Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

The NOAEL for reproductive toxicity was 1000 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-04-01 to 2014-10-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Health Effects guidelines, OPPTS 870.3550, Reproduction/ Developmental Toxicity Screening Test. EPA 712-C-00-367, July 2000
Commission Regulation (EC) No. 440/2008, L 142, Appendix Part B, May 30, 2008
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no
Justification for study design:
According to guideline
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Test System
Species/strain: healthy 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: 10-11 weeks old, females: 10-11 weeks old.
Body weight at the allocation of the animals to the experimental groups:
males: 272 - 316 g (mean: 287.55 g, ± 2 0% = 230.04 – 345.06 g)
females: 185 - 216 g (mean: 200.57 g, ± 20 % = 160.46 – 240.69 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)
- The animals were kept in groups of 2 animals/ sex/ cage in IVC cages), type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 1526) (except during the mating period when one female were paired with one male and during gestation/lactation period when females were housed individually).
For mating one female was paired with one male. During post-mating period males were housed with their respective pre-mating partners.
- 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. No animal arriving at BSL showed
pathological signs. Before the first administration all animals used for the study were weighed. Mean body weight of the pairwise kept 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. Each animal was marked with its identification number by individual ear tattoo.

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
water
Details on exposure:
Preparation of the Test Item and Control Formulations
The test item was weighed into a tarred plastic vial on a precision balance and was suspended in corn oil. Homogeneity of the test item in the vehicle was maintained by using an ultra turrax.
The vehicle was selected as suggested by the sponsor based on the test item’s characteristics.
The test item formulations were prepared freshly at least every ten days. Prepared test item formulations were stored at 2-8 °C, but warmed to room temperature before dose administration.
The vehicle was also used as control item.

Experimental Groups and Doses
According to the results of a previous dose range finding study (BSL study no. 140338) and in consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C). The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum 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 days was completed.

C 0 mg/kg bw/ day (Male No.:1-10/ Female No.:41-50)
Csup 0 mg/kg bw/ day (Male No.:81-90/ Female No.:101-110)
LD 100 mg/kg bw/ day (Male No.:11-20/ Female No.:51-60)
MD 300 mg/kg bw/ day (Male No.:21-30/ Female No.:61-70)
HD 1000 mg/kg bw/ day (Male No.:31-40/ Female No.:71-80)
HDsup 1000 mg/kg bw/ day (Male No.:121-130/ Female No.:131-140)

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 dosage related response and NOAEL. The doses were selected on the basis of data from a Dose Range Finding Study (BSL study no. 140338). The animals in the control group were handled in an identical manner to the test group subjects and received corn oil using the same volume as used for the dose groups. Due to wrong dosing, HD animals were excluded from the study on study day 7 and a supplementary group HDsup was introduced (10 males and 10 females). As these animals were taken from a different batch, additionally a supplementary control group Csup was introduced (10 males and 10 females; also shared with BSL Study No. 140337). Animals of HD group were euthanized on study day 7 without measurement of body weight, food consumption, organ weights and without clinical or macroscopic observations. Organs or tissue of these animals for possible histopathological evaluation were not preserved. Data of group HD will be filed and archived with the raw data of the study but are not reported here.

Administration of Doses
The test item formulation and vehicle were administered at a single dose to the animals by oral gavage with disposable polypropylene feeding tubes for rats (78 mm long; 3.0 mm diameter; Instech Laboratories Inc). 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.
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 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 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 after the preparation, 6 hours after the preparation (at room temperature) and another sample 10 days after the preparation (stored at 2-8°C), from high and low dose formulations (6 samples). Each sample was retained twice (sample A, sample B). The dose formulation analysis was performed by BSL BIOSERVICE Scientific Laboratories GmbH, in accordance with GLP. All formulation samples were stored at -15°C to -35 °C. The A samples were analysed after the completion of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 140289. The exact procedure was described in a phase plan which was amended to the study plan. The B samples will be retained at BSL until the analysis has been performed, and discarded after completion of the final study report.
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum 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 days was completed.
Frequency of treatment:
7 days/week
Details on study schedule:
Arrival of the Test Item: 07 January 2014
Study Initiation Date: 01 April 2014
1st Amendment to Study Plan: 10 April 2014
2nd Amendment to Study Plan: 20 May 2014
3rd Amendment to Study Plan: 18 July 2014
Delivery of Animals: 27 March 2014
Acclimatisation Period: 27 March 2014 - 02 April 2014
Experimental Starting Date: 02 April 2014
Treatment Period: 04 April 2014 – 04 June 2014
Experimental Completion Date: 04 June 2014
Completion Date of Delegated Phase (Histopathology): 25 September 2014
Completion Date of Delegated Phase (Formulation Analysis): 12 September 2014
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg Body weight/day
Basis:
nominal conc.
No. of animals per sex per dose:
In total 120 animals (60 males and 60 females) were included in the study (10 male and 10 female animals per group).
Control animals:
yes, concurrent vehicle
Details on study design:
Not available
Positive control:
No data
Parental animals: Observations and examinations:
Clinical Observations
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. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

