Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test guideline (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 30 January 2004 to 11 November 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: There are no deviations from the recommended guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.34 (One-Generation Reproduction Toxicity Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Phenyloxindole
- Physical state: yelllowish powder
- Analytical purity: 99.80%
- Lot/batch No.: CHAD 9902K
- Storage condition of test material: at dark and cool

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, TOP VELAZ, 165 00 Praha, Czech Republic
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: males 165 - 231 g, females 126 - 179 g
- Housing: Animals were housed in SPF animal room, 2 rats of the same sex in one plastic cage (40x25x20cm) containing sterilised clean shavings of soft wood.
- Diet (e.g. ad libitum): Complete pelleted diet for rats and mice in SPF breeding (ST 1 BERGMAN), producer: Mill Kocanda, Jesenice by Prague was used. Diet was sterilised before using.
- Water (e.g. ad libitum): Free access to drinking water (water ad libitum). Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Health Ministry. Water was sterilised before using.
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3ºC
- Humidity (%): 30-70%
- Air changes (per hr): 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12-hour light/12 hour dark cycle.


IN-LIFE DATES: From: 30 January 2004 To: 08 July 2004

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The concentrations of suspensions in all three-dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. The application form (suspension in olive oil) was prepared daily just before administration. The vehicle control group was administered by olive oil in the same volume per 100 g of body weight. The application form of the test substance was prepared daily before administration and during the administration it was mixed by magnetic stirrer.
Details on mating procedure:
Females (30F per dose level) were paired on two-to-one basis with males from One generation study (5M) and with males from the same treatment group of 90-day study (10M).
Examinations:
Mating period between the 11th and the 13rd week of study
Presence ofsperm: vaginal smears - each morning during the mating period
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability was monitored in olive oil suspension under continual mixing. The mixture was mixed all the time of monitoring. The measurement was performed on two concentration levels (3 and 30 g/L) in 5 time intervals (0, 30; 60 and 120 minutes and 24 hours).

The homogeneity of application form was monitored by the analyses of olive oil suspension in three sampling spots (the bottom, the middle and the surface of container content). Measusement was performed on iwo concentration levels (3 g/L and 30 g/L). The mixture was mixed all the time of monitoring.
Duration of treatment / exposure:
133 days
Frequency of treatment:
Seven days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
30, 100 and 300 mg/kg body weight/day
Basis:
actual ingested
No. of animals per sex per dose:
90-day study: ten males and ten females in each group
One generation study: ten males from 90-day study + five additional males and 20 mothers recruiting from thirty females of mating group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses for the were chosen with respect to the results of 28-day toxicity study.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: Mortality/viability


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: females were weighed on the first day of exposure and weekly thereafter, mated females were weighed on days 0, 7, 14,21 of gestation, and during lactation on days 1, 4, 7, 14, 21.


FOOD CONSUMPTION: Yes
- Time schedule: weekly; during the mating period the analysis of food consumption was suspended; food consumption of mated females was measured on gestation days 0, 7, 14, 21 and during lactation on days 1, 4, 7, 14 and 21.


WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly


Sperm parameters (parental animals):
In all males of all groups surviving to scheduled necropsy the sperm parameters were examined: sperm motility and sperm morphology.
Litter observations:
PARAMETERS EXAMINED:
The following parameters were examined in F1 offspring:
- Number of live pups: daily. On day 4 after birth the size each litter was adjusted by eliminating extra pups to yield, as nearly as possible, four males and four females per litter. Whenever the number of males or female pups prevented to have four of each sex per Iitter, partial adjustment (for example, five males and three females) was done. Adjustments were not applied to litters of less than eight pups.
- Body weight of pups: per litter - 1st and 4th day; individually - 7th, 14th and 21st day
- Behaviour of pups: 1st, 7th, 14th and 21st day. Development of pups - individual parameters and behavioural abnormalities, was observed in
detail, in scheduled days: determination of sex, ability of sucking, growth of hair, teeth, and dugs, opening of eyes and ears.


GROSS EXAMINATION:
Pathological examination: All pups after termination of experiment on 21st day of pups age
Postmortem examinations (parental animals):
GROSS NECROPSY
Males: The five males for One generation study of each group was subjected to macroscopic examination of the cranial, thoracic and abdominal. tissues and organs. Non-pregnant females were examined only for gross findings.

Females: After sacrifice or death all females were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
Males: Samples of the following tissues and organs were collected and fixed in neutral phosphate buffered 4% formaldehyde solution for further microscopic histopathological evaluation: all gross lesions, coagulation gland (fixed in Bouin's), pituitary gland, prostate gland, seminal vesicles and
testes (fixed in Bouin's). The reproductive organs of all males of all groups were examined microscopically.

