N-isopropyl-N'-phenyl-p-phenylenediamine

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: GLP study, comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-isopropyl-N'-phenyl-p-phenylenediamine

EC Number:

202-969-7

EC Name:

N-isopropyl-N'-phenyl-p-phenylenediamine

Cas Number:

101-72-4

Molecular formula:

C15H18N2

IUPAC Name:

N1-phenyl-N4-(propan-2-yl)benzene-1,4-diamine

Details on test material:

Flexzone 3C, purity: 97.2%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

- M/F ratio per cage: 1:2

Duration of treatment / exposure:

day 6-15 day of gestation

Frequency of treatment:

daily

Duration of test:

study termination on gestation day 20

No. of animals per sex per dose:

24

Control animals:

yes

Details on study design:

Sex: female

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

62.5 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

LOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

3

Dose descriptor:

NOAEL

Effect level:

62.5 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

4

Dose descriptor:

LOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

62.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

2

Dose descriptor:

LOAEL

Effect level:

125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Mortality Data

There were no deaths.

Clinical Observations

At 125 mg/kg clinical observations were limited to pre-dosing salivation noted in 10/23 animals and soft dark faeces noted for 6/23 animals.

At 62.5 mg/kg transient noisy respiration was noted in one animal.

Body weight

There were no treatment related effects on bodyweight noted during

pregnancy.

Table: Group mean body weight (g)

Group

Dose level (mg/kg)

Day Post Coitum

0

3

6

7

8

9

10

11

12

13

14

15

18

20

1

0

269±16.8

284±20.2

300±22.2

305±23.0

311±23.5

316±21.9

323±20.9

330 ±20.9

337±22.5

344 ±22.0

351 ±22.7

360±22.9

402±27.6

436±29.9

2

12.5

270 ±14.6

287 ±15.6

300 ±14.6

303±14.8

308 ±16.1

317 ±16.6

324 ±16.5

332 ±18.2

338 ±18.9

346±19.0

354±19.2

362 ±19.7

405 ±23.3

436±27.8

3

62.5

269±17.5

285 ±18.7

299±19.8

302 ±19.4

308±20.8

314 ±19.9

320 ±20.7

329±22.0

337±21.7

344±22.7

353 ±22.6

362±22.9

401 ±28.3

431 ±32.3

4

125

265±15.2

282±15.6

297±16.5

300±16.1

307±18.5

311±19.9

321±20.2

329±18.8

338 ±20.6

346±20.4

354±21.1

361±22.2

403±25.3

431±28.1

Food Consumption

At 125 mg/kg there was a statistically significant (p < 0.001) reduction in food consumption from day 6 to day 9 of gestation which coincides with the initiation of dosing.

At 62.5 mg/kg there was also a statistically significant reduction in food consumption over this period (p < 0.01).

After day 9 of gestation food consumption in these two groups was comparable with control animals.

At 12.5 mg/kg there were no significant effects on food consumption throughout the study.

Table: Group mean food consumption

Group	Dose level (mg/kg)	Days Post Coitum
		0 -3	3 -6	6 -9	9 -12	12 -15	15 -18	18 -20
1	0	24.7±2.38	27.8 ±2.81	27.7±2.61	30.7 ±2.96	32.3±2.54	35.3 ±2.50	34.1 ±2.42
2	12.5	24.6±2.39	27.2±2.77	27.1±3.04	30.2±2.99	32.2±3.57	35.3±3.12	33.5±3.77
3	62.5	24.3±3.33	27.6±2.75	25.0**±3.27	29.1 ±3.07	31.7±2.63	35.6±3.11	35.5±3.02
4	125	24.3±2.44	27.1±2.34	23.8***±3.87	30.1 ±3.21	32.3±2.96	35.4±2.49	35.7±3.18

**significantly different from control (p<0.01)

***significantly different from control (p<0.001)

Terminal Studies

Adult Necropsy Data

There were no treatment-related macroscopic findings for adult females at necropsy.

Uterine/ Implantation Data

There were no treatment-related effects on uterine/implantation data.