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 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.




Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
No data
Litter observations:
The duration of the gestation was recorded and was calculated from day 0 of the 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 and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by tattooing. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.
Postmortem examinations (parental animals):
Pathology
All surviving male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29, while female animals were sacrificed on post-natal day 4 along with pups using an anaesthesia (ketamine/xylazin, 2:1, Pharmanovo, lot no: 24664, expiry date: 06/2015, lot no: 24863, expiry date: 10/2015 and Serumwerk, lot no: 00513, expiry date: 05/2015, lot no: 01213, expiry date: 10/2015) was used. Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities. Non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal smear as an evidence of mating. 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 preserved in modified Davidson’s Solution and transferred to 70% ethanol on the next day. 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 24 to 26 days after the end of the pairing period with no evidence of mating and for any females sacrificed on day 25 post-coitum due to non-delivery.

Organ Weights
The testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland as a whole) of all male adult animals as well as the ovaries, uterus with cervix of all female adult animals were weighed. Paired organs were weighed together.

Histopathology
A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study or which were euthanized due to morbidity. 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 examined in Csup and HDsup animals. Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at 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. The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of compliance and quality assurance statement) to the study director. The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon the completion of the study.
Postmortem examinations (offspring):
All surviving pups were sacrificed by decapitation on post-natal day 4. Dead pups and 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 absolute and relative organ weights was performed for each gender by comparing values of all groups with each other (C, Csup, LD, MD, HDsup) using a Tukey-Test (used in conjunction with ANOVA). These statistics were performed with GraphPad Prism V.6.01 software (p<0.05 was considered as statistically significant).
Reproductive indices:
There were no treatment-related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared to the respective control group. A slightly reduced copulation index (number of copulated females / number of pairs x 100) was only observed in the Csup group (90 % compared to 100 % in all other groups) and is considered to be incidental. The fertility index (number of pregnant females/ number of copulated females x 100) was also slightly reduced in the Csup group (88.9 %) compared to 100 % in all other groups. The cause of the unsuccessful copulation and/or pregnancy and/or infertility of females no. 101 and 102 of the Csup group as well as their male pairing partners (no. 81 and 82), respectively, could not be established from the reproductive organs examined histopathologically from these animals. A slightly reduced delivery index in the C group (90 %) compared to 100 % in the Csup group and the dose groups was based on pregnant female no. 41 of the C group which did not litter before the end of the study due to parturition difficulties. At necropsy one dead foetus was found in a contorted uterus. The remaining pregnant females of the C group littered normally.
Offspring viability indices:
There were no treatment-related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared to the respective control group. The viability index (no. of live offspring at day 4 / no. of live offspring at birth) X 100) was marginally lower in the Csup group (98.96 %), the LD group (99.09 %) and the HDsup group (99.17 %) when compared to 100 % in each the C and MD group. As this was attributed to the death of one pup in one single dam per group, respectively, and without dose-dependency, this finding is considered incidental.
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Mortality
No mortality occurred in the control groups or any of the dose groups during the treatment period of this study.

