Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP and guideline study (OECD TG 421), study design with limitations (e.g. low dose group with 7 pregnant females was below the minimum acceptable number of pregnant females (8 females per group) according to TG 421; because of the limited number of pregnant females used in this study (number of pregnant females: control: 8, low dose: 7, mid dose: 8, high dose group: 10) a meaningful evaluation of the potential of the substance to affect fertility, pregnancy, maternal and suckling behavior, and growth and development of F1 offspring from conception to day 4 post-partum is limited. The study should be used only for supporting reasons.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Number of pregnant females used in this study is under the minimum acceptable number of pregnant females required to assure a meaningful evaluation of the potential of the substance to affect fertility, pregnancy, maternal and suckling behavior, and growt
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Dusantox IPPD, purity: 97.73%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on mating procedure:
- M/F ratio per cage: 1:1
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
males: 14 days pre-mating, mating, 14 days postmating; females: 14-pre-mating, mating, during pregancy to day 3 of lactation
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 20, 50, 125 mg/kg bw/day
Basis:

No. of animals per sex per dose:
10 per sex and dose
Control animals:
yes, concurrent vehicle

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: sporadic clinical signs
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: clinical signs, males: body weight reduction, females: 2 aborted females

Results: F1 generation

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: sporadic pathological findings
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
125 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: postnatal decreased viability index, pathological findings (haematoma 3 pups, stomach full of pellucid liquid one pup)

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Mortality:

Males: no death occured during the study.

Body weight and body weight gain: body weights of low dose males (20 mg/kg bw) were comparabe or even slightly decreased compared to control, mid dose and high dose males showed a decrease in body weights compared to control, at study termination the body weights of high dose males were significant reduced compared to control (p<0.05)

Body weight gain: a negative body weight gain was noted in mid dose males (50 mg/kg) in week 3 of application.

Table: males terminal body weights

            Average body weight (grams/animal/week)
            Dose level
 Application period  0  20  50  125
 study termination  376.9± 23 -09  368.2±30.88  350.3±26.13  327.7±23.40*

*statistically significant (p<0.05)

Food consumption:

Males: food consumption was slightly reduced in all treated males compared to the concurrent vehicle control.

Table: food consumption, males

          Average food consumption per dose group
            Dose level
 Application period  0  20  50  125
 week1  21.8  17.37  18.03 16.89 
 week 2  22.32  20.78  17.65  17.23
 week 3 + week 4:  mating period         
 week 5  18.94  16.26  16.65  17.39
 week 6  17.97  14.86  12.86  16.18

Clinical signs:

2/10 low dose males showed red/brown secretion around nostrils, no other findings observed. 1/10 male of mid dose group showed sporadic salivation and 3/10 piloerection at week 6. The authors concluded that these findings were incidental and did not effect all males from this treatment group. Clinical signs related to test substance application were observed in high dose group males (125 mg/kg). The clinical signs observed were diarrhoea (sporadic 1/10), red or pink secretion around nose or eyes (sporadic 1 to 2/10), piloerection (sporadic 3 to 10 animals) and salivation (2 to 3 animals).

Macroscopic examination:

No changes in external surface of the body were noted in any of the evaluated males. No changes were revelaed in thoracic cavity. Local change in lungs (punctiform stratum) was noted in one male of the mid dose group. Sporadic pathological changes in abdominal cavity were observed in control males and treated males (sporadic findings: reduced testes, epididymis or seminal vesicle, changed colour of testes, marked structure of liver, changes of stomach mucosa) A higher incidence of whitish strata was noted in high dose males. The authors suggested that these findings were not treatment-related.

Table: macroscopic findings, males

          Macroscopic findings
            Dose level
 Pathological findings  0  20  50  125
 Number of examined animals  10  10  10  10
 epididymis reduced  1  1  0  0
 testes reduced  1  2  0  0
 testes prominated# whitish strata  0  1  0  4
 testes violescent# colour  1  2  0  0
 seminal vesicle reduced  0  0  0  1
 stomach haemorrhage  0  0  1  0
 lungs local changes  0  0  1  0
 liver market structure  0  0  2  0
 without changes  9  7  7  5

Organ weights: organ weights of testes,epididymis, prostate glands and pituitary glands were comparable to control. Table: organ weights, males                                                                                          

# wording study report

Organ weights: organ weights of testes,epididymis, prostate glands and pituitary glands were comparable to control.

