3(or 4)-methylbenzene-1,2-diamine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline full report, pre-GLP.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1979

Report date:

1979

Reference 2

Reference Type:

publication

Title:

Teratogenesis study of o-toluenediamine in rats and rabbits

Author:

Becci PJ, Reagan EL, Knickerbocker MJ, Barbee SJ & Wedig JH

Year:

1983

Bibliographic source:

Toxicol.Appl.Pharmacol. 71: 323-9

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

no

Remarks:

Pre-GLP

Test material

Test material information

Constituent 1

Reference substance name:

3(or 4)-methylbenzene-1,2-diamine

EC Number:

248-145-0

EC Name:

3(or 4)-methylbenzene-1,2-diamine

Cas Number:

26966-75-6

Molecular formula:

C7H10N2

IUPAC Name:

3(or 4)-methylbenzene-1,2-diamine

Details on test material:

Light brown solid crystalline.
analysis:
ortho - 97.3%
para - 0.71%
meta - 0.64%
mono - 1.28%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

All animals used in this study were sexually mature Sprague-Dawley (TAC: N (SD) FBR) rats. All were purchased from Taconic Farms, Inc.,
Germantown, NY.

All animals were individually housed in wire-mesh bottom cages in temperature controlled quarters.
Fresh tap water and ground Charles River Rat/Mouse/Hamster Formula (Agway) were available ad libitum throughout the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Beginning on day 6 of gestation and continuing daily through day 15 of gestation, the appropriate material was administered orally using a
plastic, disposable syringe and gavage needle to each pregnant rat. The test material was prepared fresh daily using corn oil as the vehicle at
concentrations necessary to be dosed on a 10 ml/kg basis. The positive control compound (Aspirin) was given as an aqueous suspension.
The amount of test material administered to each female was adjusted during gestation according to the most recent body weight. The dosage
regimen was as follows:
Group Minimum No. Treatment/Level
Pregnant Females

A 20 Corn Oil: 10 ml/kg/day
B 20 Aspirin: 250 mg/kg/day
C 20 Ortho-IDA: 10 mg/kg/day
D 20 Ortho-TDA: 30 mg/kg/day
E 20 Ortho-TDA: 100 mg/kg/day
F 20 Ortho-TDA: 300 mg/kg/day

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Female rats were mated 1:l with male rats in sufficient numbers to produce at least 120 pregnant females. Observation of the vaginal sperm
plug was considered day 0 of gestation.
One male was not allowed to impregnate more than one female per treatment group.

Duration of treatment / exposure:

day 6 through 15 of gestation

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 10, 30, 100, 300 mg/kg bw/day
Basis:
actual ingested

Details on study design:

On day 20 of gestation, all females were killed by a 5-10 minute exposure to chloroform vapours. The uterine contents of each female
were removed and the reproductive performance recorded. The urogenital tract of each female was examined for anatomical normality.
All females that died or were sacrificed moribund during the course of the study were weighed; the weights were recorded;
and all were subjected to a thorough uterine examination.

Examinations

Maternal examinations:

Body weights of females were recorded on days 0, 6, 9, 12, 15 and 20 of gestation. All animals were observed daily for changes in
general appearance and behaviour and a record maintained.

Ovaries and uterine content:

At the time of sacrifice on day 20 (or if the animal died or was sacrificed moribund) the following observations were recorded: numbers of
corpora lutea, implantation sites, resorption sites, live and dead foetuses; sex of foetuses; and body weights of foetuses. If possible, late
resorptions were weighed ad sexed.

Fetal examinations:

At the time of uterine examination, all foetuses were examined grossly for the presence of external congenital abnormalities.
One-half of the foetuses from each litter were placed in Boulin’s solution for detailed visceral examination employing the Wilson free-hand
slicing technique. Any foetus showing external abnormalities was selected for examination by this method.

The remaining foetuses were eviscerated, fixed in 70% isopropyl alcohol, macerated in a 2% potassium hydroxide solution, stained in
Alizarin-Red S dye, cleared in glycerine, and examined under low power magnification for skeletal anomalies and ossification variations.
Each fetus was processed, examined and stored for possible further examination in a manner retaining the identity of both dam number and
uterine position.

