Sodium 1-amino-9,10-dihydro-4-[5-[(2-hydroxyethyl)sulphamoyl]-3,4-xylidino]-9,10-dioxoanthracene-2-sulphonate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008, L 142, Appendix Part B, May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-​Anthracenesulfonic acid, 1-​amino-​9,​10-​dihydro-​4-​[[3-​[[(2-​hydroxyethyl)​amino]​sulfonyl]​-​4,​5-​dimethylphenyl]​amino]​-​9,​10-​dioxo-​, sodium salt (1:1)

IUPAC Name:

2-​Anthracenesulfonic acid, 1-​amino-​9,​10-​dihydro-​4-​[[3-​[[(2-​hydroxyethyl)​amino]​sulfonyl]​-​4,​5-​dimethylphenyl]​amino]​-​9,​10-​dioxo-​, sodium salt (1:1)

Constituent 2

Reference substance name:

Sodium 1-amino-9,10-dihydro-4-[5-[(2-hydroxyethyl)sulphamoyl]-3,4-xylidino]-9,10-dioxoanthracene-2-sulphonate

EC Number:

247-269-2

EC Name:

Sodium 1-amino-9,10-dihydro-4-[5-[(2-hydroxyethyl)sulphamoyl]-3,4-xylidino]-9,10-dioxoanthracene-2-sulphonate

Cas Number:

25797-81-3

Molecular formula:

C24H23N3O8S2.Na

IUPAC Name:

sodium 1-amino-4-({3-[(2-hydroxyethyl)sulfamoyl]-4,5-dimethylphenyl}amino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Specific details on test material used for the study:

Name: FAT 20033/L
Common Name: TECTILON BLUE 4R CRUDE MOIST (LAB DRIED) Acid Blue 277
Chemical Name: sodium 1-amino-9,10-dihydro-4-[5-[(2-hydroxyethyl) sulphamoyl]-3,4-xylidino]-9,10-dioxoanthracene-2-sulphonate
Batch No.: AT-PD13-359A1
CAS No.: 25797-81-3
Physical State / Colour: solid / dark blue
Storage Conditions: room temperature, protected from light
Molecular Weight of Base Form: 544.58 g/mol
Molecular Weight of Salt Form: 567.57 g/mol
Active Components: >95 %
Analysation Date: 07 January 2014
Expiry Date: 07 January 2019
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System:
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: males: 9-10 weeks old, females: 9-10 weeks old.
Body weight at the allocation of the animals to the experimental groups:
males: 248 - 281 g (mean: 267.83 g, ± 20 % = 214.26 – 321.39 g)
females: 171 - 202 g (mean: 184.33 g, ± 20 % = 147.46 – 221.19 g)
The animals were derived from a controlled full barrier-maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on
Animal Welfare: the animals were bred for experimental purposes.
The animal study was authorized by local government under file no. 55.2-1-54-2532.2-11-11.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 131113)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Animals were housed in groups of 2 animals/ sex/ cage in IVC cages (type III H, polysulphone cages) during the premating period in both males and females and during postmating period in males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period and males were returned to its original cage. Each cage was provided with Altromin saw fibre bedding (lot no. 1526)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions


Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. None of the animals showed any adverse clinical signs before the study initiation.
Before the first administration, all animals to be used for the study were weighed. Mean body weight of the group housed animals was used to assign all animals to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively, while ensuring to keep each animal with its initial cage partner if possible.
Each animal was marked with its identification number by individual ear tattoo.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test item was weighed into a tared plastic vial on a precision balance and suspended in aqua ad injectionem. Homogeneity of the test item in the vehicle was maintained by using an ultra turrax. The test item formulations were prepared once in every ten days and stored at 2-8°C. Every day before the dose administration the formulation samples were allowed to reach the room temperature and the homogeneity was ensured by vortexing the sample on vortex machine. Since little or no toxicity was anticipated for the test substance, the highest dose level was set at 1000 mg/kg bw/d corresponding to a limit dose for this study. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL. The animals in the control group were handled in an identical manner to the test group subjects and received aqua ad injectionem using the same volume as used for the dose groups. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured. The administration volume was 5 mL/kg body weight.