Females: Samples of the following tissues and organs were collected from all parental females and fixed in neutral phospliate buffered 4% formaldehyde solution (v/v): all gross lesions, cervix, ovaries, pituitary gland, utems, vagina. Ovaries, pituitary gland, uterus with oviducts and cervix were examined histopathologically.
Postmortem examinations (offspring):

GROSS NECROPSY
- Offspring found dead or killed before day 14 of lactation were be sexed and externally examined If practically possible, the stomach was examixied for the: presence of milk.
- Offspring found dead or killed on or after day 14 of lactation were sexed and subjected to external examination of the cranium, and macroscopic examination of the tlioracic and abdominal tissues and organs
- Other pups on day 21 post partum.


Reproductive indices:
Percentage mating, fertility index, conception rate, gestation index, percentage of live males at first litter check, percentage of live females at first litter check
Offspring viability indices:
Percentage of postnatal loss days 0-4 post partum, percentage of breeding loss day 5 until weaning, percentage live males at weaning, percentage live females at weaning, viability index, weaning index.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality occurred during the study period.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
MALES: The body weight increments at all dose levels and a control group were comparable and had an increasing trend.
FEMALES: The body weight increments at all treated groups and control group were comparable during the study. The body weight of mothers in all treated groups during the gestation and lactation was similar with the body weight of mothers in control group.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
MALES: Food consumption of males in all treated groups had a decreasing trend. Food consumption was similar in animals of control group and at dose levels 100 and 300 mg/kg; at dose level 30 mg/kg the food consumption was lower as compared to others.
FEMALES: Food consumption was similar in all treated groups and the control group. The food consumption in all treated groups of mothers duriiig gestation and lactation was similar with the control group.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No treatment related effects on sperm motility and morphology were observed.
Sperm morphology: Major sperm abnormalities observed in males were amorphous head, abnormal form of tail and abnormal form of neck. Lowest number of abnormalitles was found in males of dose level 300 mg/kg. In the dose level 30 and 100 mg/kg aspermia for 1 male was found out. In the control group and dose level 300 mg/kg no aspermia was recorded.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The differences among the groups were very low. The number of females pregnant is very similar at all treated group and higher against the
control group. Fertility index, gestation index and conception rate were higher in all treated groug compared to control.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Males: Examination of the external surface of the body revealed no changes. In thoracic cavity no changes were observed. In abdominal cavity the following affections were diagnosed: marked structure and discolour of liver. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered as changes of no toxicological significance.
Females: Examination of the extemal surface of the body revealed no changes. In thoracic cavity no changes were observed. In abdominal cavity the following affections were diagnosed: marked structure of liver and dilatation of uterine horns by pellucid Iiquid. Macroscopic findings did not relate to results of microscopic examinations. Above mentioned findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered as changes of no toxicological significance. Non-pregnant females were examined only macroscopically. No pathologic alterations were recorded.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Males: Incidence of affected animals is expressed in numeric form and ranged in sequence of dose levels 0-30-100-300 further in the text.
Incidence of histopathological affections of reproductive system in male genital tract was sporadic (testes dystrophy 0-0-1 -0 and epididymide vacuolation 1-0-2-0). Histopathological findings in prostate gland were more often: focal inflammation in 1-2-4-3 males, atrophy in 1-0-2-3 males and oedema in 2-0- 1-11 males. Cysts of pituitary gland wese found out in 2-1-0-0 males.
Females: Pathological affections of reproductive system in female genital tract were detected in ovary: follicular cysts 3-3-5-9, ovary luteal cysts 0-0-0-1, ovary Sertoli-like tubules 5-5-4-2. In uterus the following affections were found: uterus cystic changes 5-1 -2-2, uterus retentio deciduae 6- 1 1-7-9, cervix uteri retentio deciduae 0-3-0-2, uterus hydrometra 2-1-4-2, cervix uteri cystic changes 0-0-3-1 and cervix uteri hydrometra
2- 1-3- 1. Also some pituitary gland cysts 1-3 -2- 1 were detected.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: The series of effects observed at the dose levels 300 mg/kg/day in the One generation study (reproduction) could not be denoted as negative.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
The total number of pups was the highest at dose level 300 mg/kg. Number of pups at dose levels 30 and 100 mg/kg were very similar and also higher than at control group. On day 4 after birth the size of each litter was adjusted by eliminating extra pups to yield, as nearly as possible, four males and four females per litter, Whenever the number of male or female pups prevented to have four of each sex per litter, partial adjustment (e.g. five males and three females) was done. Adjustment was not applied to litters of less than eight pups. The number of pups per litter in all treated groups was higher than in the control group. The highest number of pups was found out in dose level 300 mg/kg.