Clinical Observations
There were no clinical signs of toxicological relevance in the dose groups, when compared to the control groups.
However, the skin of all male and female animals of the HDsup group was observed to be discoloured blue from the 7th day of treatment until the end of the study. Blue coloured faeces were noted in both males and females of the LD and the MD group from the 4th day of treatment and in both males and females of the HDsup group from the 3rd day of treatment until the end of the study, respectively. On one single occasion, blue urine was observed in one female animal of the HDsup group.Discolouration of the skin, faeces and urine was considered to be based on the properties of the blue coloured test item.
On gestation day 26, the health status of female no. 41 was moderately reduced (wasp waist, slightly reduced spontaneous activity, dark urin and bright eyes (probably anaemic). Due to parturition difficulties female no. 41 of the C group did not litter before the final planned sacrifice on gestation day 26. At necropsy, a uterine torsion was noted as likely reason for a pregnancy complication. One dead foetus was found in the uterus. Uterine torsion is a rare phenomen, but can occur incidentally.
Slight salivation was noted in one male of the MD group and one male of the HDsup group on one single day, respectively. One female of the HDsup group showed moderate salivation on two days. Moving the bedding was observed in two males of the MD group on one occasion each. The symptoms of salivation and moving the bedding were only noted transitorily in isolated animals presumably as a local reaction to the test item and thus, they are not considered to be toxicologically relevant.
Low incidences of slight clinical signs like alopecia on various body parts, nasal discharge or a crust at the cheek were noted in isolated males and/or females of the dose groups and/or the control groups. As these findings were mostly transient and seen irrespective of the groups in isolated animals, they are considered to be incidental.

Body Weight Development
In both males and females, the mean body weight increased with the progress of the study during premating, mating/postmating and gestation period in the control groups, the LD, the MD and the HDsup group. Values were within the normal range of variation of historical control data.
There were no test item-related effects of toxicological relevance noted on body weight and body weight gain in both males and females.
Generally, body weight of both the male and female Csup and HDsup group was slightly lower compared to the C, LD and MD group, respectively (up to 6 % in males and up to 8 % in females).This finding can be attributed to the fact that these supplementary animals were delivered in an extra batch.
Throughout the premating, mating and post-mating periods of male animals there were no statistically significant differences in body weights between male animals of LD or MD and C groups and between male animals of HDsup and the Csup group. Moreover, no statistically significant difference in body weight was observed in female animals between LD or MD and C groups and between HDsup and the Csup group during premating, gestation and lactation.
Due to a generally slightly lower body weight in supplementary animals (see above), a comparison in body weight gain between LD or MD and HD is not possible. However, it was found that in male animals statistically significantly lower body weight gain was observed in LD and MD groups when compared to C group (38 % and 35 %) between premating day 7 and 14. As, however, no significant difference was observed during this time between HDsup and Csup groups, this transiently lower body weight gain in dosed animals is not assumed to be related to toxic characteristics of the test item. Moreover, no decrease in body weight gain was observed in female animals treated with FAT 40171/Y TE during this time period.

Food Consumption
In correlation to the body weight and body weight change, the food consumption of the control groups, the LD, MD and HDsup group increased with progress of the study for males during premating period and for females during premating and first two weeks of gestation. In the last week of gestation food consumption of females remained roughly constant or decreased slightly. Values were within the normal range of variation for animals of this strain and age.
A slightly – but statistically significantly higher food consumption in male animals of the HDsup group, when compared to Csup group (approx. 8 %), during the second premating week is not assumed to be toxicologically relevant.
In the first week of premating, food consumption of the female MD group was slightly, but not statistically significantly, lower compared to the control groups and dose groups. This finding is based on the low food consumption of two individual females housed in one cage (no. 69 and no. 70) and therefore is considered incidental.
Besides, there were no considerable differences in food consumption of male and female animals between LD, MD and C groups and between HDsup and Csup groups during this study.