Details on mating procedure:

Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the pregnancy. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation. The cages were arranged in such a way that possible effects due to cage placement were minimized.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing concentration was analysed with respect to the target nominal concentration. Stability and homogeneity of the test item in the vehicle were analysed for the low and high dose concentrations. Samples for the nominal concentration verification were taken in study week 1 (first week of pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation/lactation) from all groups (16 samples). Samples for homogeneity analysis were taken from the top, middle and bottom of the high dose and the low dose formulation in study week 1 and 5 (12 samples). Samples for stability analysis were taken in the first week of the study [0 hours, 6 hours (at RT) and 10 days after the preparation (stored at 2-8 °C)] from high and low dose formulations (6 samples). All formulation samples were analysed on the day of sample collection and were stored at -20 °C. These samples were analysed after the completion of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 140307. The results are reported in the annex of the final report.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 54 days, i.e. during 14 days of pre-mating and 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Details on study schedule:

Arrival of the Test Item: 22 January 2014
Study Initiation Date: 22 April 2014
1st Amendment to Study Plan: 25 June 2014
2nd Amendment to Study Plan: 11 September 2014
Delivery of Animals: 29 April 2014
Acclimatisation Period: 29 April 2014 – 05 May 2014
Experimental Starting Date: 05 May 2014
Date of Behavioural Observation: 07 May 2014- 30 June 2014
Treatment Period Males: 08 May 2014 – 06 June 2014
Treatment Period Females: 08 May 2014 – 01 July 2014
Necropsies Males: 05 June 2014 and 06 June 2014
Necropsies Females: 17 June 2014 – 01 July 2014
Experimental Completion Date: 01 July 2014
Completion Date of Delegated
Phase (Histopathology): 29 September 2014
Completion Date of Delegated
Phase (Formulation Analysis): 29 September 2014

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control (Group - 1)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low Dose (Group - 2)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Mid Dose (Group - 3)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High Dose (Group - 4)

No. of animals per sex per dose:

80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
Once before the first exposure, and once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and on lactation days in 5 randomly selected females (only lactating females were evaluated) outside the home cage using a functional observational battery of tests.

BODY WEIGHT: Yes
- The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups.

FOOD CONSUMPTION:
- Food consumption was measured weekly on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: yes

Litter observations:

Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum.

Postmortem examinations (parental animals):

SACRIFICE
All male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29 or 30, while female animals were sacrificed on post-natal day 4 along with pups using an anaesthesia (ketamine/xylazin, 2:1, Pharmanovo, lot no: 24863, expiry date: 10/2015 and Serumwerk, lot no: 00513, expiry date: 05/2015) was used for the females and decapitation for the pups.

GROSS NECROPSY
Pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.
Non pregnant (79) and non mated females (41 and 78) were sacrificed on study day 26 from the day of confirmed mating and day 26 from the end of 2 weeks mating period, respectively.
All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), and all organs showing macroscopic lesions of all adult animals were preserved in 10 % neutral buffered formalin, except for testes and epididymides which were fixed in modified Davidson’s Solution for 24 hours and then transferred to 70% ethanol.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. The number of corpora lutea and implantation sites was recorded for any females sacrificed 26 days after the end of the pairing period with no evidence of mating and for any females sacrificed on day 26 post-coitum due to non-delivery.

HISTOPATHOLOGY / ORGAN WEIGHTS
A full histopathology was carried out on the preserved organs and tissues of 5 randomly selected male and female animals (only lactating females were evaluated) of the control and high dose groups which were sacrificed at the end of the treatment period.
These examinations were not extended to animals of all other dosage groups as no treatment-related changes were observed in any organ of the high dose group.
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and eosin and were examined in all animals.
All gross lesion macroscopically identified was examined microscopically in all animals..
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle for the evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
Histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). Histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology). The study phases from test site 1 and 2 was performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012). Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.

