	22	13	12	6,4 +/- 1,1
75	13	7	6	8,3 +/- 1,3
100	23	13	12	7,3 +/- 0,9
150	13	6	2	7,3 +/- 1,7
Vehicle control	37	22	20	7,3 +/- 0,7
Positive control	36	22	19	7,7 +/ 0,6

DOSAGE FORMULATIONS

Calculated dosages based upon analysis of DNT and hydroxyurea formulations are given below :

Nominal Treatment (mg/kg/day)	Theoretical Treatment Mean and Range (mg/kg/day )
Hydroxyurea (200)	185.0 (175 .2-200 .4)
DNT (14)	12.1 [8 .5-17 .1]
DNT (35)	33.8 [28 .7-41 .2]
DNT (37.5)	38.4 [15 .5-70 .4]
DNT (75)	75.0 [71 .6-81 .9]
DNT (100)	110.5 [65 .6-155 .3]
DNT (150)	157.4 [125 .6-177 .3]

Since dams received doses from as many as three individually formulated solutions during gestation and since a wide range of measured concentrations was represented within these formulations, it was not possible to confirm the actual oral exposure for individual dams. Discrepancies between dosage formulation records and the results of HPLC analyses may have been in part due to prolonged storage (6 to 11 months) prior to analysis.

Applicant's summary and conclusion

Conclusions:

DNT was not teratogenic in Fischer 344 rats even at dosages which produced significant dose-related signs of maternal and fetal toxicity .