Table: organ weights, males

                        Average organ weights
 Dose level  0     20     50     125   
   Absolute weight (g)  Relative weight  Absolute weight (g)  Relative weight Absolute weight (g) Relative weight   Absolute weight (g) Relative weight 
 Testes  3.5030  0.9285  3.3960  0.9204  3.3360  0.9546  3.4530  1.0554
Epididymis   0.6450  0.1771  0.6910  0.1878 0.6564   0.1877  0.5930  0.1797
 Pituitary glands  0.0090  0.0024  0.0091  0.0025  0.0084  0.0024  0.0082 0.0025 

no standard deviation documented, no raw data availabe

Sperm evaluation: No treatment related effect on sperm motility and sperm vitality were observed. Changes in sperm morphology was observed in the low dose group (increased incidence of flattened head and bent neck); whereas in the mid and high dose groups morphology of sperm was comparable with control. The biological relevance of the findings in the low dose group are questionable, because of the absence of a dose-response relationship.

Table: evaluation of sperm

         observations
            Dose level
 Parameters  0  20  50  125
 Sperm motility (?)  1.5  1.5  1.5  1.4
 Sperm vitality (% live sperms)  88  84  82.2  84.4
 Sperm morphology (% affected sperms)  9.9  15.7  6.7  7.3
  - Flattened head  3.5  5.7  3.1  4.0
  - Flattened head + bent neck  0 4.8   0  0
  - Bent neck  6.1  4.2  1.7  2.4
  - Bent tail  0.3  1.0  1.9  0.9

no standard deviation given, no raw data given histopathology:

no standard deviation given, no raw data given

Histopathology:

Incidences of histopathological changes of male reproduction organs were sporadic in control and low dose group, higher incidences of changes were observed in mid and high dose males. Inflammation of prostate gland was observed in control and treated animals, however, the incidences were increased in mid and high dose males (control: 1/10, low: 1/10, mid: 5/10, high 3/10). Insignificant damage of spermiogenesis was noted in control and treated males. The incidence was increased in high dose males

Atropic changes of germinative epithelium was observed in control and treated animals, however the incidences were slightly increased in high dose males (control: 1/10, low: 2/10, mid: 1/10, high: 3/10); whereas the severity of findings showed no dose dependency

control: testes: 1/10 atrophy of germinative epithelium with only Seratoli cells

low dose: testes: 1/10: dystrophy of germinative epithelium (75%), 1/10 dystrophy of germinative epithelium (5% ) (+: insignificant damage)

mid dose: testes: 1/10 sporadic dystrophy of germinative epithelium (+: insignifcant damage)

high dose: testes: 2/10 atrophy of germinative epithelium in some tubes (within 5%) (+: insignifcant damage), 1/10 degeneration of germinative epithelium (+: insignificant damage)

No atrophy of germinal epithelium was observed in a very well documented subchronic feeding study with rats (Monsanto Co.1990) after treatment with IPPD. Moreover, in a 90 day gavage study (Duslo 2009) done in the same laboratory as the screening test, atropy of the testes were seen in low incidences in control (1/10), low (1/10) and mid dose (1/10) males treated with IPPD, no atrophy of testis was observed in high dose males (0/10).

 

Table: histopathologic findings, males

        Findings
            Dose level
 Histopathological findings  0  20  50  125
 number of animals examined  10  10  10  10
 without pathologic changes  6  5  4  4
 prostate gland inflammation  1  1  5  3
 prostate gland oedema of interstitium  0  1  1  0
 epididymis aspermia or hypospermia  1  1  0
 testes focal dilation of vasa  0  1  0  0
 testes atrophic changes of germinative epithelium  1  2  1  3
 testes oedema of interstitium  1  1  0  0
 pituitary gland dystrophy  0  0  0  1
 insignificant damage of spermiogenesis  2  0  1  5
 slight damage of spermiogenesis  0  0  0  0
 moderate damage of spermiogenesis  0  1  0  0
 important damage of spermiogenesis  1  0  0  0

Females

Females: no death occured during the study.

Body weight:

Pre-mating: No significant changes in body weights were noted in treated females compared to control

Pregnancy: A slight decrease in body weight gain was noted in mid and high dose females at the end of week 1 of pregancy.At the end of week 2 a slight increase in body weight gain was noted in mid dose females.

Lactation: a slight decrease in body weight and body weight gain was noted in low, mid and high dose females compared to control.

Table: average body weights, females

          Average body weight (grams/animal/time period)
            Dose level
 Application period  0  20  50  125
 Pre-mating:Before application 178.3± 11.79 176.2±11.00  173.6±12.98 174.9±9.43
 Premating: 1st week  189.3±12.09  180.9±11.35  184.2±12.96  187.3±12.66
 Pre-mating: 2nd week  197.8±11.27  194.9±9.25  192.7±11.97  196.9±13.30
 Pregnancy day 0  209.8±8.87  199.2±10.48  199.9±17.01  206.7±12.29
 Pregnancy day 7  227.6±9.89  215.3±13.49  210.4±16.36 220.7± 10.00
 Pregnancy day 14  249.6±9.78  236.8±13.17  237.0±18.55  241.9±11.00
 Pregnancy day 20  298.3±13.47  282.0±11.00  288.8±23.01  293.0±20.60
 Day 0 of lactation  238.5±14.85  224.0±16.40  226.7±27.10  224.6±20.19
 Day 4 of lactation  257.9±11.19  238.6±15.17  241.7±27.72  242.1±21.15

Food consumption:

Pre-mating period: the food consumption of treated females were slightly lower compared to control.