CLINICAL SIGNS (OFFSPRING)
Incidence of affected animals is expressed in numeric form and ranged in sequence of dose levels 0-30-100-300 further in the text.
No differences in development of pups were observed in treated groups in comparison with control group.
Death of pups was similar in all groups:
- during 1st day after birth the 6-0-8-4 pups died in single groups
-until 4th day after birth the 1-1-5-3 pups died in single groups
- 7th and 21st day after birth no death of pups was observed

BODY WEIGHT (OFFSPRING)
The body weight increment of pups in all treated groups was similar with the control group and had an increasing trend.
The lowest birth-body weight of pups at dose level 300 mg/kg was recorded on 1st day. The body of pups on 21st day was similar in all groups.

GROSS PATHOLOGY (OFFSPRING)
The pathological examination was performed in all pups survived to scheduled necropsy. Macroscopic examination of the thoracic and abdominal tissues and organs were carried out. Sacrificed pups in all treated groups had no macroscopic pathological changes.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No effects observed at the dose level 300 mg/kg/day.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 7.8.1-1: Reproduction parameters

Dose level

Control

30 mg/kg bw/day

100 mg/kg bw/day

300 mg/kg bw/day

Males paired

15

15

15

15

Females paired

30

30

30

30

Females mated

28

26

29

27

Females pregnant

21

26

25

26

Mothers (reproduction study)

20

20

20

20

Mothers pregnant

17

16

16

17

Mothers bearing live pups

16

16

16

17

Table 7.8.1-2: Fertility parameters

Dose level

Control

30 mg/kg bw/day

100 mg/kg bw/day

300 mg/kg bw/day

Percentage mating

93.3

86.7

96.7

90.0

Fertility index

70.0

86.7

83.3

86.7

Conception rate

75.0

100.0

86.2

96.3

Gestation index

94.1

100.0

100.0

100.0

Applicant's summary and conclusion

Conclusions:
It Is possible to conclude, that the test substance Phenyloxindole has predominantly positive effects on organism of experimental animals, especially on some reproduction parameters: increased number and body weight of pups, decreased incidence of abnormalities of sperms. The series of effects observed at the dose levels 300 mg/kg/day in the One generation study could not be denoted as negative. Based on the results of the study, the NOAEL for the One generation study (reproduction) was established as 300 mg/kg body weight/day.
Executive summary:

The test substance, Phenyloxindole, was tested in combined Repeated Dose 90-day Oral Toxicity/One-Generation Reproduction Study in rats. The male rats exposed for ten weeks during the 90-day toxicity study were used for mating with the females exposed for two weeks at the same dose level during the sne-generation seproduction study. The treatment period for the males and females in 90 -day toxicity study was at least 13 weeks (10 weeks of pre-mating, at least 1 day of mating, 3 weeks of post coitum). The treatment period for the females in one-generation reproduction study was at least 8 weeks (2 weeks of pre-mating, at least 1 day of mating, 3 weeks of post-coitum - gestation, 3 weeks of post partum - lactation). For the one-generation reproduction study 120 female rats were used (30 females for each dose level). The test substance was administered in olive oil by stomach tube; oral application was made daily. The 4 groups of animals were included in the study - 3 treated groups (doses 30, 100 and 300 mg/kg body weight/day) and one control group (vehicle only). Clinical observation and health status control were performed daily. The body weight, food consumption were measured weekly as well as the detailed clinical observation was carried out. Water consumption was measured twice a week. Vaginal smears were prepared daily after pairing (until presence 0-6 spermatozoa), observations of sperms (motility, morphology), and parameters relevant to pups were also carried out. During the study there were observed no treatment related changes in rnortality, health condition, body weight, food consumption, sperm motility and morphology, development of pups and pathological examination of pups. Treatment-related fíndings were as follows: At all dose levels increased body weight of parents after mating, higher fertility index, conception rate and gestation index were recorded. Increased incidence of retentio deciduae in uterus and cervix uteri and cysts of ovary were observed in mothers of all dose levels. Increased number of pups and their body weight on day 21 were recorded. Decreased incidence of sperm abnormalities in males was recorded. Based on the results the NOAEL for the One generation study (reproduction) was established as 300 mg/kg body weight/day.