Pathology- Macroscopic Findings
However, at necropsy a treatment-related, dose-dependent macroscopic finding consisted of dark and/or blue discolouration of several organs in males and females of the MD and HDsup group as well as of blue discolouration of the whole organism in males of the HDsup group. Regarding the LD group, kidneys of 5 females were also discoloured dark. Since the marcroscopic finding of dark/blue/dark blue discolouration was presumably due to the dark blue colour of the test item used in this study, the blue discoloured organs were not subjected to microscopic examination. For dark discoloured organs, no histological correlate was seen at histopathological examination.
Female no. 41 of the C group did not litter before the end of the study (gestation day 26) presumably because of parturition difficulties. At necropsy, one dead foetus was found in a contorted uterus which was assumed to be the reason for the pregnancy complication. Furthermore, a node (20x10mm) was seen in the uterus. The left ovary, oviduct and cervix could not be found. The spleen was enlarged and the liver discoloured yellowish. The macroscopic findings and the resulting deteriorated health condition of female no. 41 are presumably based on a spontaneous uterine torsion which is considered incidental. The few other gross lesions recorded were considered to be within the range of normal background alterations, which may be seen in rats of this strain, age and this type of study. Testes were enlarged in one male of the Csup group and one testis was missing in one male of the C group. There was a yellow spot/nodule at the epididymis in each one male of the C and Csup group which is considered to correspond to spermatic granuloma, a lesion known to occur spontaneously in rats of this strain and age. Few further gross lesions were observed in single males of control or dose groups such as small epididymis, small or enlarged seminal vesicles, enlarged coagulating glands and prostate or pelvic dilation of the kidney. In females, further findings consisted of a small thymus in one female of the C group, discoloured pale kidneys in 3 females of the Csup group and discoloured pale adrenal glands in one female of the MD group. The distribution of those findings among the groups is considered to be incidental, reflecting the usual individual variability.

Organ Weight
In females, there were no statistically or biologically significant effects on the absolute and relative organ weights in the dose groups, when compared to the respective control group. In males, there was a slightly, but statistically significantly higher mean weight of epididymides in the MD group compared to the C group (absolute weight: 15 %; relative weight: 20 %). As no significant difference was noted between the Csup and HDsup group and without following a dose response pattern this is considered to be incidental. Mean relative weight of testes was also slightly, but statistically significantly higher in the MD group (114 % of controls) compared to the C group. Without dose dependency this is considered to be incidental. There were no effects of toxicological relevance on the mean weight of prostates.

Histopathology
Under the conditions of this study, the test item FAT 40171/Y TE produced no histological evidence of toxicological properties in the organs and tissues of the reproductive system; i.e. testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, uterus with cervix, and vagina. In addition, as a result of the detailed examination of the testes, it was judged that there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure. The cause of the unsuccessful pregnancy and/or infertility of female no. 101 and 102 of the Csup group and their male pairing partners no. 81 and 82, respectively, could not be established from the reproductive organs examined from these animals. Organs and tissues with the macroscopic finding of blue/dark blue discolouration were not subjected to microscopic examination, as this finding was due to the dark blue colour of the test item used in this study. All other gross lesions recorded were considered to be within the range of normal background alterations.

Dose Formulation Analysis
Concentration analysis of formulation samples was determined in study weeks 1, 3, 5 and in the last week for all dose groups. The mean recoveries observed in the LD, the MD and the HDsup group were 95.3 %, 92.3 % and 89.2 % of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as mean of measured concentration did not differ from nominal concentration by more than 20 %. Stability of formulation samples was investigated in study week 1 for the LD and the HDsup group. After 6 hours of storage at room temperature, recovery compared to starting value was 101.6 % and 99.7 % for LD and HDsup group, respectively. After 10 days of storage at 2-8 °C, recovery compared to starting value was 104.4 % and 100.0 % for LD and HDsup group, respectively. All samples were stable, as concentration after storage did not differ from start value by more than 20 %. Homogeneity of formulation samples was determined in study week 1 and 5 for the LD and the HD group. The mean recovery observed for the LD group was 93.6 % and 92.0 % of the nominal value and 87.2 % and 91.3 % of the nominal value for HDsup dose group. The coefficients of variation of the different sampling locations (top, middle, bottom) were 1.0 % and 3.4 % in LD dose group and each 0.3% (in study week 1 and 5) in HDsup dose group. All samples were homogenous, as COV was below 20 %.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Remarks on result:
other: Generation: P + F1
Clinical signs:
not examined
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:
no effects observed
Histopathological findings:
not examined
Litter Data
There were no effects of toxicological relevance on litter data including total number of pups born, number of live pups, still births and runts on PND 0 as well as number of male pups, number of female pups and sex ratio on PND 0 and PND 4 of the dose groups, when compared to the respective control groups. There were no statistically significant changes noted for these litter data.

