Registration Dossier

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Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Nov 2012 to 19 Sep 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Health Effects guidelines, OPPTS 870.3550, Reproduction/ Developmental Toxicity Screening Test. EPA 712-C-00-367, July 2000 [
Deviations:
no
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)
Limit test:
no

Test material

Constituent 1
Reference substance name:
FAT40549/A
IUPAC Name:
FAT40549/A
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: FAT 40549/A
Batch No.: TV1
Physical State: powder
Storage Conditions: at room temperature
Purity: 40.5%
Date of Analysis: 15.03.1996
Expiry Date: 29.09.2014
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.



Specific details on test material used for the study:
Name: FAT 40549/A
Batch No.: TV1
Physical State: powder
Storage Conditions: at room temperature
Purity: 40.5%
Date of Analysis: 15.03.1996
Expiry Date: 29.09.2014

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 (non-pregnant and nulliparous).
Age at the start of the treatment period: males: 10 - 11 weeks old, females: 10 - 11 weeks old.
Body weight at the allocation of the animals to the experimental groups
males: 253 - 296 g (mean: 279.77 g, ± 20% = 223.82 – 335.73 g)
females: 173 - 209 g (mean: 194.44 g, ± 20% = 155.55 – 233.33 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.

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. 1039)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at
regular intervals)
- The animals were housed individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (Lot. No. 160812) except during
mating period when individual male and female rats were cohabited.
- Adequate acclimatisation period (at least five days)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured on weekly basis.
Details on mating procedure:
Mating was performed in 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. 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 minimised.
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 was analysed for the low and high dose concentrations.

Samples for the nominal concentration verification was taken in study week 1 (first week of pre-mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation) from all groups (16 samples).

Samples for homogeneity analysis was 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 was taken in the first week of the study, 0 hours after the preparation (at room temperature), from high and low dose formulations (4 samples).
All formulation samples were stored at -20°C until the analysis was performed.
Duration of treatment / exposure:
Males: 28 days; Females: approx. 54 days
Frequency of treatment:
7 days/ week
Details on study schedule:
Arrival of the Test Item: 09 May 2012
Study Initiation Date: 26 November 2012
1st Amendment to Study Plan: 17 December 2013
2nd Amendment to Study Plan: 18 February 2013
3rd Amendment to Study Plan: 26 February 2013
4th Amendment to Study Plan: 19 April 2013
Experimental Starting Date: 04 December 2012
Experimental Completion Date: 28 January 2013
Completion Date of Delegated Phase (Histopathology): 14 August 2013
Completion Date of Delegated Phase (Formulation Analysis): 22 July 2013



Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group I - Control group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group II - Low Dose Group
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group III - Mid Dose Group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group IV: High Dose Group
No. of animals per sex per dose:
10 males and 10 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Preparation of the animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females.

Dosage
The animals were treated with the test item formulation or vehicle on 7 days per week for a period of minimum 28 days for males and for a period of maximum 54 days for females, 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.
The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. 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 (sterile water) the same volume as used for the treatment groups.

Administration of doses: The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured on weekly basis.

Mating: Mating was performed in 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. 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 minimised.

Clinical observation: General clinical observations were made at least once a day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

Body weight and food Consumption: 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. Any animals prematurely sacrificed were weighed prior to the sacrifice.
Food consumption was measured 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.

Pathology
Gross necropsy
The males were sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days) and the surviving females were sacrificed on the respective post-natal day 4 by using an anesthesia (e.g. ketamine/xylazin). Surviving pups were killed on post-natal day 4 by decapitation.
Dead pups and pups sacrificed on day 4 post-partum or shortly thereafter were carefully examined externally for gross abnormalities.
Non-pregnant or the non copulated females were sacrificed on day 26 from the day of sperm-positive vaginal smear as an evidence of mating or from the last day of mating period.
All animals were subjected to a detailed gross necropsy which included 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 before transferring it to 10% neutral buffered formalin.
The number of implantation sites and corpora lutea was recorded for each parental females at necropsy.


