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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
10.9.2012-15.4.2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
(see Overall remarks)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
Details on test materials:
Name of test material (as cited in study report): Solvent Yellow 124
Substance type: organic
Physical state: liquid
Appearance: dark yellow viscous liquid

Composition of test material, percentage of components:
- Analytical purity: 90.0 % (w/w)
- Impurities (identity and concentrations):
4´-[2-((hydroxy)ethyl)ethylamino]azobenzene 3.0 % (w/w)
1,1-bis(N-ethyl[4-(phenylazo)phenyl]aminoethan-2-oxy)ethan 2.5 % (w/w)
- Unknown impurities 4.5 % (w/w)

Lot/batch No.: S2408
Expiration date of the lot/batch: Unlisted
Stability under test conditions: stable
Storage condition of test material: During the study the test substance was stored in glass bottle at laboratory temperature.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF Breeding VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760118
- Age at study initiation: 10 weeks – on arrival
- Weight at study initiation: (P) Males: cca 284 g; Females: cca 186g

- Selection of animals: random selection according to the internal rule – at the beginning of the study the weight variation of animals in groups of each sex should not exceed + 20% of the mean weight
Identification of animals: the animals were identified by the colour marks on their fur, each cage were marked with the number of animals, sex, number of cage, name and dose level of the test substance

- Fasting period before study: no
- Housing: SPF conditions – 2 rats of the same sex in one cage in pre-mating period, during mating period – one male and one female in one cage, pregnant females – individually, offspring – with mother, satellite animals - 2 rats of the same sex in one cage
-Bedding: sterilized soft wood shavings

- Diet (e.g. ad libitum): complete pelleted diet for rats and mice in SPF breeding (ST BERGMAN, manufacturer: Ing Miroslav Mrkvička – Výroba krmných směsí, Mlýn Kocanda No. 19, 252 42 Jesenice u Prahy. Diet was sterilised before using and was analysed for nutrients (once a year) and bacteriologically examined (every two months) on a regular basis)
- Water (e.g. ad libitum): drinking tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3°C
- Relative humidity (%): 30-70%
- Air changes (per hr): approximately 15 air changes per hour

STUDY TIME SCHEDULE
Administration: 24. 10. – 18. 12. 2012 (details below Duration of treatment..)

- Photoperiod (hrs dark / hrs light): light: 12 hour light/12 hour dark

STUDY TIME SCHEDULE
Experimental part: 10.9.2012-15.4.2013
Administration: 24. 10. – 18. 12. 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
The test substance solution was administered to the stomach by gavage. Oral way of administration was chosen according to the guideline and it was approved by sponsor. The animals were treated 7 days per week at the same time (8.00 – 10.00 am). The vehicle control group was administered by olive oil in the same volume.

PREPARATION OF DOSING SOLUTIONS:
The test substance was weighted into glass beaker and the beaker was replenished by olive oil. The solution was mixed by magnetic stirrer (650 rpm) for 60 minutes and then it was mixed continually during administration. The application form was prepared daily just before administration.

- Concentration in vehicle: The concentrations of solutions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.
- Lot/batch no. (if required): 5211201(Dr. Kulich Pharma, s.r.o.)





Details on mating procedure:
- M/F ratio per cage: 1 : 1
- Length of cohabitation:
Animals were mated from the 15th day of study (see below Duration of treatment).

- Proof of pregnancy: Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found.
- After successful mating each pregnant female was caged (how): SPF conditions – pregnant females – individually, offspring – with mother
- Any other deviations from standard protocol: see Overall remarks - with no impact on condition of animals and course of study



Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity were determined by evaluation of absorbance measured at maximum of the test substance absorbance spectra by spectrophotometric method. The method was developed at the test facility (Analytical Group I).
Duration of treatment / exposure:
Parental males:
1st day – 14th day (pre-mating) → 28th day (mating) → 42nd day of study
Parental females:
1st day – 14th day (pre-mating) → 28th day (mating) → gestation → lactation → day 4 post partum
Non-pregnant females (with evidence of copulation):
1st day – 14th day (pre-mating) → 28th day (mating) → 25th day after confirmed mating (max. 54th day of study)
Non-pregnant females (without evidence of copulation):
1st day – 14th day (pre-mating) → 28th day (mating) → 54th day of study
Satellite males:
1st day → 42nd day (administration) → 56th day (observation)
Satellite females:
1st day → 42nd day (administration) → 56th day (observation)
Frequency of treatment:
7 days per week at the same time
Details on study schedule:
- Age at mating of the mated animals in the study: 10 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
10 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
30 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12 females and 12 males per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels for study were determined on the basis of results of a dose-range finding study
(14 days, dose levels 0, 10,30 and 100 mg/kg bw/day)

- Animal assignment random

- Other:
Positive control:
None

Examinations

Parental animals: Observations and examinations:
HEALTH CONDITION CONTROL
All rats were observed pre-experimentally to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. During the administration period they were examined for behaviour changes each day before, during application and immediately after application.