Statistics:

Incidences of occurrence were expressed as percent, and comparisons between the negative control and test groups were made using either
95% confidence intervals for proportions or by computation of exact probabilities. Continuous data were analyzed using a one-way completely
random classification analysis of variance for fixed effects. Differences were deemed significant when the probability of rejecting the null
hypothesis when true was less than 0.05. The least significant difference test was then employed to determine which test group(s) differed from
the negative control. All comparisons were made using the dam or litter as the unit of observation rather than the foetus.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Reduced body weight gain at 300mg/kg/day

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

1

Dose descriptor:

NOEL

Effect level:

> 300 mg/kg bw/day

Basis for effect level:

other: teratogenicity

2

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: fetotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

On day 20 of gestation, the treated females were killed and subjected to uterine examination. The reproductive performance of treated dams is summarized in Table 1. There were no significant differences in pregnancy, implantation, numbers of live foetuses, numbers of dead foetuses or numbers of resorptions per dam between any test level and the vehicle control. There were increases in the total numbers of resorption sites in the ortho-TDA groups; however, the percentage of dams affected was not increased when compared to the vehicle control. The data showed a significant reduction in foetal weight in dams treated at 300 mg/kg.

Dam body weights during gestation are summarized in Table 2. Females administered 300 mg/kg ortho-TDA exhibited significantly lower body weights on days 12, 15 and 20 of gestation when compared to the negative control. In addition these animals showed a significantly smaller weight gain through gestation.  Body weights of females treated at 10, 30 or 100 mg/kg/day did not differ from the vehicle control.

Foetuses were evaluated for skeletal anomalies, and the results are summarized in Table 3. The abnormalities noted in foetuses whose dams received ortho-toluene diamine at 10, 30, 100 or 300 mg/kg/day were generally variations rather then malformations. Skeletal examinations showed a significant increase in litters with missing sternebrae, incompletely ossified vertebrae, and incomplete skull closure at the 300 mg/kg level. There was a significant increase in the percentage of litters with incomplete vertebrae at 100 mg/kg also. These findings would probably indicate a generalized retardation of maturation due to maternal toxicity rather than a frank teratogenic response.

The results of soft tissue examinations in the foetuses are summarized in Table 4. The data showed a significant increase in litters with foetuses exhibiting a hemorrhagic abdomen in the 10, 100 and 300 mg/kg groups. There was however no dose-related increase among test groups which would suggest the finding was related to mechanical damage rather than treatment with o-TDA. Other soft tissue findings did not differ significantly from the control in frequency of occurrence.

Treatment with 250 mg/kg/day of acetylsalicylic acid (aspirin) on days 6 through 15 of gestation as the positive control resulted in many noteworthy findings. Aspirin significantly increased the percentage of dams with resorption sites, reduced the number of live foetuses per dam, and reduced fetal weights (Table 1). This in turn resulted in significantly lowered dam body weights on day 20 and smaller weight gains during gestation (Table 2). Examinations of foetuses revealed several skeletal abnormalities as well as soft tissue anomalies (Tables 3 and 4).

.

Table 1. Average Litter Reproduction Data of Rats

	Control(2) Corn oil 10 ml/kg	Aspirin 250 mg/kg	Ortho-Toluene Diamine
			10 mg/kg	30 mg/kg	100 mg/kg	300 mg/kg
No. females mated	25	33	33	25	33	33
..
Pregnancies
Total No.	22	25	22	22	25	25
Wastage(1)	0	2	0	0	0	1
To term (20 days)	22	23	22	22	25	24
..
Implant sites
Total No.	235	235	241	223	276	269
Average/Dam	10.7	10.2	11.0	10.1	11.0	11.2
..
Live Fetuses
Total No.	227	128	223	200	264	246
Average/Dam	10.3	5.6*	10.1	9.1	10.6	10.3
M/F	116/111	65/63	121/102	94/106	131/133	120/126
Average Wt. (g)	3.91	2.76*	3.94	3.97	3.77	3.19*
..
Resorptions
Total No.	8	106	18	23	12	22
Dams Affected	6	20*	5	6	8	8
Dams w/all resorbed	0	6	1	1	0	0
..
Dead Fetuses
Total No.	0	1	0	0	0	1
Dams affected	0	1	0	0	0	1
Dams w/all dead	0	0	0	0	0	0

1. Dam died or aborted; data not included below.

2. All materials administered orally days 6-15 of gestation

* Significantly different from control (p less than 0.05)

.