Organ weight
The testes and epididymides of all male adult animals as well as the ovaries, uterus with cervix of all female adult animals were weighed.
Paired organs were weighed separately. Organ weights of animals found dead were not taken. Additional organs of animals which died during the course of the study were preserved: all gross lesions, brain, stomach, small and large intestines (including Peyer´s patches), liver, thymus, spleen, lung, urinary bladder, lymph nodes (mesenteric and axillary), trachea, kidneys, adrenal glands, heart.

Histopathology
Histopathology was not carried out on the preserved organs and tissues of animal (animal 126) which died during the study.
Testes, epididymides, ovaries, uterus with cervix, vagina and accessory sex organs (prostate, seminal vesicle with coagulating gland) will be examined in C and HD animals. Macroscopic changes were evaluated in all study animals.
A detailed qualitative examination of the testes was made taking into account the tubular stages of the spermatogenic cycle at the evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides. The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory Propath UK Ltd, Willow Court, Netherwood Road, Hereford HR2 6JU, Great Britain (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory KALEIDIS – Consultancy in Histopathology (test site for histopathology), 6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, H&E staining and scientific slide evaluation was performed according to the corresponding SOP’s of the test sites.



Examinations

Parental animals: Observations and examinations:
Body weight, food consumption, clinical signs, pathology, organ weight (reproductive organs), histopathology (reproductive organs)
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not Examined
Litter observations:
The duration of gestation was recorded and was calculated from day 0 of pregnancy. 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 and sexed. Litters were weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups
were identified by marking with tattoo. In addition to the observations on parent animals, any abnormal behavior of the offspring was recorded.
Postmortem examinations (parental animals):
yes
Postmortem examinations (offspring):
not examined
Statistics:
For statistical analysis one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was carried out to reveal any
differences between control- and test groups. Statistical analysis was performed with GraphPad Prism (version V) software and p<0.05 was
considered as statistical significants.
Reproductive indices:
Copulation, fertility, delivery indices
Offspring viability indices:
yes

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were few clinical signs recorded in control and treated group animals during the study period. The clinical signs recorded were red/ dark nasal discharge, alopecia, moving the bedding, salivation, piloerection, abnormal breathing and reduced spontaneous activity. These findings were observed transiently in few isolated animal of control or treated groups. These findings were not considered to be of toxicological relevance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female rat of MD group (No. 126) was found dead during the early treatment period (on mating/ postmating day 8). As its lung was distended and dark discoloured, its cause of death was assumed to be due to gavaging error.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In males and females, there were no effects on body weight and body weight change throughout the study period in treated groups when compared with controls. However, there was statistically significant decrease in body weight change in male HD group on 1st week of premating. This decrease was due to considerably lower weight gain of 2 isolated males (animals 101 and 107), which with the progress of the study showed weight gain which were in the normal range of variation. Hence, the finding was not associated with treatment.
There was also statistically significant increase in body weight change in male LD and HD groups on 2nd week of mating/ postmating days. In the absence of dose response pattern the changes were not considered to be due to treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Description (incidence and severity):
In males and female, there was no effect on food consumption during the study period. The statistical analysis of data revealed no significant changes between the treated and control groups.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Reproductive organs:
Yellow pigment in interstitial macrophages was seen at a minimal or mild degree in the testis and epididymis in all males and at a minimal degree in the prostate gland in 2/10 males of HD group. Minimal yellow pigment was also noted in the epididymis of two males of LD group and evaluated due to macroscopic findings. These changes were considered to represent test item deposition in macrophages.
No test item-related histological findings were noted in the other male and in the female reproductive organs.
Reproductive organs of most control and high dose females showed typical post-partum histomorphology. The number of large ovarian corpora lutea was not essentially different between control animals and animals of HD group.
One control female, one female of LD group and one female of HD group did not show any indication of recent pregnancy at terminal sacrifice. Histomorphology in the control and high dose female showed physiological sexual cycling, the female of the LD group was not evaluated histologically. The decedent female also did not show any indication of pregnancy, which was related to its early death.