Table: Summary of adult performance

	Dose level mg/kg
	0	12.5	62.5	125
Number of females in group	24	24	24	24
Number pregnant	24	24	24	23
Number died/killed before day 20	0	0	0	0
Number not pregnant	0	0	0	1
Number with total litter loss	0	0	0	0

Table: Group mean data uterine/implantation

Group	Dose level (mg/kg)	Number of animals	Number pregnant	Number of Corpora Lutea	Number of Live Fetuses	% Male Fetuses
1	0	24	24	18.4	15.0	49.4
2	12.5	24	24	18.5	15.2	48.1
3	62.5	24	24	18.6	13.8	46.8
4	125	24	23	20.1	16.1	45.9

Table: Group mean litter data

		Embryonic/Foetal deaths			Implantation loss%		Total litter weight (g)	Mean foetal weight (g)
Group	Dose level (mg/kg)	Early	Late	Total	Pre	Post
1	0	0.54	0.00	0.54	14.9	3.4	55.7	3.72
2	12.5	0.83	0.00	0.83	12.6	5.2	59.2	3.91
3	62.5	0.96	0.00	0.96	20.3	7.5	52.9	3.80
4	125	0.48	0.09	0.57	15.7	3.8	62.4	3.88

Foetal Data

Throughout all groups there were three foetuses with significant structural anomalies.

At 12.5 mg/kg one foetus, (dam 35, foetus 1) had an encephalocele with a further foetus (dam 44, foetus 7) having an interventricular septal defect. At 0 mg/kg one foetus (dam 14, foetus 5) had an interventricular septal defect, an

atretic left atrium and mitral valve, right sided aorta, aortic arch and ductus arteriosus, no brachiocephalic trunk, no pulmonary trunk with a modified ductus arteriosus exiting from the right ventricle.

There were no intergroup differences in the incidence of foetal external findings.

Table: Incidence of foetal external findings

Dose level (mg/kg)	0	12.5	62.5	125
Number of foetuses examined	361	364	332	370
Total Number of fetuses affeted	2	4	7	1
Total number of litters affected	2	4	5	1
Group mean per litter (%)	0.6	1.2	3.4	0.3

Table: Group summary of foetal skeletal development

Dose level (mg/kg)	0	12.5	62.5	125
Number of foetuses examined	174	175	159	182
Number of rips: 13/13	153F/24L/89%	152F/24L/87.2%	143F/24L/90.1%	154F/23L/82.7%
13/14	16F/9L/8.3%	13F/10L/7.3%	8F/7L/4.3%	16F/12L/9.6%
14/14	5f/3L/2.5%	10F/7L/5.4%	7F/6L/5.6%	12F/6/L7.7%
Number of fully ossified sternebrae <4	5F/5L/2.8%	11F/4L/6.8%	11F/9L/8.5%	4F/4L/2.9%
4	69F/19L/38.0%	54F/18L/32.4%	52F/21L/36.2%	75F/21L/40.7%
>4	99F/24L/59.2%	107F/23L/60.8%	94F/20L/55.3%	103F/L22/56.4%
Number of ost lumbar vertebral centra <7	8F/6L/4.5%	8F/5L/4.1%	12F/7L/9.6%	7F/5L/3.6%
Number of ost lumbar vertebral centra >7	166F/24L/95.5%	167F/24L/95.9%	146F/24L/90.4%	174F/23L/96.4
Number of post lumbar vertebral arches <5	26F/12L/16.0%	27F/12L/16.7%	33F/13L/19.6%	23F/12L/12.3
Number of post lumbar vertebral arches >5	148F/24L/84.0%	148F/24L/83.3%	125F/23L/80.4%	158F/23L/87.7%
Number of metacarpals/ Metatarsals 0/0	0F/0L/-	0F/0L/-	1F/1L/2.1%	0F/0L/-
5/6	0F/0L/-	0F/0L/-	1F/1L/0.6%	0F/0L/-
6/6	2F/2L/1.1%	3F/3L/1.4%	2F/2L/2.9%	4F/3L/1.9
6/7	0F/0L/-	1F/1L/0.6%	0F/0L/-	2F/2l/1.1%
7/7	0F/0L/-	2F/1L/1.2%	0F/0L/-	0F/0L7-
4/8	0F/0L/-	1F/1L/0.5%	0F/0L/-	0F/0L/-
6/8	132F/23L/77.0%	110F/21L/65.1%	108F/21L/67.6%	114F/23L/62.0%
7/8	2F/1L/0.9%	3F/3L/1.5%	3F/3L/1.7%	4F/4L/2.3%
8/8	38F/11L/21.1%	48F/14L/27.0%	41F/14L/24.6%	57F/18L/32.7%
8/10	0F/0L/-	5F/1L/2.6%	1F/1L/0.6%	0F/0L/-
Fontanelle size: small	7F/5L/4.3%	21F/7L/12.8%	18F/7L/13.7%	8F/3L/4.0%
Fontanelle size: medium	166F/24L/95.2%	150F/23L/85.8%	132F/21L/77.9%	174F/23L/96.0
Fontanelle size: large	1F/1l/0.5%	3F/2L/1.4%	9F/4L/8.4%	0F/0l/-

