N,N'-bis(1,4-dimethylpentyl)-p-phenylenediamine

Toxicity to reproduction

Administrative data

Endpoint:

three-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

acceptable documented study report, which meets basic scientific principles, but study with limitations (mortality of parental animals in all study groups was excessively high throughout the study; a large number of treated and control F0 animals were reported as possibly having respiratory infection)

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1980

Reference 2

Reference Type:

publication

Title:

Chronic toxicity and reproduction studies on rubber antizonants (substituted para-phenylenediamines)

Author:

Stevens, M., W.; et al.

Year:

1981

Bibliographic source:

Toxicologist, 1, 58

Reference 3

Reference Type:

other company data

Title:

Unnamed

Year:

1981

Materials and methods

Principles of method if other than guideline:

other: early three-generation study with limitations

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-bis(1,4-dimethylpentyl)-p-phenylenediamine

EC Number:

221-375-9

EC Name:

N,N'-bis(1,4-dimethylpentyl)-p-phenylenediamine

Cas Number:

3081-14-9

Molecular formula:

C20H36N2

IUPAC Name:

N1,N4-bis(5-methylhexan-2-yl)benzene-1,4-diamine

Details on test material:

Santoflex 77

Test animals

Species:

rat

Strain:

other: Charles River CD® albino rats

Sex:

male/female

Administration / exposure

Route of administration:

oral: feed

Vehicle:

acetone

Details on mating procedure:

M/F ratio per cage: 1:2

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

8 males and 16 females per group were administered test diet for 11 weeks; admnistration continouded through mating, gestation and lactation for two successive litters.

Frequency of treatment:

daily

Details on study schedule:

8 males and 16 females per group were administered test diet for 11 weeks; admnistration continouded through mating, gestation and lactation for two successive litters (F1a, F1b). Groups of 8 males and 16 females were retained at weaning from the second litters of each dose level as parental animals for the succeeding generation.

Doses / concentrations

Remarks:

Doses / Concentrations:
30, 100, 300 (ca. 2.25, 7.5, 22.5 mg/kg bw/day)
Basis:

No. of animals per sex per dose:

8 males per group, 16 females per group

Control animals:

yes, plain diet

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

Fertility

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects on fertility

Remarks on result:

other: Generation: F0, F1, F2 (migrated information)

2

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: transient and slight body weight gain reduction during the study

Remarks on result:

other: Generation: F0, F1, F2 (migrated information)

3

Dose descriptor:

LOAEL

Remarks:

parental toxicity

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight and bidy weight gain reduction, reduced liver and kidney weights

Remarks on result:

other: Generation: F0, F1, F2 (migrated information)

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Diet Preparation

For the first 4 preparations of the test diets, a pre-mixture of test material/basal diet was employed which had been prepared with the

first set of test diets and stored for use in the following 3 weeks. It was noted that this pre-mixture had discolored prior to the fourth weeks preparation. Thereafter, the material was not pre-mixed with basal diet until the day of the test diet preparation. It should also be noted that acetone was employed as a solvent for the test material of a concurrent Monsanto Company project, and acetone

was added to the basal diet of the common control animals beginning with the filth week of diet preparation.

Body Weights

Males and females of the 300 ppm test group exhibited statistically signifcant reduced body weight gains throughout each parental generation of the investigation in comparison to those of the control group. No consistent weight gain reductions were noted for the animals fed either 30 or 100 ppm SANTOFLEX 77. Final body weights were lower for parental animals of the highest dose group.