Other organs:
According to the study plan, other organs were only examined in case of macroscopic observations made at necropsy.
In the kidney, mild or moderate amounts of yellow pigment were observed in the corticotubular epithelium, in 10/10 males and 3/3 females of HD group. Minimal or moderate amounts of yellow pigment were also seen in 2/10 males and 1/1 female of MD group. This change was considered to be caused by deposition of the coloured test item. In addition, in 2/10 males of HD group, a minimal degree of corticotubular degeneration/regeneration was noted. The change was minimal only, but as reversibility could not be assessed, an adverse character of it could not be completely excluded.
In the mesenteric lymph node, minimal sinus histiocytosis, partially yellow pigmented, was seen in 5/6 males of HD group and evaluated for macroscopic change. Minimal multifocal yellow pigment was also observed in the thymus of one female of MD group.

No test item-related histopathological lesions were noted in the other miscellaneous organs evaluated in this study.
Organs of the female decedent of the MD group were not evaluated histologically, and its cause of death could therefore not be determined.
No test item-related histopathological lesions were noted in the other miscellaneous organs evaluated in this study.
Other effects:
not examined

Reproductive function / performance (P0)

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

Details on results (P0)

Day of sacrifice
All male animals were killed after minimum 28 days of application (half of the males of each group were killed on day 29 and rest half on day 31).
Non pregnant or the non copulated females were killed on the day 26 from the day of sperm positive vaginal smear as an evidence of mating or from the last day of mating period. Lactating females along with pups were sacrificed on respective post natal day 4.



Clinical observation and mortality
One female rat of MD group (No. 126) was found dead during the early treatment period (on mating/ postmating day 8). As its lung was distended
and dark discoloured, its cause of death was assumed to be due to gavaging error.
There were few clinical signs recorded in control and treated group animals during the study period. The clinical signs recorded were red/ dark nasal discharge, alopecia, moving the bedding, salivation, piloerection, abnormal breathing and reduced spontaneous activity. These findings were
observed transiently in few isolated animal of control or treated groups. These findings were not considered to be of toxicological relevance.

Body weight and body weight change
In males and females, there were no effects on body weight and body weight change throughout the study period in treated groups when compared with controls. However, there was statistically significant decrease in body weight change in male HD group on 1st week of premating. This decrease was due to considerably lower weight gain of 2 isolated males (animals 101 and 107), which with the progress of the study showed weight gain which were in the normal range of variation. Hence, the finding was not associated with treatment.
There was also statistically significant increase in body weight change in male LD and HD groups on 2nd week of mating/ postmating days. In the
absence of dose response pattern the changes were not considered to be due to treatment.

Food consumption
In males and female, there was no effect on food consumption during the study period. The statistical analysis of data revealed no significant changes between the treated and control groups.

Precoital interval and duration of gestation
There were no treatment related effect observed on the duration of gestation and precoital interval in the treated groups when compared with controls.
All females in control and treated groups showed evidence of copulation during 14 days mating period. Successful mating of females resulted in pregnancy rate as follows, Control 90%, LD group 90% MD group 100% and HD group 100%.
One control female (No. 48) and one female of LD group (No. 114) did not show any indication of recent pregnancy at terminal sacrifice. There was no dose relationship, and this was therefore considered to be unrelated to treatment. One female of HD group (No. 135) did not get copulated during the 14 days mating period, but histomorphology showed physiological sexual cycling. Hence, the treatment related effect was not considered.




Gross pathology
At terminal sacrifice, organ discoloration was seen in all dose groups in a dose-related manner. Yellow/light/orange/red discoloration was noted,
mostly in the mesenteric lymph node, testis and/or kidney, in all males of MD or HD group and in one female of MD group and three females HD
group. In LD group, only kidney was yellow or light discoloured in six males, but in none of the females. These color changes were considered to be caused by the color of the test item itself.
Other macroscopic organ findings were very few and not considered to be test item-related, including yellow spot(s) at the epididymis of a small
number of control and treated males, which were histologically confirmed to be spermatic granuloma, a findings seen occasionally in untreated male rats of this strain and age.