Postmortem examinations (offspring):

Pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.6.01 software (p<0.05 was considered as statistically significant).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Isolated incidences of nasal discharge, alopecia on various body parts, moving the bedding, regurgitation, slight to moderate piloerection, aggressiveness, chromodacryorrhea at the right eye, slightly reduced spontaneous activity and emisis (blue mouth and nose) in a few control and/or dose group animals was observed. There were also discoloured blue faeces observed in male and female animals of high dose group which could be attributed to the colour of the test item. During the weekly detailed clinical observation, no significant changes or differences between the groups were observed.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In females, marginally lower (2-6%) group mean body weight during gestation and lactation period and body weight gain during gestation period was observed in high dose group when compared to the controls. As this difference was marginal, values were within the range of biological variation and this effect on body weight development was attributed to low body weight values from single high dose female (no. 80) in which no live pups were born, no toxicological relevance could be attributed to this effect on female body weight development.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Marginally lower group mean food consumption from gestation day 7 to post-natal day 4 was observed in high dose group when compared to the controls. As this difference was correlated with body weight development it is not considered to be no toxicologically relevant.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males and females, there were no adverse effects of toxicological relevance on parameters of haematology and blood coagulation parameters in dose groups when compared with corresponding control. However, in males there was marginally but statistically significantly higher reticulocyte count in LD and HD group when compared with the controls. As the mean value was in the range of historical control data and in the absence of dose dependency, the finding was not considered to be toxicological relevant.
In females, statistical analysis of haematology and blood coagulation data revealed statistically significantly higher eosinophils in LD group (0.62 %) when compared with the controls (0.16%). As the mean value was in the range of historical control data and due to lack of dose dependency, this effect was not considered to be toxicologically relevant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males and females, there were no adverse changes of toxicological relevance for clinical biochemistry in dose groups when compared to the controls. However, statistically significantly higher creatinine in HD males (26.80) and statistically significantly lower potassium in HD females (3.45) was observed when compared with the corresponding controls.
Since all mean and most individual values of clinical biochemistry of males and females were within the historical control range, the effect on creatinine in male HD group and potassium in female HD group was not considered to be an adverse effect due to the treatment.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

In males statistically significantly lower body temperature was observed in the last week of treatment in MD and HD group when compared with the controls. As this difference was marginal and overall there was no effect on the general health condition, body weight development and food consumption of these animals this effect on body temperature is not considered to be adverse.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic alteration of various organs due to bluish /greenish discolouration was considered to be the coloration derived from the unique colour of the test item and/or the colour being formed by the test item mixing with the intestinal contents. However, there was no histologic alteration correlated with macroscopic appearances.
Discoloured kidney of some animals including the control animals was probably due to the status of blood removal at the necropsy. There were no histologic abnormalities which might be related to macroscopic coloration. All other gross lesions recorded were within the range of normal background alterations which may be recorded in animals of this strain and age, or were considered to be incidental macroscopic appearances that did not correlate with microscopic changes.

Micropscopically, no histomorphologic evidence of toxicological properties observed in any organs and tissues including the male reproductive organs (testes, epididymides, prostate and seminal vesicles) and the female reproductive organs (ovaries, uterus with cervix and vagina).
In addition, as a result of the detailed examination of the testis, it was judged that there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure.
All findings recorded in the organs and tissues examined in this study were within the range of normal background lesions which may be recorded in animals of this strain and age. In conclusion, no histomorphological evidence of toxicity in any organs and tissues of male and females was established up to 1000 mg/kg bw/d.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Description (incidence and severity):

Except for confirmation of mating

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no effects of FAT 20033/L on the copulation, fertility and delivery indices of the dose groups when compared to the control. The copulation index and fertility index in control and dose groups were 90 % in control, 100 % in LD and MD 100 and 80 % in HD group. However, there was slight reduction in the delivery index of the HD group (87.5 %) when compared with the controls. This reduction in delivery index of the HD group was attributed to one female (no. 80) without litter but a number of implantation sites noted at necropsy. This finding was considered to be incidental and without toxicological significance. There was no effect on the viability index in dose group when compared to the control group.

