Sodium 6-amino-5-[[2-[(cyclohexylmethylamino)sulphonyl]phenyl]azo]-4-hydroxynaphthalene-2-sulphonate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The no observed adverse effect level (NOAEL) of FAT 20033/K TE is considered to be 1000 mg/kg bw/d for reproduction toxicity in males and females.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22-04-2014 to 08-10-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Inadvertently, the behavioural observations before the first treatment were not performed in few animals

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Limit test:

no

Specific details on test material used for the study:

Name: FAT 20003/K TE
Common Name: TECTILON RED 2B CRUDE MOIST (LAB DRIED)
Chemical Name: sodium 6-amino-5-[[2-[(cyclohexylmethylamino) sulphonyl]phenyl]azo]-4-hydroxynaphtalene-2-sulphonate
Batch No.: AT-0026132600
CAS No.: 32846-21-2
Physical State / Colour: solid / brown
Storage Conditions: room temperature, protected from light
Molecular Weight of Base Form: 517.60 g/mol
Molecular Weight of Salt Form: 540.59 g/mol
Active Components: > 90%
Date of Analysis: 07 January 2014
Expiry Date: 20 December 2018.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat is the preferred rodent species.

Sex:

male/female

Details on test animals or test system and environmental conditions:

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: 10-11 weeks old, females: 10-11 weeks old.

Body weight at the allocation of the animals to the experimental groups:
males: 255 - 287 g (mean: 273.78 g, ± 20 % = 219.02 – 328.53 g)
females: 166 - 202 g (mean: 182.78 g, ± 20 % = 146.22 – 219.33 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.
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 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.

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

corn oil

Details on exposure:

The test item was weighed into a tared plastic vial on a precision balance and suspended in corn oil. 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 room temperature and the homogeneity was ensured by vortexing the sample on vortex machine.

The following doses were evaluated:
Control: 0 mg/kg bw/d
Low Dose: 100 mg/kg bw/d
Medium Dose: 300 mg/kg bw/d
High Dose: 1000 mg/kg bw/d

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 doses were selected on the basis of data from a 14 day Dose Range Finding Study (BSL study no. 140257)
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. 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 and 10 days after the preparation (stored at room temperature)], from high and low dose formulations (4 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. 140317.

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.

Frequency of treatment:

7 days a week.

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: 25 July 2014
Delivery of Animals: 06 May 2014
Acclimatisation Period: 06 May 2014 – 13 May 2014
Experimental Starting Date: 14 May 2014
Date of Behavioural Observation: 14 May 2014- 27 June 2014
Treatment Period Males: 15 May 2014 – 12 June 2014
Treatment Period Females: 15 May 2014 – 28 June 2014
Necropsies Males: 12 June 2014 and 13 June 2014
Necropsies Females: 24 June 2014 – 29 June 2014
Experimental Completion Date: 29 June 2014
Completion Date of Delegated Phase (Histopathology): 29 September 2014
Completion Date of Delegated Phase (Formulation Analysis): 29 September 2014

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low dose (LD)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Medium dose (MD)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose (HD)

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

Positive control:

None

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

Oestrous cyclicity (parental animals):

None

Sperm parameters (parental animals):

None

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes]
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities]

GROSS EXAMINATION OF DEAD PUPS:
[yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

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
Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.

Non-pregnant (48 and 73) and non-mated females (71) were sacrificed on study day 26 from the day of confirmed mating and 26 day 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 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
The wet weight of the organs (liver, kidneys, adrenals, testes, epididymides, prostate with seminal vesicles and coagulating glands,
ovaries, uterus with cervix, thymus, thyroid/parathyroid glands, spleen, brain, pituitary gland, heart) of 5 males and 5 females randomly
selected from each group was recorded as soon as possible. Paired organs were weighed together. In addition reproductive organs
of all animals were weighed.

The following tissues (brain -cerebrum, cerebellum and pons, spinal cord, liver, kidneys, adrenal glands, stomach, small and large intestines
(including Peyer´s patches), thymus, thyroid, spleen, lung and trachea, mammary glands, gross lesions, skin, heart, ovaries (females) , uterus with cervix (females), vagina (females), testes (males), epididymides (males), prostate and seminal vesicles with coagulating glands as a whole (males), urinary bladder, lymph nodes (mesenteric and axillary), peripheral nerve (e.g. sciatic nerve) with skeletal muscle, bone with bone marrow (sternum), pituitary gland, oesophagus of the same selected animals from each group were preserved in 10% neutral buffered formalin except eyes, testes and epididymides that were fixed in Modified Davidson’s Fixative for approximately 24 hours before they were transferred to 70% ethanaol.

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).