Organ weight
In males and females, the organ weight data (absolute and relative to terminal body weight) showed no changes considered to be of toxicological relevance. The statistical analysis of data indicated no significant changes in treated groups when compared to the corresponding control group.

Histopathology
Reproductive organs:
Yellow pigment in interstitial macrophages was seen at a minimal or mild degree in the testis and epididymis in all males and at a minimal degree in the prostate gland in 2/10 males of HD group. Minimal yellow pigment was also noted in the epididymis of two males of LD group and evaluated due to macroscopic findings. These changes were considered to represent test item deposition in macrophages.

No test item-related histological findings were noted in the other male and in the female
reproductive organs.

Reproductive organs of most control and high dose females showed typical post-partum
histomorphology. The number of large ovarian corpora lutea was not essentially different between control animals and animals of HD group.

One control female, one female of LD group and one female of HD group did not show any indication of recent pregnancy at terminal sacrifice. Histomorphology in the control and high dose female showed physiological sexual cycling, the female of the LD group was not evaluated histologically. The decedent female also did not show any indication of pregnancy, which was related to its early death.

Other organs:
According to the study plan, other organs were only examined in case of macroscopic
observations made at necropsy.

In the kidney, mild or moderate amounts of yellow pigment were observed in the corticotubular epithelium, in 10/10 males and 3/3 females of HD group. Minimal or moderate amounts of yellow pigment were also seen in 2/10 males and 1/1 female of MD group. This change was considered to be caused by deposition of the coloured test item. In addition, in 2/10 males of HD group, a minimal degree of corticotubular degeneration/regeneration was noted.

In the mesenteric lymph node, minimal sinus histiocytosis, partially yellow pigmented, was seen in 5/6 males of HD group and evaluated for macroscopic change. Minimal multifocal yellow pigment was also observed in the thymus of one female of MD group.

No test item-related histopathological lesions were noted in the other miscellaneous organs evaluated in this study.

Organs of the female decedent of the MD group were not evaluated histologically, and its cause of death could therefore not be determined.

No test item-related histopathological lesions were noted in the other miscellaneous organs evaluated in this study.



Dose formulation analysis
Concentration analysis of formulation samples was determined in study week 1, 3, 5 and 7 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 105.4%, 103.1% and 103.4% of the nominal concentration, respectively.

Stability of formulation samples was investigated in study week 1 for LD and HD dose groups. After 6 hours storage at room temperature recovery
compared to starting value was 100.8% and 98.4%.
Homogeneity of formulation samples was determined in study week 1 and 5 for LD and HD dose groups. The mean recovery observed for LD dose group was 108.8 and 103.2% of the nominal value and 104.2 and 104.7% for HD dose group.

The coefficients of variation of the different sampling locations (top, middle, bottom) were 0.6 and 14.2 % in LD dose group, and 4.8 and 2.2% in HD
dose group.



Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects seen

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:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

Litter weight data:
There were no treatment related changes noted on group mean litter weight, total litter weight, male litter weight and female litter weight measured on PND 0 and PND 4. There were no statistically significant difference noted between the treated and control groups.

Pre and post natal data:
There were no statistically significant difference noted for group means of corpora lutea, implantation sites, live pups born on PND 0, percent pre implantation loss and percent post implantation loss in the treated groups when compared with corresponding controls. However, the mean value of % post implantation loss was slightly higher in HD group, but the mean corpora lutea, implantation sites and live pups on PND 0 being comparable between the treated groups and the corresponding controls and also in the absence of statistical significance, the increase in percent post implantation loss was not considered to have toxicological relevance.

Litter data:
There were no treatment related effect observed on the number of male pups, number of female pups, sex ratio, live pups, still birth and runt on PND 0 and total number of live pups and sex ratio on PND 4. There were no statistically significant difference noted between the treated and control groups.

Reproductive indices:
The copulation index, fertility index and viability index remained unaffected due to treatment in treated groups compared to corresponding control.
All pregnancies resulted in normal births and therefore delivery index remained unaffected in all treated groups.