Litter Weight Data
There were no statistically or biologically significant effects on the litter weight data including pup mean weight, total litter weight, male and female litter weight on PND 0 and PND 4 of the dose groups, when compared to the respective control groups. Values were within the normal range of variation for animals of this strain.

Precoital Interval and Duration of Gestation
There were no statistically or biologically significant effects on the duration of precoital interval and the duration of gestation in the dose groups, when compared to the respective control groups. Values were within the normal range of variation of historical control data.

Pre- and Post-Natal Data
There were no statistically or biologically significant effects on the number of corpora lutea, number of implantation sites, number of live pups (PND 0 and PND 4) and percentage of pre- and post-implantation loss in the dose groups, when compared to the respective control groups. A slight tendency towards a higher number of corpora lutea in the HDsup group (14.40) compared to the respective Csup group (12.75) is considered incidental, as no dose dependency was observed.
The HDsup group was noted to have a slightly higher percentage of pre-implantation loss compared to the other groups (HDsup: 20 %, C: 7 %, Csup: 10 %, LD: 12 %, MD: 4 %). Values were within the range of historical control data and lacked statistical significance and dose-dependency. Thus, this effect cannot be attributed to the treatment with the test item.

Reproductive Indices
There were no treatment-related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared to the respective control groups. A slightly reduced copulation index (number of copulated females / number of pairs x 100) was only observed in the Csup group (90 % compared to 100 % in all other groups) and is considered to be incidental. The fertility index (number of pregnant females/ number of copulated females x 100) was also slightly reduced in the Csup group (88.9 %) compared to 100 % in all other groups. The cause of the unsuccessful copulation and/or pregnancy and/or infertility of females no. 101 and 102 of the Csup group as well as their male pairing partners (no. 81 and 82), respectively, could not be established from the reproductive organs examined histopathologically from these animals. A slightly reduced delivery index in the C group (90 %) compared to 100 % in the Csup group and the dose groups was based on pregnant female no. 41 of the C group which did not litter before the end of the study due to parturition difficulties. At necropsy one dead foetus was found in a contorted uterus. The remaining pregnant females of the C group littered normally. There were no treatment-related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared to the respective control groups. A slightly reduced copulation index (number of copulated females / number of pairs x 100) was only observed in the Csup group (90 % compared to 100% in all other groups) and is considered to be incidental. The fertility index (number of pregnant females/ number of copulated females x 100) was also slightly reduced in the Csup group (88.9 %) compared to 100 % in all other groups. The cause of the unsuccessful copulation and/or pregnancy and/or infertility of females no. 101 and 102 of the Csup group as well as their male pairing partners (no. 81 and 82), respectively, could not be established from the reproductive organs examined histopathologically from these animals. A slightly reduced delivery index in the C group (90 %) compared to 100 % in the Csup group and the dose groups was based on pregnant female no. 41 of the C group which did not litter before the end of the study due to parturition difficulties. At necropsy one dead foetus was found in a contorted uterus. The remaining pregnant females of the C group littered normally. The viability index (no. of live offspring at day 4 / no. of live offspring at birth) X 100) was marginally lower in the Csup group (98.96 %), the LD group (99.09 %) and the HDsup group (99.17 %) when compared to 100 % in each the C and MD group. As this was attributed to the death of one pup in one single dam per group, respectively, and without dose-dependency, this finding is considered incidental.

Pup Survival Data
There were no statistically or biologically significant effects on the survival of the pups from PND 1 through PND 4 in the dose groups, when compared to the control group. A marginally higher mean mortality of pups between PND 1 and PND 4 was observed in the Csup group (1.04 %), the LD group (0.91 %) and the HDsup group (0.83 %) compared to 0.0 % in the C and MD group. As this outcome did not achieve statistical significance and did not show a dose response pattern, it is considered incidental and not related to the test item. Regarding female no. 103 of the Csup group and female no. 58 of the LD group, one pup was missing on PND 1, respectively. The loss of pups was attributed to cannibalism by the dam. One pup of female no. 132 of the HDsup group was found dead on PND 1.