Table 2. Dam Body Weights (g) During Gestation(1)

Day Treatment^(2)	0	6	9	12	15	20	Weight gain g
Control	215.3	236.5	238.1	249.5	260.5	321.8	106.5
Corn oil	+/-2.5	+/-2.7	+/-3.0	+/-3.1	+/-3.7	+/-5.0	+/-3.7
(10 ml/kg)	(22)	(22)	(22)	(22)	(22)	(22)	(22)
..	..	..	..	..	..	..	..
Aspirin	217.9	239.1	236.1	249.5	257.1	290.7*	73.5*
(250 mg/kg)	+/-2.5	+/-3.2	+/-3.4	+/-3.7	+/-4.1	+/-6.7	+/-6.2
..	(23)	(23)	(23)	(23)	(23)	(23)	(23)
..	..	..	..	..	..	..	..
Ortho-TDA^(3)	216.7	234.7	236.4	249.9	263.1	322.5	106.0
(10 mg/kg)	+/-3.3	+/-3.4	+/-3.2	+/-3.1	+/-3.7	+/-5.1	+/-3.7
..	(22)	(22)	(22)	(22)	(22)	(22)	(22)
..	..	.	.	.	.	.	.
Ortho-TDA	214.8	237.0	237.1	249.5	261.2	317.4	102.6
(30 mg/kg)	+/-2.6	+/-2.7	+/-2.5	+/2.5	+/-2.6	+/-5.5	+/-4.7
..	(22)	(22)	(22)	(22)	(22)	(22)	(22)
..	.	.	.	.	.	.	.
Ortho-TDA	218.7	238.9	235.9	250.6	262.4	326.0	107.2
(100 mg/kg)	+/-2.9	+/-2.9	+/-2.8	+/-2.7	+/-2.8	+/-3.7	+/-2.3
..	(25)	(25)	(25)	(25)	(25)	(25)	(25)
..	. .	.	.	.	.	.	.
Ortho-TDA	216.6	237.0	228.7	233.6*	244.5*	305.4*	88.8*
(300 mg/kg)	+/-2.6	+/-2.7	+/-3.0	+/-3.7	+/-4.7	+/-5.2	+/-5.0
..	(24)	(24)	(24)	(24)	(24)	(24)	(24)

(1) All values are means +/-SEM for the number of pregnancies in ( ).

(2) All materials administered orally days 6-15 of gestation.

(3) Ortho-TDA = Ortho-toluene diamine

* Significantly different from control (p less than 0.05)

.

Table 3. Summary of Skeletal Findings in Fetuses(1)

Findings	Corn Oil(2) 10 ml/kg	Aspirin 250 mg/kg	Ortho Toluene Diamine
			10 mg/kg	30 mg/kg	100 mg/kg	300 mg/kg
No. of fetuses examined (at term)	120/22	67/17	117/21	105/21	134/25	129/24
...
Sternebrae
Incomplete ossification	54/19	63/17	59/19	53/18	71/22	111/24
Bipartite	1/1	1/1	-	-	2/1	1/1
Missing	1/1	20/11*	1/1	-	3/2	10/8*
..
Ribs
Fused/split	-	6/4*	-	-	-	-
More than 13; Rudimentary	34/16	16/8*	21/13*	24/12	30/14	22/15
More than 13; Extra	2/2	39/11*	-	2/2	4/2	2/2
..
Vertebrae
Incomplete ossification	22/11	56/17*	24/12	23/13	40/22*	49/20*
..
Skull
Incomplete closure	2/2	8/4*	1/1	-	2/2	8/6*
Missing	-	2/1	-	-	-	1/1
..
Extremities
Incomplete ossification	-	3/3*	-	-	-	4/2
..
Miscellaneous
Hyoid; missing	1/1	6/4*	-	-	-	-
Hyoid; reduced	1/1	8/5*	-	-	1/1	4/3

(1) Numerator = number of fetuses affected; Denominator = number of litters affected.

(2) All materials administered orally days 6-15 of gestation

* Significantly different from control (p less than 0.05)

.

Table 4. Summary of Soft Tissue Abnormalities of Fetuses

Treatment(1)/Findings	Fetuses	Litter
	No. affected / No. observed
A Corn Oil 910 ml/kg)
Hemorrhagic Abdomen	3/120	3/22
..
B Aspirin (250 mg/kg)
Spina Bifida	4/61	3/17*
Encephalomeningocele	10/61	5/17*
Gastroschisis	1/61	1/17
Hemorrhagic Abdomen	5/61	3/17
..
C Ortho-TDA(2) (10 mg/kg)
Hemorrhagic Abdomen	8/106	8/21*
One pup appears squatty	1/106	1/21
..
D Ortho-TDA (30 mg/kg)
Hemorrhagic Abdomen	2/95	2/20
..
E Ortho-TDA (100 mg/kg)
Gastroschisis	1/120	1/25
Hemorrhagic Abdomen	8/130	8/25*
..
F Ortho-TDA (300 mg/kg)
Encephalomeningocele	1/115	1/24
Gastroschisis	1/115	1/24
Hemorrhagic Abdomen	14/115	10/24*
Pups small	3/115	2/24

(1) All materials administered orally days 6-15 of gestation

(2) Ortho-TDA = Ortho-toluene diamine

* Significantly different from control (p less than 0.05)