Clinical signs:

no effects observed

Description (incidence and severity):

In males and females, there were no clinical signs of toxicological relevance in any of the dose groups when compared to the control. However, there were isolated incidences of alopecia on various body parts, slight crust behind left eye, moving the bedding, red nasal discharge, regurgitation, slight to severe salivation, abnormal breathing, and moderate piloerection observed in a few control and/ or dose group animals. Discoloured red faeces was observed in all male and female animals of all dose groups and could be attributed to the colour of the test item. Moving the bedding and salivation in all HD animals and few MD animals is assumed to be a local effect and not considered to be systemic effect due to the test item administration.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in the control or any of the dose groups during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In males, no test item related adverse effect was observed on body weight and body weight gain during the entire study duration. However, in HD group marginally lower (1-6%) absolute body weight was observed during the mating and post mating periods and on the day of terminal sacrifice, when compared to the controls. This was associated with a statistically significantly lower body weight gain during day 1-7 of premating period and overall period from premating day 1 to post-mating day 14 in male HD group animals when compared to the controls. As this difference was marginal and values were within the range of biological variation and therefore no toxicological relevance could be attributed to this effect on male body weight development.

In females, there were no adverse effects of FAT 20003/K TE on body weight and body weight gain in dose groups when compared to the controls, throughout the study period. A statistical analysis of data revealed no statistically significant difference between the dose groups and corresponding control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Throughout the study period, no test item related adverse effect FAT 20003/K TE on food consumption was observed. In males and females of all dose groups when compared to the corresponding controls. However, statistically significantly lower food consumption in males and female HD group was observed in first week of the treatment (Day 1-7) when compared to the controls. As this difference was marginal and correlated with body weight development it is not considered to be adverse. In correlation to the body weight and body weight gain, the food consumption in both male and female animals was increased after first week of the treatment in all dose groups including control group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

In males and females, there were no adverse effects of FAT 20003/K TE on haematology and blood coagulation parameters in dose groups when compared with the corresponding control. However, in males there was marginally but statistically significantly higher monocyte count in HD group and eosinophil count in LD group was observed when compared with the controls. As the mean values were in the range of historical control data and in the absence of dose dependency, the finding was not considered toxicologically relevant.

In females, statistical analysis of haematology and blood coagulation data revealed no statistically significant differences in dose groups when compared with the controls and all mean and individual values were within the historical control range.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In males and females, no adverse effects of FAT 20003/K TE on clinical biochemistry were observed in dose groups when compared to the controls. However, in females, a statistically significantly lower ASAT level in LD and HD groups and AP level in LD group was observed when compared with the controls. As all serum levels of ASAT and AP were within the respective historical control range, they are not considered toxicologically relevant.

All other values of clinical biochemistry of males and females were within the historical control range and no statistically significant differences were found between dose groups and control group.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no signs of toxicological relevance in urine parameters of males and females dose groups when compared to the controls.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. However, statistically significantly higher body temperature in last week of treatment was observed in MD and HD group when compared with the controls. As this difference was marginal, the finding was not considered toxicologically relevant.

Before treatment, a statistically significantly higher count of supported rearing was observed in females of all dose groups when compared with the controls. As this difference was marginal and observed before the initiation of treatment, this reflects no adverse effect of the test item.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All 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 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.
No histomorphological evidence of toxicological properties in any organs and tissues of male and females were established up to 1000 mg/kg bw/d.

Other effects:

not specified

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

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No toxicologically relevant adverse effects were seen

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

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

not examined

Ophthalmological 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

Litter Data:
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. All group mean and individual values for various litter data parameters in the dose groups were comparable with the controls.

Litter Weight Data:
Statistical analysis of litter weight data revealed no treatment related effect of FAT 20003/K TE 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.

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. 56 from LD group that had no litter but revealed implantation sites at the time of necropsy on gestation day 26.
The majority of females in control and treatment groups showed evidence of copulation during the 14 day mating period except one female from HD group (no. 71) that did not show evidence of mating through vaginal smear and was sacrificed 26 days after the completion of the mating period. There were also two females (48 and 73) from control and HD group which did show evidence of mating in the form of sperm positive vaginal smears but had no litters and were sacrificed on gestation day 26.
Successful mating resulted in 9/10, 10/10, 10/10 and 8/10 pregnancies in C, LD, MD and HD groups, respectively.

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, the 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 in LD group, without achieving statistical significance, compared to the control group. This increase was attributed to one female (no. 56) which showed implantation sites at necropsy on gestation day 26 and in other female (no. 55) there were no live pups but all 9 pups born were dead (still birth). Due to lack of dose dependency, the higher percent post implantation loss in LD group was not considered to be related to administration of the test item.