Pup External Findings
No gross external abnormalities of toxicological relevance were observed in any of the groups. A dark snout observed in one single pup of the C group, one single pup of the Csup group and two pups of the LD group is considered incidental.
There were few incidental further external findings such as a wound in two pups of the C group and one pup of the MD group as well as a swollen eye in one single pup of the LD group and a cold pup with pale skin in the MD group. These findings are considered to be spontaneous and not related to the test item.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Reproductive effects observed:
not specified
Conclusions:
On the basis of this reproduction/ developmental toxicity screening test with the read across substance, FAT 40171/Y TE in male and female Wistar rats with dose levels of 100, 300 and 1000 mg/kg bw/ day the following conclusions can be made:
No adverse effects of FAT 40171/Y TE were found at dose levels of 100, 300 and 1000 mg/kg bw/day. Thus, the NOAEL of FAT 40171/Y TE in this study is considered to be 1000 mg/kg bw/day.

Executive summary:

The aim of this study was to assess the possible effects of the read across substance, FAT 40171/Y TE on male and female fertility and embryofetal development after repeated dose administration in Wistar rats. The test item was administered daily in graduated doses to 3 groups of test animals (low dose: LD, medium dose: MD and high dose: HD), 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 days were completed. Animals of one additional control group were handled identically as the dose groups but received aqua ad injectionem, the vehicle used in this study. The control group was shared with BSL Study No. 140337. The 4 groups comprised 10 male and 10 female Wistar rats. On day 7 HD animals were excluded from the study due to wrong dosing and a supplementary group HDsup was introduced (10 males and 10 females). As these animals were taken from a different batch, additionally a supplementary control group Csup was introduced (10 males and 10 females; also shared with BSL Study No. 140337). 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, 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 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. 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 treatment day 29 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females were sacrificed on day 26.

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.

A full histopathological evaluation of the tissues was performed on Csup and HDsup animals. Only organs and tissues of the other dosage groups showing changes in the high dose group were examined. Any gross lesions macroscopically identified were examined microscopically in all animals. The following doses were evaluated:

Control: 0 mg/kg body weight

Control sup: 0 mg/kg body weight

Low Dose:100 mg/kg body weight

Medium Dose: 300 mg/kg body weight

High Dose sup: 1000 mg/kg body weight

The test item formulation was prepared at least every ten days. Prepared test item formulations were stored at 2-8 °C, but warmed to room temperature before dose 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 28 days. Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight.

Summary Results

No mortality occurred in the control groups or any of the dose groups during the treatment period of this study. There were no clinical signs of toxicological relevance in the dose groups, when compared to the control groups. However, the skin of all male and female animals of the HDsup group was observed to be discoloured blue from the 7th day of treatment until the end of the study. Blue coloured faeces were noted in both males and females of the LD and the MD group from the 4th day of treatment and in both males and females of the HDsup group from the 3rd day of treatment until the end of the study, respectively. Discolouration of the skin and the faeces was considered to be based on the properties of the blue coloured test item. In both males and females, the mean body weight increased with the progress of the study during premating, mating/postmating and gestation period in the control groups, the LD, the MD and the HDsup group. Values were within the normal range of variation of historical control data. There were no test item-related effects of toxicological relevance noted on body weight and body weight gain in both males and females. There were no biologically significant effects on food consumption during the treatment period in both males and females of the dose groups, when compared to the respective controls. There were no effects of toxicological relevance on litter data including total number of pups born, number of live pups, still births and runts on PND 0 as well as number of male pups, number of female pups and sex ratio on PND 0 and PND 4. There were no statistically or biologically significant effects on the litter weight data including pup mean weight, total litter weight, male and female litter weight on PND 0 and PND 4. There were no statistically or biologically significant effects on the duration of precoital interval and the duration of gestation in the dose groups, when compared to the control groups. There were no statistically or biologically significant effects on the number of corpora lutea, number of implantation sites, number of live pups (PND 0 and PND 4) and percentage of pre- and post-implantation loss in the dose groups, when compared to the control groups. There were no treatment-related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared to the control groups. There were no statistically or biologically significant effects on the survival of the pups from PND 1 through PND 4 in the dose groups, when compared to the control group. No gross external abnormalities of toxicological relevance were observed in any of the groups. There were no macroscopic findings of toxicological relevance in any of the groups. Dose-dependent macroscopic findings related to the test item were dark and/or blue discolouration of several organs in males and females of the MD and HDsup group as well as of blue discolouration of all visible organs and tissues in males of the HDsup group. Regarding the LD group, kidneys of 5 females were also discoloured dark. Since the marcroscopic finding of dark/blue/dark blue discolouration was presumably due to the dark blue colour of the test item used in this study, the blue discoloured organs were not subjected to microscopic examination. For dark discoloured organs, no histological correlate was seen at histopathological examination. The test item produced no histological evidence of toxicological properties in the organs and tissues of the reproductive system; i.e. testes, epididymides, prostate, seminal vesicles, coagulating glands, ovaries, uterus with cervix, and vagina.