Details on results (P0)

Pre- and Post-Natal Data:
The group mean number of corpora lutea, number of implantation sites, number of live pups born on PND 0 and 4, percentage of pre-implantation loss remained unaffected due to the treatment with test item when compared with the control group. However, there was higher post-implantation loss observed in HD group without achieving statistical significance compared to the control group. This increase was attributed to incidental finding that one female (no. 80) which showed implantation sites at necropsy and there were no live pups born. As this post implantation loss was within the historical control data range, the higher percent post implantation loss was not considered to be an adverse effect of treatment with test item.

Litter Data:
No treatment related effect of FAT 20033/L was observed on total number of pups born, number of males, number of male and females, sex ratio, live pups, still birth and runt on PND 0 and number of males, number of females, sex ratio and total number of live pups on PND 4. However, there was slightly lower litter size observed in HD group without achieving statistical significance compared to the control group. This decrease was attributed to incidental finding that one female (no. 80) which showed implantation sites at necropsy and there were no live pups born. As this mean litter size was within the historical control data range (5.91-15.46), it is not considered to be an adverse effect of treatment with test item.

Precoital Interval and Duration of Gestation:
No test item related effect was observed on precoital interval and duration of gestation and values were comparable between the dose groups and controls. All pregnancies resulted in normal births except female No. 80 from HD group was not littered and showed implantation sites noted at the time of necropsy on gestation day 26.
Majority of females in control and treatment groups showed evidence of copulation during the 14-day mating period except two females from MD group (No. 63 and 67) and 1 female from HD group (76) did not show evidence of mating through vaginal smear. However, the animals were pregnant and littered normally. There were also two females (41 and 78) which did not show evidence of mating during the 14-day mating period and were sacrificed on day 26 from the end of 2 weeks mating period. Successful mating resulted in 9/10 pregnancies in the control group, in 10/10 pregnancies in LD and MD and in 8/10 pregnancies in the HD group.

Pup Survival Data:
There was no test item related effect of FAT 20033/L on the survival of the pups from PND 0 through PND 4 in dose groups when compared to the control. However, one pup of control group female no. 49 (pup no. 6) and one pup from LD group female no. 57 (pup no. 4) was missing on PND 1 and 2, respectively. These missing pups were presumably cannibalised by the respective dam. Since a control animal was affected in 1 of 2 incidences, this cannibalism is not considered to be test item related.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Thus, the no observed adverse effect level (NOAEL) of FAT 20033/L is considered to be 1000 mg/kg/d for systemic toxicity and also for reproduction/ developmental toxicity in males and females.

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

Pup External Findings:
No treatment related gross external findings were observed in the pups of any of the dose groups on PND 0 and 4. However, following few external findings were observed:
In control females, one pup from female 45 (pup No. 1) was observed for dark head and two pups from female 48 (pup No. 1 and 2) were found cold to touch at the time of observation on PND 0. In LD females, one pup from female 56 (pup No. 4) had a dark head on PND 0. In MD females, one pup from female 62 and 63 (pup no. 8 and 1, respectively) had a dark snout, 4 pups from female 65 (pup no. 1-4) had dry skin and one pup of this dam had a dark snout on PND 0.
All these findings were considered to be spontaneous in nature and not related to the treatment with test item.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

There were no adverse toxicological effects in the adult males and females or significant reproductive and developmental effects at any administered dose. Thus, the no observed adverse effect level (NOAEL) of FAT 20033/L is considered to be 1000 mg/kg bw/day for systemic toxicity and also for reproduction/ developmental toxicity in males and females.

Executive summary:

The aim of this study was to assess the possible effects of FAT 20033/L on male and female fertility and embryofetal development after repeated dose administration in Wistar rats. This study was conducted in accordance to OECD TG 422 (GLP). The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 or 29 days were completed. Animals of an additional control group were handled identically as the dose groups but received aqua ad injection, the vehicle used in this study. The 4 groups comprised 10 males and 10 female Wistar rats.

