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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

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:

1989

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

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, assume 100% active ingredient

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: 77PD contained in the diet

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

92 to 93 days (depending on the day of necropsy)

Frequency of treatment:

daily (7 days per week)

Doses / concentrations

Remarks:

Doses / Concentrations:
males: 0, 100, 250, 500 ppm, females: 0, 250, 500, 750 ppm
Basis:

No. of animals per sex per dose:

10 per dose and sex

Control animals:

yes, plain diet

Results and discussion

Effect levels

1

Dose descriptor:

NOEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male

2

Dose descriptor:

LOAEL

Effect level:

250 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: slight body weight reduction (-5% at study termination), reduced body weight gain (-9%)

3

Dose descriptor:

LOAEL

Effect level:

250 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: slight body weight reduction (-11% body weight at study termination), body weight gain reduced (-23%)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Dietary Analysis:

Dietary analysis conducted during the course of this study indicate that the treated animals were presented diets which were homogeneously prepared at the intended dietary concentrations.

The nominal concentrations of Santoflex 77 in rat diet assayed were 100 ppm, 250 ppm, 500 ppm and 750 ppm for Groups II, III, IV and V, respectively. A homogeneity analysis perfomed on Groups II and V yielded mean and standard deviation values of 98.0% + 2.21 and 101% + 1.25, respectively. Analysis performed during the study yielded mean and standard deviation

values of 104% + 5.10, 99.8% + 3.29, 100% + 1.34 and 102% + 2.99 for Groups II, 111, IV and V, respectively.

Mortality:

All of the control and treated animals survived the duration of the study

Physical Observations:

Clinical observations were the type commonly seen in rodent studies. Isolated occurrences of nasal discharge, chromodacryorrhea, lacrimation, alopecia and scabs were noted in 1 or 2 males and females. These are not unusual and were not attributed to treatment.

Ophthalmoscopic Examination:

There were no ophthalmoscopic findings noted following three months of treatment which were attributed to the administration of the test substance.

Body Weights:

Mean body weights and mean body weight gains were reduced in the males treated at the 250 and 500 ppm dose levels and in the females treated at the 250, 500 and 750 ppm dose levels.

Mean body weight in grams

Males

Dose                           control               100 ppm              250 ppm              500 ppm  

weeks 1                  211.8 ± 14.0     214.2 ± 13.5      211.7 ± 14.9           207.0± 17.0

weeks 2                  267.2 ± 15.6     269.0 ± 16.6      263.8 ± 20.1          252.9 ± 23.4

weeks 3                  307.3 ± 18.1     305.8 ± 19.5      298.3 ± 24.5           285.1 ± 30.4

weeks 4                  341.9 ± 19.6     338.0 ± 27.1      327.8 ± 30.2           310.5 ± 36.5

weeks 5                  377.5 ± 22.3     368.4 ± 34.1     359.5 ± 34.6           336.7 ± 41.9

weeks 6                  398.5 ± 24.4     389.5 ± 34.6     376.9 ± 36.5           349.2* ± 36.8

weeks 7                  424.7 ± 24.4     417.3 ± 35.9      401.6  ± 41.5          373.1*± 40.7

weeks 8                  440.2 ± 27.0     433.3 ± 43.1      414.0  ± 44.7          386.9*± 42.9

weeks 9                  433.6 ± 29.1     430.1 ± 44.1      406.8  ± 45.0          382.9*± 40.4

weeks 10                456.8 ± 29.1    450.0 ± 48.4     429.2 ± 47.4          399.4*± 46.2

weeks 11                467.3 ± 32.7     466.6 ± 51.2      439.5  ± 48.0         407.3*± 45.2

weeks 12                473.9 ± 31.4     478.8 ± 50.4      452.5  ± 47.7         417.6*± 47.7

weeks 13                482.6 ± 27.3     489.1 ± 55.7      456.2  ± 43.7         422.1*± 48.4

terminal weights %:       100%                     101%              95%                     88 %