MORTALITY
Twice daily

CLINICAL OBSERVATION
Males and Females
All rats were observed daily during the administration period.
This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day
(11.00 – 13.00 p.m.) – at the time of expectation of maximal effect of the test substance. Animals were observed in natural conditions in their cages.

DETAILED CLINICAL OBSERVATION
This observation was carried out before the first application and then weekly. At the first part of observation the behaviour of animals in the cage was monitored: piloerection, posture, position of eyelids, breathing, tonic or clonic movements, stereotypes or bizarre behaviour.
The second part was the observation during the removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

BODY WEIGHT
The body weight of animals was recorded on automatic balances with group mean computing module on specified days. All animals were weighed immediately before euthanasia too.
males - weekly
females - weekly in pre-mating and mating period, during pregnancy 0., 7th, 14th, 20th day, during lactation 0. or 1st, 3rd and 4th day
Weight increment was computed as a mean per group (in grams). Non-pregnant females (females without parturition) were not included in calculation of means in pregnancy and lactation period.

FOOD CONSUMPTION
In a specified day the remainder of pellets was weighed in each cage, the new food was weighed out and the food consumption for the previous week was computed.
In males mean values were calculated for each week of the study (except the mating period). Food consumption for animal/day was calculated from mean values of each group. The same way of calculation of mean food consumption was used for females in pre-mating period. In pregnancy and lactation period mean individual values (grams/animal/day) were calculated for each week of the study. Mean food consumption for each group was calculated from individual values. Nonpregnant females (females without parturition) were not included in calculation of mean food consumption in pregnancy and lactation period.
Food conversion in % (weight increment/food consumption x 100) was calculated for animals of repeated dose toxicity part of study. In pre-mating period the food consumption and conversion of females was calculated from values of all females. (Numbers of females for repeated dose toxicity part of study were chosen at the end of study).

WATER CONSUMPTION
The drinking water consumption was recorded in satellite males and females. The mean values in groups (water consumption per animal and per day) were calculated for each week of the study.
Sperm parameters (parental animals):
In all males (except the satellite group) the following sperm parameters were examined: sperm motility and sperm morphology.
Sperm motility
Sperm samples were taken from one epididymis and sperm motility was assessed from these samples. The motility of sperm was determined by microscopic examination of the prepared sperm suspension.
The result of observation was evaluated subjectively according to following grades: 1 - fast progressive motility, 2 - slow progressive motility, 3 - no progressive motility, 4 - non-motile sperm.

Sperm morphology
Sperm samples were taken from one epididymis and sperm morphology was assessed from these samples. A smear from the sperm suspension was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination.
All deviations – e.g. broken tail, abnormal form of tail, double head, amorphous head, and abnormal form of neck – were recorded.
Litter observations:
All pups were observed in natural conditions in their cages daily during the lactation. Changes in behavioural abnormalities were recorded. Detailed examination of each litter was performed as soon as possible after delivery (day 0 or 1 post-partum) and the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.
Postmortem examinations (parental animals):
SACRIFICE
Parental males: 43th day of study
Parental females: 4th day of lactation
Non-pregnant females: 55th day of study or 26th day after confirmed mating

GROSS NECROPSY
During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
Statistics:
The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis (the raw data were used for statistical analysis). This statistical analysis was used for the results of body weight, results of haematology, blood biochemistry, urinalysis, biometry of organs and selected reproduction parameters – number of live born pups, number of corpora lutea, number of implantations, mean weight of pup on the 0./1st day and mean weight of pup on the 4th day. Males/females from control group were compared with males/females from three treated groups. Satellite males/females from control group were compared with satellite males/females from treated group.
Reproductive indices:
Male mating index= (number of males with confirmed mating / number of males cohabited) x 100
Female mating index = (number of sperm-positive females / number of females cohabited) x 100
Male fertility index = (number of males impregnating a females / number of males cohabited) x 100
Female fertility index = (number of pregnant females / number of sperm-positive females) x 100
Gestation index = (number of females with live born pups / number of pregnant females) x 100
Survival index = (number of live pups on day 4 post partum* / number of pups born alive+) x 100

* without still born pups (dead pups with anaerial lungs)
+ with dead pups with aerial lungs
Offspring viability indices:
Pre-implantation loss Number of corpora lutea – number of implantations
Post-implantation loss Number of implantations – number of live births
Post-natal loss Number of live births – number of alive at postnatal day 4

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Females consuption decreased
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Females consuption decreased
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

MORTALITY
There were no unscheduled deaths during the whole study.