F:number of foetuses in category

L:number of litters in category

%:group mean per litter

Skeletal findings:

At 125 mg/kg there were statistically significant effects on the incidence of skeletal findings. At this dose level there was an increased incidence of irregularly and incompletely ossified cranial and facial bones (p < 0.001 - P < 0.05). There was also an increased incidence of no ossification of hyoid (p < 0.001), unilaterall bilateral wavy ribs (p < 0.05) and semi bipartite vertebral centra (p < 0.01). At 62.5 mg/kg there was a statistically significant increase in incomplete ossification of more than one cranial bone (p < 0.05).

At 12.5 mg/kg there was a statistically significant increase in incomplete ossification of more than one facial bone (p < 0.05).

remarks: The only adverse effect noted in the foetuses and related to treatment was a retardation of ossification in the high dose group (125 mg/kg bw/d); and a low percentage of wavy ribs, 3.8% compared with 0.6% in the controls. Wavy ribs are a commonly observed effect in studies, especially in presence of maternal toxicity, and normally disappears post-natal and is not considered as a malformation but as a variation. There are no adverse effects in low and mid dose pups.

Table:Group incidence of foetal skeletal findings

Dose level (mg/kg)	0	12.5	62.5	125
Number of foetuses examined	174	175	159	182
Total Number of fetuses affeted	72	98	86	124
Total number of litters affected	22	23	22	23
Group mean per litter (%)	40.3	53.3	51.3	69.5
Incomplete ossification of more than one cranial bone	12F/10L/ 6.9%	13F/9L/ 7.5%	25*F/11L/15.2 %	35**F/16L/17.8%
incomplete ossification of more than one facial bone	2F/2L/ 1.1%	10*F/5L/5.3%	6F/6L/5.1%	19**F/10L/10%
irregular ossification of one cranial bone	1F/1L/0.5%	2F/2L/1.0%	3F/3L/1.7%	8*F/7L/ 4.8%
irregular ossification of one facial bone	0F/0F/-	4F/4L/ 1.9%	3F/3L/ 1.5%	9**F/6L/ 5.0%
irregular ossification of more than one facial bone	3F/3L/ 1.6%	4F/4L/ 2.1%	7F/5L/ 3.8%	19**F/8L/9.7%
no ossification of hyoid	16F/11L/9.4%	27F/13L/14.4%	24F/12L/ 13.8%	43***F/16L/22.3%
unilateral/bilateral wavy ribs	1F/1L/0.6%	0F/0L/-	2F/2L/ 1.1%	7*F/5L/3.8%
one thoracic vertebral centrum semi-bipartite	10F/6L/5.6%	14F/7L/ 7.3%	18F/9L/9.7%	30**F/14L/16.4%

*: significantly different from control (p<0.05)

**: significantly different from control (p<0.01)

***: significantly different from control (p<0.001)

F:number of foetuses in category

L:number of litters in category

%:group mean per litter

Visceral Findings:

The overall incidence of visceral findings for foetuses was considered comparable for all groups. Intergroup variations for specific visceral findings were not considered to be significant.

Table: Group incidence of foetal visceral findings

Dose level (mg/kg)	0	12.5	62.5	125
Number of foetuses examined	187	189	172	188
Total Number of fetuses affeted	61	64	66	75
Total number of litters affected	23	23	23	19
Group mean per litter (%)	33.0	34.7	39.6	37.2