Females

Dose                           control               250 ppm              500 ppm              750 ppm  

weeks 1                  174.0 ±  8.2     169.2 ± 7.3        171.5 ± 10.0           166.7 ± 9.2

weeks 2                  197.7 ± 10.5     190 ± 9.7       193.4 ± 11.0          185.2 ± 13.0

weeks 3                  213.6 ± 11.9     203 ± 12.3      203.0 ± 10.6           195.1* ± 14.3

weeks 4                  230.6 ± 12.8     217.8 ± 13.8      214.7 ± 12.8           207.6* ± 23.5

weeks 5                  246.0 ± 17.4     230.0 ± 15.4     228.0 ± 15.7           221.5* ± 21.4

weeks 6                  254.3 ± 14.3     234.0* ± 15.5    233.3* ± 15.3         228.4** ± 20.7

weeks 7                  265.7 ± 19.1     246 ± 18.2       239.3* ± 14.6          234.7**± 24.1

weeks 8                  274.3 ± 26.3     248.6*± 16.0     242.0** ± 12.6        233.3**± 21.9

weeks 9                  272.1 ± 18.9     247.3*± 15.0      245.1**± 12.8        235.1**± 22.1

weeks 10                282.0 ± 16.2    258.8*± 14.6     256.8 *± 14.4          244.6**± 24.3

weeks 11                289.9 ± 17.3     260.5**± 14.5    257.9**± 12.7         246.3**± 24.7

weeks 12                294.2 ± 18.3     267.5*± 16.5      262.9**± 17.1         252.5**± 24.8

weeks 13                300.2 ± 17.0     268.5**± 16.8     265.9**± 15.7        256.1**± 26.4

terminal weights %:       100%                     89%              89%                           85 %

*significant different from control (p<0.05)

** significant different from control (p< 0.01)

Mean body weight gain calculated from week 0 through week 13:

Males

Dose                                 control        100 ppm       250 ppm       500 ppm            

Mean weight gain g            + 292              +298       +266              +232**      

Mean weight gain %              100%            102%      91%                80%

Females

Dose                                 control        250 ppm       500 ppm       750 ppm            

Mean weight gain g            + 138              +107**       +107**          +94**  

Mean weight gain %              100 %            77.5%         78%              68%

* *significant differenr from control (p<0.01)

The authors concluded that the differences noted in males at 250 and 500 ppm and in females at 250 ppm, 500 ppm and 750 ppm were attributed to the administration of the test substance.

Food Consumption:

In general with the exception of Week 1, the mean food consumption values of the treated males and females were comparable to control throughout the study. During Week 1 the mean food consumption values of the males in Groups III (250 ppm) and IV (500 ppm) and of the females in Groups III (250 ppm), IV (500 ppm) and V (750 ppm) were significantly reduced compared to controls. These reduced mean food consumption values were attributed to the administration of the test substance, possibly due to poor initial palatability.

Test Substance Intake:

Mean test substance intake values calculated based on individual body weight and food consumption data and nominal dose levels were as follows:

Santoflex 77 (mg/kg bw/day)

Dose       100 ppm       250 ppm              500 ppm       

Males            6.6              15.9                   32.4            

Dose       250 ppm       500 ppm              750 ppm       

Females        18.1              36                  54.5       

Laboratory Studies:

Hematology

The mean hematology data of the treated males and females was unremarkable. There were no findings which were attributed to the administration of the test substance.

Clinical Chemistry:

Several clinical chemistry parameters exhibited statistically significant differences from control which were attributed to the administration of the test substance. These included:

Males:

• Glutamic oxaloacetic transaminase mean serum level: were significantly reduced in the 100 ppm (p<0.05), 250 ppm and 500 ppm (p<0.01) males at Month 1.5 but not at Month 3 (1.5 months: 100 ppm: 108 ± 15, 250 ppm 96 ± 14, 500 ppm 90 ± 16 vs. control 136 ± 32 IU/l)
• Calcium²⁺ was slightly increaed in males of the 250 ppm and 500 ppm groups at 1.5 months but not at 3 months (1.5 months: 250 ppm: 9.9 ± 0.3, 500 ppm: 9.9 ± 0.2 mg/dl vs. control . 9.5 ± 0.3 mg/dl)
• Creatinine was significant increased (p<0.05) at 100 ppm but not in the higher dose groups at 3 months (100 ppm 0.7 + 0.1 vs. control 0.6 + 0.1 mg/dl)
• Alkaline phosphatase was significant (p<0.05) increased in males of the 500 ppm group at 3 months (500 ppm: 83 ± 17 vs. control 63 ± 14 IU/l)

Females:

• Serum Glutamic Pyruvic transaminase mean serum level: were significantly reduced (p<0.01) in females at 750 ppm at 1.5 months (750 ppm: 17 + 3 vs. control 27 + 10 IU/l) and in 500 ppm (p<0.05) and 750 ppm (p<0.01) females at 3 months (500 ppm: 23 + 4, 750 ppm: 21 + 3 vs. control 30 + 8 IU/l)
• Fastin glucose was significant reduced (p<0.05) in 750 ppm females at 1.5 months but not at 3 months (750 ppm: 95 + 11 vs. 110 + 11 mg/dl)
• total Bilirubin was significant reduced (p0.01) in females of the 500 ppm group at 1.5 months, but not at 3 months (500 ppm: 0.2 + 01 vs. control 0.4 + 0.1 mg/dl)
• Blood Urea Nitrogen: significantly elevated (p<0.01) in the Group V (750 ppm) females at 3 months (20.2 + 2.6 vs. control 15.3 + 2.2 mg/dl)
• Alkaline Phosphatase: significantly elevated (p<0.05) in Group V (750 ppm) females at 3 months (67 + 21 vs. control 40 + 9 IU/l)

The significance of the changes noted in serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase are unclear. Other sporadic differences from control (some statistically significant) were observed in the clinical chemistry data. However, there were no treatment-related findings noted in any of the other clinical chemistry parameters.

Terminal Organ and Body Weights and Organ/Body Weight Ratios:

Several organs in the treated males and females exhibited alterations in mean absolute and/or relative (to body weight or to brain weight) weight data as follows:

a) The mean brain to body weight ratio was increased in the Group IV (500 ppm) males.

b) The mean testes/epididymides to body weight ratio of the Group IV (500 ppm) males were increased.

c) The mean liver weight of the Group IV (500 ppm) males was reduced.

d) The mean brain to body weight ratios were increased in the females in Groups III (250 ppm), IV (500 ppm) and V (750 ppm).

e) The mean liver to body weight ratios of the females in Groups III (250 ppm), IV (500 ppm) and V (750 ppm) were increased.

f) The mean kidney/brain weight ratios of the Group IV (500 ppm) and Group V (750 ppm) females were slightly reduced.

g) The mean spleen to body weight ratios of the Group III (250 ppm), Group IV (500 ppm) and Group V (750 ppm) females were slightly increased.

These alterations noted in absolute and relative (to body weight and brain weight) organ weight data were generally consistent with the reductions noted in terminal body weights. These organ weight changes were considered secondary effects and were not considered indicative of significant organ toxicity.

Pathology

Various tissue alterations were observed at the time of necropsy in males and females of all groups. Microscopic assessment of the tissues revealed no histomorphological evidence of a treatment relationship. Many of the "gross lesions" were iatrogenic and associated with the orbital bleeding technique. Reddened foci in the lungs were due to inhalation of blood which had accumulated in the nasopharyngeal region. Reddened tracheobronchial lymph nodes were also associated with the bleeding technique and were related to erythrophagocytosis. Enlarged submandibular lymph nodes were often noted. The enlargement was associated with lymphoid hyperplasia and/or plasma cell hyperplasia which commonly occurs in the nodes receiving drainage from the oropharyngeal region. Fluid in the uterus, noted frequently, is a normal physiological change associated with estrus. It does not represent a pathological process. Various other miscellaneous tissue changes were observed but were not representative of a treatment effect.

Histopathology:

Microscopic evaluation of the tissues revealed no histomorphological evidence of a treatment induced or related effect. Tissues from the dietary controls were comparable histologically with those from the treated rats. Various tissue alterations were encountered; however, none was considered to have been treatment-related. All occurred with approximately equal frequency and degree in control rats as in treated animals. Occasional subintimal mineralized concretions were observed in the pulmonary veins of numerous rats. These concrements were not treatment associated but were thought to have been possibly induced through dietary imbalances of vitamin D and/or calcium. All other tissue findings were sporadic in occurrence and were changescommonly encountered in rats of this age and strain.

Under the conditions of this test it was concluded that Santoflex 77 when administered via the diet at levels of up to 500 ppm to males and 750 ppm to females for 3 months causes no histomorphological tissue alteration.

Based on the body weight data generated during this 13 week study, the authors concluded a no effect level (NOEL) for Santoflex 77 of 100 ppm in males. A no effect level was not established for the females based on reduced body weights observed at the 250 ppm dose level.