F0 Group	Dietary level(ppm)	Mean final body weight (g)	% from control
Males	None	522	100%
I	30	547	105%
II	100	517	99%
III	300	453	87%
F1
Control	None	497	100%
I	30	537	108%
II	100	453	91%
III	300	379*	73%
F2
Control	None	461	100%
I	30	475	103%
II	100	500	109%
III	300	378	82%
Females
F0 Group
Control	None	309	100%
I	30	304	98%
II	100	308	100%
III	300	270**	87%
F1
F2	-	-	-
Control	None	293	100%
I	30	296	101%
II	100	271	93%
III	300	251**	86%

Food consumption

Food intake was determinated in the F0 generation in 3 intervals in the pre-mating period (weeks 4, 5, and 9) for 5 males and

5 females per group. No differences in food intake was noted in week 4 and 5 (pre-mating period) in control and treatment groups. However, the observations were made that animals of the 300 ppm group continously "kicking" out their dietary mixtures. It was suggested that some spillage of food occured in this dose group. At week 9 animals of the 300 ppm group showed reduced food intake compared to the lower dose groups and control. The authors suggested poor palatability of the 300 ppm dietary mixture.

(Note: for the first week of feed intake measurement (pre-mating week 4) the diets were prepared without the use of the pre-mixture of test material/basla diet that had previously been tested.

F0 generation food consumption

Group	ppm	Food concumption mean value g/per rat/day
weeks (pre-mating)		4	5	9
Males
Control	None	22	22	25
T-I	30	24	25	23
T-II	100	18	21	23
T-III	300	22	20	20
Females
Control	none	17	17	17
T-I	30	18	16	18
T-II	100	17	17	17
T-III	300	17	16	13

Organ weights:

For the high dose animals, kidney weights were reduced in the F0, F1 and F2 generations and liver weights were reduced for the F1 and F2 generation in high dose group animals. no other statistical differences in organ weights were noted in F0, F1 or F2 parental animals in any treatment group

Mortality:

Mortality of parental animals was high throughout the study. However, these mortalities occurred in all study groups and were not considered related to treatment.

Table: Mortality rate parental animals

Goup	ppm	Mortality data (total)
Generation		F0	F1	F2
Males
Control	None	2/8	6/8	2/8
T-I	30	5/8	2/8	2/8
T-II	100	3/8	2/8	6/8
T-III	300	3/8	2/8	4/8
Females
Control	None	-	4/16	1/16
T-I	30	-	3/16	2/16
T-II	100	-	5/16	2/16
T-III	300	-	5/16	3/16

Note: Postmortem pathologic evaluation revealed as common findings in the lungs lesions associated with chronic murine pneumonia, suppurative bronchopneumonia and absesses. The lessions of chronic murine pneumonia were present in most animals of the sacrificed, moribund sacrifice and death groups of animals of the F0, F1 and F2 generations. However, lesions of suppurative bronchopneumonia, graded as moderately severe to severe, were present in many animals that died in these three generation study.

The lesions of suppurative bronchopneumoia present were similar to those decribed for bacterial diseases of rats and they were superimposed on the lesions of chronic murine pneumonia. The authors suggested that severity of the suppurative bronchopneumonia lesions was considered to be related to the most probable cause of death for many of the animals that died. In many of the animals that died with severe lesions of suppurative bronchopneumonia in the lung, they also had lesions of centrilobular necrosis of hepatocytes in the liver; centrilobular necrosis in the liver is often associated with hypoxic conditions. The authors suggested that hypoxia may have resulted in these animals from severe bronchopneumonia changes in the lung.

Clinical observations:

No treatment-related clinical signs were noted in any of the treated animals. A number of treated and control F0 animals were reported as possibly having respiratory infection (as discussed above).

Gross necropsy.

Gross necropsy observations of F0, F1 and F2 adults sacrificed after weaning of their second litters did not reveal any adverse effects related to test material adimistration. For the high dose animals, kidney weights were reduced in the F0, F1 and F2 generations and liver weights were reduced for the F1 and F2 generation in high dose group animals. no other statistical differences in organ weights were noted in F0, F1 or F2 parental animals in any treatment group.

Histological observations.

Microscopic examination of tissues from selected F0, F1 and F2 parental animals and F3b pups from control and high dose groups and from selected animals from low and mid dose groups, revealed no abnormalities associated with treatment.

Reproductive data

Reproductive parameters of treated animals were comparable to control, for parental animals of the F0, F1, F2 generation.

Mating indices, fertility indices, and the incidence of partuition were comparable between control and treated groups throughout the study.