Pregancy: food consumption of treated females was slightly lower compared to control

Lactation: food consumption of treated females was lower ( low: -22%, mid: -32%, high dose: -21%) compared to control

Table: average food consumption, females

          Average food consumption per dose group (gram/animal/day)
            Dose level
 Application period  0  20  50  125
 pre-mating: 1st week  15.06  12.12  12.61  12.72
 pre-mating: 2nd week  15.70  14.32  11.87  13.73
 pregnancy day 7  15.69  14.04  12.47 14.92 
 pregancy day 14  20.98  18.29  15.47  17.39
 pregancy day 20  21.13  16.33  19.01  18.23
 day 4 of lactation  32.95  25.85  22.42  26.01

Clinical signs:

No clinical signs were noted in females of the low dose group throughout the study. In females of the mid dose group sporadic occurrence of piloerection, secretion around nostrils or closed eyes were observed from week 4 of application. Clinical signs were noted in high dose females from week 2 of application. The clinical signs observed were piloerection (10/10), red or sorrel secretion (3/10) around nostrils, hunch posture2/10, flabby body (2/10) or apathy (1/10).

Macroscopic examination:

Examination of the external surface of the body revealed no changes. In thoracic cavity no changes were observed. No changes of abdominal cavity were noted in control animals and animals of the low and mid dose group. Sporadic changes were observed at 125 mg/kg. One female of the high dose group had a cyst around ovary. Dilatation of uterus was observed in one female of the control group and in two females of the low dose group.

Table: macroscopic findings, females

          Macroscopic findings
            Dose level
 Pathological findings  0  20  50  125
 Number of animals  10  10  10  10
 Uterus dilatation  1  2  0  0
 ovary cyst  0  0  0  1

Organ weights (females)

Nonpregnant females and females with abortion were used for calculation of means and evaluation of organ weights.

No treatment related effects were noted in organ weights.

Table: organ weights, females

                      Average organ weights
 Dose level  0     20     50     125   
   Absolute weight (g)  Relative weight  Absolute weight (g)  Relative weight Absolute weight (g) Relative weight   Absolute weight (g) Relative weight 
 Ovaries  0.1000  0.0388 0.0964   0.0405  0.1105  0.0454  0.0988  0.0417
 Uterus  0.6913  0.2682  0.5700  0.2392 0.6350   0.2614  0.5875  0.2508
 pituitary gland  0.0125  0.0049  0.0125 0.0052   0.0116  0.0047  0.0124  0.0052

Histopathology:

In reproductive system in ovaries and uterus sporadic changes were noted. Folicular cysts of ovaries were observed in low incidences in treated animals (low: 2/10, mid: 1/10, high: 1/10). Luteal cysts were observed in one low and one mid dose group female. Hyperplasia of interstitial glands were noted in 1 control female and two high dose females; degeneration of follicles were seen in one mid dose female. In uterus dystrophy of ateries attended by infiltration of histiocytes were observed in two high dose females.Hydrometra of uterus were noted in one control and one low dose female.

Offspring:

Number of pups and sex ratio:

the total number of pups and average number of pups per litter in all treatment groups were comparable to control at parturition. Four day after parturition a signifcant decrease in number of pups and average number of pups per litter were noted in the high dose group (death of two litters). No treatment related effects were observed in sex ratio, however a slight decrease in females were observed at 50 mg/kg bw/d.

Table: number and sex ratio of pups

                        Number of live pups and sex (average per litter)
 Dose level  0     20     50     125   
 Day of study  total number(average) number of males and females   total number (average) number of males and females  total number (average)  number of males and females  total number (average)  number of males and females 
 0 parturition (first check of litter)  81 (10.13  4.75M/5.38F  69 (9.86) 4.86M/5.00F  66 (8.25)   5.25M/3.00F  89 (11.13)  5.00M/6.25F
 day 4 of lactation  81 (10.13)  4.75M/5.38F  69 (9.68)  4.86M/5.00F  65(8.13)  5.13M/3.00F  53(8.83)  4.17M/4.67F

F: females, M: males

Body weight

The average body weight of pups at 20 and 50 mg/kg bw/d was comparable to control. A slight decrease of average body weight was observed at 125 mg/kg bw at study termination.