Pup survival data:
The survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treated groups compared to control. However, 1 pup (pup no. 7) of animal 131 (HD group), 2 pups (pup no. 1, 3) from animal 49 (C group) and 2 pups (pup no. 11, 12) of animal 119 (LD group) were missing between PND 1 and 2. The missing pups were assumed to be cannibalized by the dam. However, these findings were in isolated animals and hence were considered to be incidental.

Pup external findings:
There were no treatment related gross external findings observed in pups of the treated groups on PND 0 and 4. However, there were few findings namely dark head/ snout, injury (on neck/ snout), dark and swollen hindlimb and discoloured/ dry skin in few isolated pups of control or treated groups. These were considered to be spontaneous and incidental in nature.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No toxicity observed upto 1000 mg/kg

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the data generated from this reproduction/ developmental toxicity screening test with FAT 40549/A, the no observed adverse effect level (NOAEL) for male animals is considered to be 300 mg/ kg body weight/ day and for females animals the NOAEL is considered to be 1000 mg/kg body weight/ day. The NOAEL for reproduction/ developmental toxicity is considered to be 1000 mg/ kg body weight/ day.
Executive summary:

The aim of this study was to assess the possible effects of FAT 40549/A on male and female fertility and embryofetal development after repeated dose administration in Wistar rats. This study was conducted in accordance with OECD test guideline 421 in a GLP certified laboratory.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a minimum treatment period of 28 days for males and a maximum treatment period of 54 days for females, 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.

Animals of control group were handled identically as the dose groups but received aqua ad injectionem, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats.

During the period of administration, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically. Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals. After 14 days of treatment 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. After the confirmation of the mating, females were separated and housed individually. 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 days 29 and 31 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females were sacrificed on day 26 from the day of confirmed mating. The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. Pups sacrificed on post natal day 4 and those found dead, were carefully examined for gross external abnormalities. The testes and epididymides of all male adult animals as well as the ovaries, uterus with cervix of all female adult animals were weighed. Paired organs were weighed separately. Organ weights of animals found dead were not taken. Testes, epididymides, ovaries, uterus with cervix, vagina and accessory sex organs (prostate, seminal vesicle with coagulating gland) were examined in C and HD animals. Histopathology of non pregnant females was evaluated in control and HD group. Macroscopic changes were evaluated in all study animals.

The following doses were evaluated: Control: 0 mg/kg body weight, Low Dose: 100 mg/kg body weight, Medium Dose: 300 mg/kg body weight, High Dose: 1000 mg/kg body weight.

The test item formulation was prepared freshly on each day of administration. The test item was dissolved in aqua ad injectionem and administered daily during 14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 3 in females. Males were dosed for a minimum dosing period of 28 days (half males of each group were treated 28 days and rest half males treated for 30 days). Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight.

Summary Results: One female rat of MD group (No. 126) was found dead during the early treatment period (mating/ postmating day 8). As its lung was distended and dark discoloured, its cause of death was assumed to be due to gavaging error.

There were few clinical signs recorded in control and treated group animals during the study period, which were not considered to be of toxicological relevance.

In males and females, there were no effects on body weight and body weight change throughout the study period in treated groups when compared with controls.

In males and females, there was no effect on food consumption during the study period. The statistical analysis of data revealed no significant changes between the treated and control groups.

There were no treatment related effect observed on the number of male pups, number of female pups, sex ratio, live pups, still birth and runt on PND 0 and total number of live pups and sex ratio on PND 4.

There were no treatment related changes noted on group mean litter weight, total litter weight, male litter weight and female litter weight measured on PND 0 and PND 4.

There were no treatment related effects observed on the duration of gestation and precoital interval in the treated groups when compared with controls.

There were no effects on copulation in females of control and treated groups during 14 days mating period.