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. The cause of the unsuccessful pregnancy and/or infertility of female no. 101 and 102 of the Csup group and their male pairing partners no. 81 and 82, respectively, could not be established from the reproductive organs examined histopathologically from these animals. Concentration of FAT 40171/Y in formulation samples was determined in study weeks 1, 3, 5 and in the last week for all dose groups. The mean recoveries observed in the LD, the MD and the HDsup group were 89.2 %, 92.3 % and 95.3 % of the nominal concentration, respectively. Stability of formulation samples was investigated in study week 1 for the LD and the HD group. After 6 hours of storage at room temperature, recovery compared to starting value was 101.6 % and 99.7 % for LD and HD group, respectively. After 10 days of storage at 2-8 °C, recovery compared to starting value was 104.4 % and 100.0 % for LD and HD group, respectively. Homogeneity of formulation samples was determined in study week 1 and 5 for the LD and the HD group. The mean recovery observed for the LD group was 93.6 % and 92.0 % of the nominal value and for HDsup dose group 87.2 % and 91.3 % of the nominal value. The coefficients of variation of the different sampling locations (top, middle, bottom) were 1.6 % and 3.4 % in LD dose group and each 0.3 % (in study week 1 and 5) in HDsup dose group.

On the basis of this reproduction/developmental toxicity screening test with FAT 40171/Y in male and female Wistar rats with dose levels of 100, 300 and 1000 mg/kg body weight/ day the following conclusions can be made:

No adverse effects of FAT 40171/Y were found at dose levels of 100, 300 and 1000 mg/kg body weight. Thus, the NOAEL of FAT 40171/Y in this study is considered to be 1000 mg/kg body weight.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Currently no study assessing the adverse effects of Reactive Orange 35 on reproduction and development are available. However, the read-across substance, Reactive Black 039 pentasodium salt was assessed for such effects in a study conducted according to the OECD Guideline 421. The aim of this study was to assess the possible effects of the read across substance, FAT 40171/Y on male and female fertility and embryofetal development after repeated dose administration in Wistar rats.The test item was administered daily in graduated doses to 3 groups of test animals (low dose: LD, medium dose: MD and high dose: HD), 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 days were completed. Animals of one additional 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.

The following doses were evaluated:

Control: 0 mg/kg bw/day

Control sup: 0 mg/kg bw/day

Low Dose: 100 mg/kg bw/day

Medium Dose: 300 mg/kg bw/day

High Dose sup: 1000 mg/kg bw/day

No adverse effects of FAT 40171/Y on reproduction and developmental parameters were found at dose levels of 100, 300 and 1000 mg/kg bw/day. Thus, the NOAEL of FAT 40171/Y in this study is considered to be 1000 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

The NOAEL for developmental toxicity was 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Good quality GLP study
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

On the basis of this reproduction/developmental toxicity screening test with read across substance FAT 40827/B in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg/d the following conclusions can be made: Treatment with FAT 40827/B by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg revealed no parental toxicity up to 1000 mg/kg. Reproduction toxicity was observed at 1000 mg/kg and developmental toxicity was noted at 300 mg/kg. Hence, the read across test substance FAT 40827/B can be regarded as not classified for reproduction /developmental toxicity. Based on this data for similar substance, the target test substance can be considered as not classified for reproduction /developmental toxicity.