HEALTH CONDITION CONTROL
Males
In treated males of the lowest dose level no significant changes of health condition before, during and immediately after application of the test substance were observed. In all males of the middle dose level the anaemia of ear skin (in the 5th week of application) and yellowish colour of ear skin (in the 6th week of application) were observed. At the highest dose level the following changes were observed: anaemia of ear skin from the 1st to the 2nd week of the study and yellow colour of ear skin from the 2nd to the 6th week of study.

Females
In treated females of the lowest dose level no significant changes of health condition before, during and immediately after application of the test substance were observed. In all females of the middle dose level the anaemia of ear skin (in the 5th week of application) and yellowish colour of ear skin (in the 6th week of application) were observed. At the highest dose level the following changes were observed: anaemia of ear skin from the 1st to the 2nd week of the study and yellow colour of ear skin from the 2nd to the 6th week of study.


CLINICAL SIGNS
Males
At the lowest dose level no clinical changes after application of the test substance were observed. In all males of the middle dose level the yellowish colour of ear skin (in the 5th and 6th week of application) were observed. In two males at this dose level salivation was recorded in the last week of application. In all males at the highest dose level the increased activity and yellowish colour of skin, fur and visible mucous membranes were observed from the 3rd to the 6th week of study. At the highest dose level the salivation was observed in part of males from the 4th to the 6th week of application.

Females
No clinical changes after application of the test substance were observed in females at the lowest dose level. In all females of the middle dose level the yellowish or yellow colour of ear skin (from the 5th to the 8th week of application) were observed. In all females at the highest dose level the increased activity and yellowish colour of skin, fur and visible mucous membranes were observed from the 2nd to the 8th week of study. At the highest dose level the salivation was observed in part of females from the 5th to the 8th week of application.


BODY WEIGHT AND BODY WEIGHT INCREMENT

Males
The statistical analysis of the data revealed no significant intergroup differences.
The body weight of males of all dose levels was similar compared to control.

Females
Pre-mating mating period
The mean body weight increments of the control females and females at all dose levels were well balanced in the pre-mating period. Statistically significant differences were not detected.
Pregnancy
Females without parturition (non pregnant or aborted females) were not included in the evaluation of mean body weight increments during pregnancy.
The mean body weight increment of treated mothers at all dose levels was analogous to control mothers. Statistically significant differences were not detected.
Lactation
Only mothers (females with live pups born) were included in the evaluation of body weight increments during lactation period.
The mean body weight increments of treated mothers at all dose levels and control mothers were similar. Statistically significant differences were not detected.

FOOD CONSUMPTION
Parental males
The food conversion males of all dose levels were variable in comparison with control without treatment related effect.


Females
Pre-mating period
The mean food consumption of females at all dose levels was balanced with control females in the 1st week of pre-mating period. In the 2nd week the food consumption of females of the middle and the highest dose levels was decreased.
Pregnancy
Females without parturition (non pregnant or aborted females) were not included in the evaluation of food consumption during pregnancy.
Dose-dependent effect (decrease) manifested till 0-7 days of pregnancy than the mean food consumption of mothers at all dose levels was analogous to control mothers.
Lactation
Only mothers (females with live pups born) were included in evaluation of food consumption during lactation period.
The mean food consumptions of mothers of the all dose levels were slightly decreased than in control females.

OBSERVATION OF SPERM
Sperm motility was quite the same in control males and treated males. Presence of “non-motile sperms” was not detected.
Significantly increased presence of morphologically changed sperms was not detected in any dose level.

REPRODUCTIVE PERFORMANCE
Treated females of all dose levels were mated. Evidence of copulation was not found in one female at the control group, at the middle and highest dose levels and in two females at the lowest dose level.
The number of females achieving pregnancy in treated females was balanced with the control group. Abortion was not occurred. The duration of mating of females and duration of pregnancy of all dose level it was similar to the control.
The number of females bearing live pups and females with live pups at day 4 after parturition in females at all dose levels was similar to the control.
The numbers of corpora lutea in females of all dose levels were similar to the control. The number of implantation was slightly decreased at the middle and highest dose levels. The numbers of live pups at birth and at day 4 after parturition in females at the lowest and middle dose levels were conformable to the control group. At the highest dose level these numbers were slightly decreased against control.
No significant differences of mating and fertility indexes were observed. Survival and gestation indexes of treated groups were analogous to control.
Slightly increased pre-implantation losses were recorded at the highest dose level. Post-implantation losses and post-natal losses were similar to the control group.

BIOMETRY OF REPRODUCTIVE ORGANS
Males
The statistical analysis of the data revealed no significant intergroup differences in relative weight of reproductive organs and pituitary gland.
Absolute and relative weights of organs were similar in treated and control males.
Females
The statistical analysis of the data did not reveal significant intergroup differences in absolute and relative weights of ovaries, uterus and pituitary gland. Absolute and relative weights of examined organs were conformable to the control group.
Nonpregnant females and females with abortion were not used for calculation of means and evaluation of biometry results.