Table Reproductive parameters

Group	Dietary level ppm	Litter	Mating Fraction	Mating Index %	Fecundity Fraction	Fecundity Index
F0
C	none	F1A	16/32	50	15/16	93.8
C	none	F1B	13/20	65	13/13	100
T-I	30	F1A	18/36	50	16/18	88.9
T-I	30	F1B	13/20	65	13/13	100
T-II	100	F1A	16/23	69.6	16/16	100
T-II	100	F1B	15/23	65.2	15/15	100
T-III	300	F1A	18/39	46.2	15/18	83.3
T-III	300	F1B	12/15	80	12/12	100
F1
C	none	F2A	15/51	29.4	14/15	93.3
C	none	F2B	13/31	41.9	13/13	100
T-I	30	F2A	15/41	36.6	15/15	100
T-I	30	F2B	14/24	58.3	14/14	100
T-II	100	F2A	16/37	43.2	14/16	87.5
T-II	100	F2B	13/27	48.1	12/13	92.3
T-III	300	F2A	15/47	31.9	13/15	86.7
T-III	300	F2B	15/29	51.7	13/15	86.7
F2
C	none	F3A	17/47	36.2	15/17	88.2
C	none	F3B	16/48	37.2	13/16	81.3
T-I	30	F3A	14/48	29.2	12/14	85.7
T-I	30	F3B	14/32	43.8	12/14	85.7
T-II	100	F3A	19/47	40.4	15/19	78.9
T-II	100	F3B	14/25	56	14/14	100
T-III	300	F3A	15/33	45.5	14/15	93.3
T-III	300	F3B	13/23	56.5	13/13	100

Table Reproductive parameters

Group	Dietary level ppm	Litter	Fertility Fraction male	Fertility Fraction % male	Fertility Fraction Female	Fertility Fraction % female
F0
C	none	F1A	7/7	100	15/16	93.8
C	none	F1B	5/5	100	13/13	100
T-I	30	F1A	7/7	100	16/16	100
T-I	30	F1B	4/4	100	13/13	100
T-II	100	F1A	7/7	100	16/16	100
T-II	100	F1B	5/5	100	15/15	100
T-III	300	F1A	8/8	100	15/16	93.8
T-III	300	F1B	5/5	100	12/12	100
F1
C	none	F2A	5/5	100	14/16	87.6
C	none	F2B	4/4	100	13/13	100
T-I	30	F2A	7/7	100	15/16	93.8
T-I	30	F2B	6/6	100	14/14	100
T-II	100	F2A	6/7	85.7	14/15	93.3
T-II	100	F2B	5/5	100	12/12	100
T-III	300	F2A	6/6	100	13/15	86.7
T-III	300	F2B	5/5	100	13/13	100
F2
C	none	F3A	6/7	85.7	15/16	93.8
C	none	F3B	5/7	71.4	13/15	86.7
T-I	30	F3A	7/9	87.5	12/15	80
T-I	30	F3B	5/5	100	12/12	100
T-II	100	F3A	6/7	85.7	15/16	93.8
T-II	100	F3B	4/4	100	14/14	100
T-III	300	F3A	7/7	100	14/15	93.3
T-III	300	F3B	8/8	100	13/14	92.9

Table Reproductive parameters

Group	Dietary level ppm	Litter	Incidences Parturition Fraction	Incidences Parturition %
F0
C	none	F1A	15/15	100
C	none	F1B	12/13	92.3
T-I	30	F1A	13/16	81.3
T-I	30	F1B	12/13	92.3
T-II	100	F1A	15/16	93.8
T-II	100	F1B	13/15	86.7
T-III	300	F1A	13/15	86.7
T-III	300	F1B	11/12	91.7
F1
C	none	F2A	14/14	100
C	none	F2B	13/13	100
T-I	30	F2A	15/15	100
T-I	30	F2B	14/14	100
T-II	100	F2A	13/14	92.9
T-II	100	F2B	12/12	100
T-III	300	F2A	12/13	92.3
T-III	300	F2B	13/13	100
F2
C	none	F3A	15/15	100
C	none	F3B	13/13	100
T-I	30	F3A	12/12	100
T-I	30	F3B	12/12	100
T-II	100	F3A	14/15	93
T-II	100	F3B	14/14	100
T-III	300	F3A	7/7	100
T-III	300	F3B	8/8	100

 

Offspring:

Clinical signs:

No treatment-related clinical signs were noted in offspring of treated animals.