Successful mating of females resulted in pregnancy rate as follows, Control 90%, LD group 90% MD group 100% and HD group 100%. One control female (No. 48), one female of LD group (No. 114) and one female of HD group (No. 135) did not show any indication of recent pregnancy at terminal sacrifice. There was no dose relationship, and this was therefore considered to be unrelated to treatment. There were no treatment related changes noted for group means of corpora lutea, implantation sites, live pups born on PND 0, percent pre implantation loss and percent post implantation loss in the treated groups when compared with corresponding controls.

The copulation index, fertility index and viability index remained unaffected due to treatment in treated groups compared to corresponding control. All pregnancies resulted in normal births and therefore delivery index remained unaffected in all treated groups. The survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treated groups compared to control.

There were no treatment related gross external findings observed in pups of the treated groups on PND 0 and 4. At terminal sacrifice, organ discoloration was seen in a dose-related manner, in all dose groups. Yellow/light/orange/red discoloration was noted, mostly in the mesenteric lymph node, testis and/or kidney, in all males of MD or HD group and in one female of MD group and three females HD group. In LD group, only kidney was yellow or light discoloured in six males, but in none of the females. These colour changes were considered to be caused by the colour of the test item itself. Other macroscopic organ findings were very few and not considered to be test item-related, including yellow spot(s) at the epididymis of a small number of control and treated males, which were histologically confirmed to be spermatic granuloma, a findings seen occasionally in untreated male rats of this strain and age. In males and females, the organ weight data (absolute and relative to terminal body weight) showed no changes considered to be of toxicological relevance.

The statistical analysis of data indicated no significant changes in treated groups when compared to the corresponding control group. Histologically, minor degrees of yellow pigment in interstitial macrophages were seen in the testis and epididymis in all males and in the prostate gland in 2/10 males treated at 1000 mg/kg/day. Minimal yellow pigment was also noted in the epididymis of two males treated at 100 mg/kg/day, presenting gross lesions. No test item-related histological findings were noted in the other male and in the female reproductive organs. One control female, one female treated at 100 mg/kg/day and one female treated at 1000 mg/kg/day did not show any indication of recent pregnancy at terminal sacrifice. As there was no dose relationship, this was considered to be unrelated to treatment. In the kidney, mild or moderate amounts of yellow pigment were observed in the corticotubular epithelium, in all males and some females treated at 1000 mg/kg/day, as well as in a low number of animals evaluated at 300 mg/kg/day. This change was considered to be caused by deposition of the coloured test item.

In addition, in 2/10 males treated at 1000 mg/kg/day, corticotubular degeneration/regeneration was noted. The change was minimal only, but as reversibility could not be assessed, an adverse character of it could not be completely excluded.

Furthermore, at 1000 mg/kg/day only, minimal sinus histiocytosis, partially yellow pigmented, was seen in the mesenteric lymph node of 5/6 males evaluated for macroscopic change. Minimal multifocal yellow pigment was also observed in the thymus of one female treated at 300 mg/kg/day. These changes were considered to represent test item deposition and to be non-adverse, as there was no indication of other structural changes or functional impairment of these organs.

Concentration analysis of formulation samples was determined in study week 1, 3, 5 and 7 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 105.4%, 103.1% and 103.4% of the nominal concentration, respectively. Stability of formulation samples was investigated in study week 1 for LD and HD dose groups. After 6 hours storage at room temperature recovery compared to starting value was 100.8% and 98.4%. Homogeneity of formulation samples was determined in study week 1 and 5 for LD and HD dose groups. The mean recovery observed for LD dose group was 108.8 and 103.2% of the nominal value and 104.2 and 104.7% for HD dose group. The coefficients of variation of the different sampling locations (top, middle, bottom) were 0.6 and 14.2 % in LD dose group, and 4.8 and 2.2% in HD dose group.

Based on the data generated from this reproduction/ developmental toxicity screening test with FAT 40549/A, the no observed adverse effect level (NOAEL) for male animals is considered to be 300 mg/ kg body weight/ day and for females animals the NOAEL is considered to be 1000 mg/kg body weight/ day. The NOAEL for reproduction/ developmental toxicity is considered to be 1000 mg/ kg body weight/ day.