The following doses were evaluated:

Control (C):                   0        mg/kg bw/day

Low Dose (LD):            100     mg/kg bw/day

Medium Dose (MD):     300     mg/kg bw/day

High Dose (HD):           1000   mg/kg bw/day

 

The test item was suspended in aqua and dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight. During the period of administration, the animals were observed each day for signs of toxicity. At the conclusion of the test, surviving animals were sacrificed and observed macroscopically. Body weight and food consumption were measured weekly. Haematological and clinical biochemistry evaluations were performed on blood samples, urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males and females from each group. Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behavior observations were performed in the week before the treatment and at the end of the study. After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum. The males were sacrificed after completion of the mating period on treatment days 29 and 30 and the females along with their pups were sacrificed on post-natal day 4. Non-pregnant females were sacrificed on day 26. Pups sacrificed on post-natal day 4 were carefully examined for gross external abnormalities. A full histopathological evaluation of the tissues was performed of 5 randomly selected male and female animals of the control and high dose groups. Gross lesions from all groups were examined by light microscopy. Furthermore, testes, epididymides, prostate, seminal vesicles with coagulating glands, ovaries, uterus with cervix and vagina were examined in all animals and special emphasis was made on the stages of spermatogenesis in testes and histopathology of interstitial cell structure. Organs and tissues listed above were preserved in 10% neutral buffered formalin, except for testes and epididymides which were fixed in modified Davidson’s fixative.

 

Summary results:

No mortality occurred in the control or any of the dose groups during the study period. No clinical signs of toxicological relevance in dose group.

During the weekly detailed clinical observation, no significant changes or differences between the groups were found. No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. In males and females, there were no adverse effects of toxicological relevance on body weight and body weight gain and food consumption observed in dose groups when compared to the controls. In males and females, there were no adverse effects of toxicological relevance observed on haematology, blood coagulation and clinical biochemistry parameters in dose groups when compared to the corresponding controls. There were no effects of toxicological relevance observed on urine parameters of males and females dose groups when compared to the control. No gross lesions were observed in this study those could be attributed to treatment with the test item. All gross lesions were within the range of normal background alterations which may be recorded in animals of this strain and age or were considered to be incidental macroscopic findings that did not correlate with microscopic changes.

In males and females, there were no statistically significant differences observed on absolute and relative organ weights of the dose groups when compared with the corresponding controls. Micropscopically, no histomorphologic evidence of toxicological properties of FAT 20033/L was observed in macropscopic findings, any organs and tissues including the male reproductive organs (testes, epididymides, prostate and seminal vesicles) and the female reproductive organs (ovaries, uterus with cervix and vagina). All findings recorded in the organs and tissues examined in this study were within the range of normal background lesions which may be recorded in animals of this strain and age. No histomorphological evidence of toxicological properties of FAT 20033/L was found in any organs and tissues of male and females were established up to 1000 mg/kg bw/day. No treatment related effect was observed on total number of pups born, number of male and females, sex ratio, live pups, still birth and runt on PND 0 and number of males, number of females, sex ratio and total number of live pups on PND 4. Statistical analysis of litter weight data revealed no treatment related effect on group mean litter weight, total litter weight, male litter weight and female litter weight on PND 0 and PND 4 when compared with the controls. There was no test item related effect observed on the pre-coital interval and the duration of gestation in dose groups when compared to the controls. The group mean number of corpora lutea, number of implantation sites, number of live pups born on PND 0 and 4, percentage of pre-implantation loss remained unaffected after treatment with test item, when compared with the control group. There was higher post-implantation loss observed in HD group that is not considered as test item related. No effects of FAT 20033/L were observed on the copulation, fertility and delivery indices of the dose groups when compared to the control. The copulation index and fertility index in control and dose groups were 90 % in control, 100 % in LD and MD 100 and 80 % in HD group. However, there was slight reduction in delivery index of HD group (87.5 %) was observed when compared with the controls. This finding was considered to be incidental and without toxicological significance. No test item related effect of FAT 20033/L was observed on the survival of the pups from PND 0 through PND 4 in dose groups when compared to the control. No treatment related gross external findings were observed in the pups of any of the dose groups on PND 0 and 4. On the basis of the above findings, the following conclusions can be made: There were no adverse toxicological effects in the adult males and females or significant reproductive and developmental effects in pups at any administered dose. Thus, the no observed adverse effect level (NOAEL) of FAT 20033/L is considered to be 1000 mg/kg bw/day for systemic toxicity and also for reproduction/ developmental toxicity in males and females.