Observations:

No treatment-related gross pathologic alterations or external structural malformation were noted in offspring of treated animals. One F1 generation pup (F1b litter, 300 ppm group) exhibited several loops intestine protruding from stomach when initially observed following parturition. It was suspected that this was an injury caused by the dam. A single F3a control stillborn pup displayed exenephaly and clef palate. A single F3a 30 ppm stiborn pup displayed bilateral ablepharon, excencephaly, and protruding tongue. All other fetuses obtained were judged to be free of major externally noted structural malformation.

Delivered pups:

The number of pups delivered and weaned by treated and control animals was comparable.

Survival indices:

The number of pups surviving to weaning in the F1b, F2a, F2b, F3a and F3b high dose litters and the number surviving in F2a, F3a and F3b mid dose litters was reduced. Survival indices for these litters were reduced accordingly. survival indices calculated for the low dose group were comparable to control.

Pups Body weights:

Body weights of weanlings from the high dose group were significant reduced for F1b males and for F2 and F3 litter males and females. Pup weights were slightly reduced for mid-dose group litters.

Table: Number of pups delivered

Group	Dietary level ppm	Litter	Number of pups delivered total	Number of pups delivered mean	Number of pups stillborn total	Number of pups stillborn mean	Number of pups  viable total	Number of pups  viable mean
F0
C	none	F1A	185	12.3	3	0.2	180	12
C	none	F1B	124	10.3	1	0.1	123	10.3
T-I	30	F1A	151	10.3	1	0.1	150	11.5
T-I	30	F1B	155	12.9	1	0.1	153	12.8
T-II	100	F1A	171	11.4	0	0	171	11.4
T-II	100	F1B	153	11.8	2	0.2	151	11.6
T-III	300	F1A	135	10.4	3	0.2	132	10.2
T-III	300	F1B	132	12.0	5	0.5	127	11.5
F1
C	none	F2A	133	9.5	8	0.6	124	8.9
C	none	F2B	113	8.7	4	0.3	109	8.4
T-I	30	F2A	124	8.3	10	0.7	113	7.5
T-I	30	F2B	140	10.0	10	0.7	129	9.2
T-II	100	F2A	119	9.2	1	0.1	117	9.0
T-II	100	F2B	116	9.7	2	0.2	114	9.5
T-III	300	F2A	108	9.0	2	0.2	106	8.8
T-III	300	F2B	104	8.0	0	0	104	8.0
F2
C	none	F3A	109	7.3	9	0.6	100	6.7
C	none	F3B	126	9.7	3	0.2	122	9.4
T-I	30	F3A	91	7.6	2	0.2	89	7.4
T-I	30	F3B	100	8.3	1	0.1	99	8.3
T-II	100	F3A	124	8.9	1	0.1	123	8.8
T-II	100	F3B	114	8.1	5	0.4	109	7.8
T-III	300	F3A	127	9.1	3	0.2	123	8.8
T-III	300	F3B	141	10.8	12	0.9	129	9.9