Table: average body weights pups

                     Average body weight (grams)
 Dose level  0     20     50     125   
 Day of study  Litter Pup Litter Pup Litter Pup Litter Pup
 0 parturition (first check of litter)  60.94  6.03  59.53  5.80 55.63 6.00  63.09 5.44
 Day 4 of lactation  105.71  10.58  102.43  10.57  86.36  10.72 80.57   9.11

Mortality:

No death occured in control. Four still born pups occurred in 1 low dose dam. 9 dead pups 82 from dam #122, 7 from dam #130) were observed at parturition in the mid dose group (50 mg/kg bw/d); 5 of the 9 were still born pups. One pup died within lactation period. In the high dose group two aborted females were noted, two dams had still born pups (2 per dam), one pup from another dam died after birth. Two litters from the high dose group died during lactation period; in addition 10 pups from another dam of the high dose group died.

No differences in development were observed in pups from control and treated animals.

Macroscopic examination

Gross pathological examinations were performed for all pups. All died new-born pups were examined for presence of air in lungs and divided in stillborn and live birth pups. No pathological findings were recorded in control and low dose pups. Sporadic pathological findings were made in mid dose group pups (stomach without milk 4 pups). Changes were observed in pups of the highest dose group (haematoma 3 pups, stomach full of pellucid liquid one pup).

Reproduction parameters:

All females from control and treamtent groups showed evidence of copulation. The number of pregant females in control and low and high dose females were comparable. The number of pregant females in the high dose group was slightly higher as compared to control. Duration of mating was comparable in control and treated females. The duration of pregancy in the low and mid dose group was comparable to control.

high dose group: An increase in pregnancy period in high dose group females was discussed by the authors (control: 22.25 days, low: 22.67 days, 22.71 days, high dose: 22.75 days). The relevance of the finding is questionable, because the number of pregnant rats evaluated was very low (6 to 8 females per group, for single females the duration of pregnancy could not be determinate). In addition, no historical control data are given.

Pre- implantation loss and post-implantation loss were comparable in control and treated animals. A significant decrease in viability index was observed in pups of the high dose group resulted from high post partum mortality in high dose pups.

Table: reproduction data

             Reproduction data
 Evaluated parameters           Dose level
   0  20  50  125
 Pairs started (N)  10  10  10  10
 Females showing evidence of copulation (N)  10  10  10  10
 Females archieving pregnancy (N)  8  8  10
 Pregnancy < 21 days (N)  1  0  0  0
 Pregnancy 22 days (N)  4  3  3
 Pregnancy > 23 says (N)  3  3  4  6
 Duration of pregnancy not determined (remark:difficulties in test performance?)  0  1  1  0
 Average duration of pregnancy (days)  22.25  22.67  22.71  22.75
 Females with live pups born (N)  8  7  8  8
 Females with live pups at day 4 after parturition (N)  8  7  8  6
 Corpora lutea/pregnant females (average)  22.25  19.57  15.75  18.10#
 Implants/pregnant females (average)  11.25  10.86  10.5  11.30
 Live pups/mother at birth (average)  10.13  9.86  8.25  11.13
 Live pups/mother at day 4 after parturition (average)  10.13  9.86  8.13  8.83
 Abnormal pups        
Mother with 0 (N)   8  7  7  6
 Mother with 1 (N)  0  0  0  0
 Mother with > 2 (N)        
             Fertility parameters
 Percentage of mating (%)  100  100  100  100
 Fertility index  80  70  80  100
 Conception index  80  70  80  100
 Gestation index  100  100  100  80
 Percentage of postnatal loss  0  0  7.6  41.6
 Viability index 100   100  92.9 58.9 
 Loss of offspring        
 Pre-implantation (corpora lutea minus implants)        
 Pregant females with 0 -5 (N)  0  2  5  3
 Pregnant females with 6 - 10 (N)  3  2  0  5
 Prenant females with 11 - 15 (N)  4  3  3  2
 Pregnant females with >16 (N)  1  0  0  0
 Pre-natal/post-impantations (impants minus live births)        
 Pregnant females with 0 (N)  4  4  3  3
 Pregnant females with 1 (N)  2  1  0  1
 Pregnant females with 2 (N)  0  1  3
 Pregnant females with > 3 (N)  2  1  2  2
 Post-natal (live births minus alive at post-natal day 4)        
 Pregnant females with 0 (N)  8  7  6  5
 Pregnant females with 1 (N)  0  0  1  0
 Pregnant females with 2 (N)  0  0  0  0
 Pregnant females with > 3 (N)  0  0  1  3

#  (value calculated from raw data)

Applicant's summary and conclusion