GROSS PATHOLOGY
Males: No macroscopical findings were recorded in 12-0-0-0 males.
In 12-12-12-12 males no macroscopical changes were observed in reproductive organs and pituitary gland.
All treated males showed yellowish or yellow colouring of some tissue: yellowish or yellow colour of fatty tissue in 0-12-12-12 males, yellowish or yellow colour of ear skin in 0-0-12-12 males, yellow colour of hair in 0-0-0-12 males and yellow colour of mucous membrane in forestomach in 0-0-0-12 males.
Females:
The incidence of affected females is expressed in numeric form and ranged in sequence of the dose levels of 0-10-30-100 mg/kg/day further in the text.
No macroscopical changes of reproduction organs and pituitary gland were recorded in 12-12-12-12 females.
No macroscopical findings were recorded in 12-0-8-5 females.
All treated females showed yellowish or yellow colouring of some tissue: yellowish or yellow colour of fatty tissue in 0-12-12-12 females, yellowish or yellow colour of ear skin in 0-0-4-12 females, yellow colour of hair in 0-0-0-12 females and yellow colour of mucous membrane in forestomach, stomach or yellow colour of chyme in 0-2-7-12 females.

HISTOPATHOLOGY
The incidence of affected males is expressed in numeric form and ranged in sequence of the dose levels 0-10-30-100 mg/kg/day further in the text.
In 12-12-12-12 males no histopathological changes were detected in testes, epididymis, coagulation glands and seminal vesicles.
Focal chronic inflammation was recorded in prostate gland of 1-1-1-1 males. Cysts in pituitary gland were recorded in 2-0-0-0 males.

In all females no histological changes were detected in ovaries, uterus, vagina and pituitary gland. In reproductive organs only the changes related to previous pregnancy were found.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
> 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
slightly decreased
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Number and Sex Ratio of Pups
The statistical evaluation of the number of live born pups/per litter was performed. No statistically significant intergroup differences were recorded.
The total numbers of live pups and mean number of pups per litter at the dose level 30 and 100 mg/kg/day were slightly decreased in comparison with the control.
In sex ratio no significant differences were recorded in treated groups.

DEVELOPMENT (OFFSPRING)
Presence of stillborn pups was not recorded at any groups of females.
Mortality of pup (one female) in lactation period was detected at the lowest dose level.
No differences in development of pups were observed at the control and treated groups.

BODY WEIGHT (OFFSPRING)
The statistical evaluation of mean weight of pup on the 0./1st day and mean weight of pup on the 4th day was performed. No statistically significant intergroup differences were recorded.
The mean weight of litter was slightly decreased at all treated groups.

GROSS PATHOLOGY (OFFSPRING)
The macroscopic examination was performed in all pups. In examined pups of control and at the lowest dose level no pathological findings were recorded. At the middle and highest dose levels all examined pups showed yellow colour of fatty tissue, subcutis and milk in stomach. Intensity of colouring was more marked at the highest dose level.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL (No Observed Adverse Effect Level) for the REPRODUCTION and DEVELOPMENT was established higher than 100 mg/kg body weight/day.
Executive summary:

The test substance, Solvent Yellow 124, was tested for reproduction and sub-acute toxicity using the OECD Test Guideline No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on March 22nd 1996.

Results of Reproduction Part of study

 

The oral administration of Solvent Yellow 124 to rats by gavage, the dose levels 10, 30 and 100 mg/kg/day, did not cause mortality.

The course of mating, pregnancy and lactation of parental animals, number of females achieving pregnancy, spermiogenesis and sperm parameters,biometry of reproductive organs and pituitary gland, macroscopical and microscopical structure of reproductive organs and pituitary gland of parental animals and sex ratio of pups were not adversely affected by the test substance treatment. The slight intergroup differences were considered to be of no toxicological significance.

Male ability to produce sperm that can fertilise eggs and female ability to achieve pregnancy was not significantly changed – number of females achieving pregnancy was similar in control and treated groups. The total number of live pups and mean number of pups per litter were decreased in high-dose females. Pre-implantation losses were slightly increased in females of the dose level 100 mg/kg/day but the pos-implantation and post-natal development of pups were not influenced.

Evaluation of pup weight revealed no effect on intrauterine pup growth attributable to test substance. The mean pup weight at birth was similar at all dose levels and control group. The slightly decreased mean weight of litter in treated groups was related with the decreased number of pups per litters. The body weight increments of pups at the treated groups from the birth to the 4th day after parturition were similar to the weight increment of pups in control group.