Surviving of pups during lactation

Group	Dietary level ppm	Litter	Number of pups lactation day 1 total	Number of pups lactation day 1 mean	Number of pups lactation day 4 total	Number of pups lactation day 4 mean	Number of pups lactation day 21 total	Number of pups lactation day 21 mean
F0
C	none	F1A	174	11.6	162	10.8	104	6.9
C	none	F1B	120	10.0	113	9.4	86	7.2
T-I	30	F1A	150	11.5	145	11.2	113	8.7
T-I	30	F1B	152	12.7	150	12.5	104	8.7
T-II	100	F1A	170	11.3	169	11.3	122	8.1
T-II	100	F1B	151	11.6	150	11.5	101	7.8
T-III	300	F1A	130	10.0	114	8.8	102	7.8
T-III	300	F1B	126	11.5	99	9.0	53	4.8
F1
C	none	F2A	121	9.3	118	9.1	98	7.5
C	none	F2B	109	8.4	85	6.5	50	3.8
T-I	30	F2A	112	7.5	106	7.1	93	6.2
T-I	30	F2B	127	9.1	122	8.7	94	6.7
T-II	100	F2A	109	8.4	94	7.2	55	4.2
T-II	100	F2B	111	9.3	101	8.4	68	5.7
T-III	300	F2A	101	8.4	74	6.2	46	3.8
T-III	300	F2B	100	7.7	70	5.4	48	3.7
F2
C	none	F3A	100	6.7	87	5.8	76	5.1
C	none	F3B	118	9.1	90	6.9	73	5.6
T-I	30	F3A	89	7.4	87	7.3	72	6.0
T-I	30	F3B	98	8.2	98	8.2	90	7.5
T-II	100	F3A	122	8.7	85	6.1	51	3.6
T-II	100	F3B	107	7.6	84	6.0	53	3.8
T-III	300	F3A	108	7.7	70	5.0	23	1.6*
T-III	300	F3B	124	9.5	59	4.5	37	2.8

*significant different (p<0.05)

Table: Survival indices pups F1, F2, F3 generation

Group	Dietary level ppm	Litter	Live birth index	24 -h survival index	4 -day survival index	21 -day survival index
F0
C	none	F1A	97.3	96.7	90.0	76.5
C	none	F1B	99.2	97.6	91.9	81.1
T-I	30	F1A	99.3	100	96.7	92.5
T-I	30	F1B	98.7	99.3	98	86.7
T-II	100	F1A	100	99.8	98.8	85.3
T-II	100	F1B	98.7	100	99.3	86.3
T-III	300	F1A	97.8	98.5	86.4	97.3
T-III	300	F1B	96.2	99.2	78.0	65.4
F1
C	none	F2A	97.6	95.2	89.4	86.7
C	none	F2B	96.5	100	78	61.7
T-I	30	F2A	91.1	99.1	93.8	91.2
T-I	30	F2B	92.1	98.4	94.6	81.0
T-II	100	F2A	98.3	93.2	80.3	64.8
T-II	100	F2B	98.3	97.4	88.6	70.8
T-III	300	F2A	98.1	95.3	69.8	63.9
T-III	300	F2B	100	96.2	67.3	68.6
F2
C	none	F3A	91.7	100	87	89.4
C	none	F3B	96.8	96.7	73.8	83.9
T-I	30	F3A	97.8	100	97.8	83.7
T-I	30	F3B	99	99	99	92.8
T-II	100	F3A	99.2	99.2	69.1	62.2
T-II	100	F3B	95.6	98.2	77.1	64.6
T-III	300	F3A	96.9	87.8	56.9	32.9
T-III	300	F3B	91.5	96.1	45.7	66.1

Body weight of pups at lactation day 21

Group	Dietary level ppm	Litter	Mean body weight (g) lactation day 21
F0			male	female
C	none	F1A	38	35
C	none	F1B	39	35
T-I	30	F1A	38	36
T-I	30	F1B	38	35
T-II	100	F1A	36	33
T-II	100	F1B	31**	30
T-III	300	F1A	36	33
T-III	300	F1B	32*	30
F1
C	none	F2A	40	36
C	none	F2B	35	34
T-I	30	F2A	35**	35
T-I	30	F2B	39	36
T-II	100	F2A	35	33
T-II	100	F2B	31	28*
T-III	300	F2A	30**	25**
T-III	300	F2B	27**	27**
F2
C	none	F3A	39	35
C	none	F3B	36	34
T-I	30	F3A	43	41**
T-I	30	F3B	41**	39*
T-II	100	F3A	36	36
T-II	100	F3B	32	31
T-III	300	F3A	30**	28*
T-III	300	F3B	25**	24**

* significant different (p<0.05)

** significant different (p<0.01)