Reproductive Indices:
There were no test item effects observed on the copulation, fertility and delivery indices of the dose groups when compared to the control. The copulation index was 100 % in Control, LD, MD groups and 90 % in the HD group. The fertility index was 90 % in control, 100 % in LD and MD 100 and 80 % in HD group. The delivery index was 100 % in control, MD, HD group and 80 % in LD group.
There was no test item related effect on the viability index in dose group when compared to the control. However, reduction in viability index was observed in LD (86.11%) and HD (87.50%) group and attributed to one female (no. 52 and 74), respectively in which, all pups were either cannibalized or found dead before PND 4. This finding was considered to be incidental and without any toxicological significance.


Pup Survival Data:
There was no test item related effect on the survival of the pups from PND 0 through PND 4 in dose groups when compared to the control. However, one pup from LD group female 52 (pup no. 1) and one pup from LD group female 59 (pup no. 5) was missing on PND 1. In HD group, from female 74, seven pups (1, 2, 4, 5, 7, 8, 9) were missing on PND 3-4 and 4 pups (3, 6, 10, 11) were found dead on PND 2-3.
These missing pups were cannibalised by their dam. Since this incidence of cannibalism was observed in just three females without showing any dose dependency and was within the rat cannibalism rate, this incidence was not considered to be test item related. Furthermore, in HD group, mortality and cannibalism were observed in just one female litter and in remaining 7 litters with pups, no mortality or cannibalism was observed. Therefore, this cannibalism and mortality in HD group was considered as an isolated incidence and not related to the treatment with the test item.

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. 8) was found cold to touch at the time of observation on PND 0.
In LD females, all 8 pups from female 51 had dry skin, one pup from female 52 (pup no. 1) had a small head, seven pups from female 54 (pup no. 1, 3, 5-6 and 9-11) had dry skin, one pup from female 54 (pup no. 7) had a dark snout and all 9 pups from female 59 were found cold to touch and no milk was visible in the stomach on PND 0.
In the MD group, all 10 pups from female 64 were found cold to touch at the time of observation on PND 0, two pups from female 66 (pup no. 2 and 11) had a dark snout, one pup from female 67 (pup no. 1) had a hematoma at the base of the tail and one pup from female 70 (pup no. 1) was found cold to touch at the time of observation and had a dark spot on the abdomen on PND 0. There was also one pup from female 69 (pup no. 10) with a dark head on PND 4.
In HD females, one pup from female 76 (pup no. 1) had a dark head and shoulders on PND 0 and all pups from female 72 were found cold to touch on PND 2.
All these findings were considered to be spontaneous in nature and not related to the treatment with test item.

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 toxicologically relevant adverse effects were seen.

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

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/K TE is considered to be 1000 mg/kg bw/d 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 20003/K TE on male and female fertility and embryofetal development after repeated dose administrationin Wistar rats. The test was performed according to OECD test guideline 422.

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 corn oil, the vehicle used in this study. The 4 groups comprised 10 male and 10 femaleWistarrats.

The following doses were evaluated:

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

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

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

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

The test item formulation was once in every ten days and stored at room temperature. The test item was suspended in corn oil and dose volumes were adjusted individually based on weekly body weight measurements. Theadministration volume was 5 mL/kg body weight.

During the period of administration, the animals were observed each day for signs of toxicity. At the end 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. Haematological and clinical biochemistry evaluations were performed on blood samples collected at terminal sacrifice from five males and five randomly selected lactating females from each group. 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. 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 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.

 

Dead pups and pups sacrificed on postnatal 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.

 

Summary Results:

- No mortality occurred in the control or any of the dose groups during the study period.

- In males and females, there were no clinical signs of toxicological relevance in dose group.

- During the weekly detailed clinical observation, no significant clinical findings 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 of FAT 20003/K TE 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 of FAT 20003/K TE on urine parameters of males and females dose groups when compared to the control.

- No gross lesions were observed in this study that could be attributed to treatment with FAT 20003/K TE. 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. However, statistically significantly higher group mean absolute ovary weight was observed in LD females when compared with the controls. Due to a lack of dose dependency and consistency, this effect on ovary weight in LD females was of no toxicological relevance.

 

- Microscopically, no histomorphology evidence of toxicological properties 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 in any organs and tissues of male and females were established up to 1000 mg/kg bw/d.

- 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 precoital 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 were unaffected after treatment with FAT 20003/K TE, when compared with the control group. However, there was higher post-implantation loss in LD group without achieving statistical significance compared to the control group. Due to lack of dose dependency, the higher percent post implantation loss in LD group was not considered to be an adverse effect of treatment with the test item.

 

- No effects of test item were observed on the copulation, fertility, delivery and viability indices of the dose groups when compared to the control.

- No test item related effect 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.

- Dose formulation analysis for nominal concentration, stability and homogeneity revealed that all dose formulations were stable and homogenous at the time of application throughout the study period.

 

Conclusion

Based on this combined repeated dose oral toxicity and reproduction/developmental toxicity screening test with FAT 20003/K TE in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg/d 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/K TE is 1000 mg/kg/d for systemic toxicity and also for reproduction/ developmental toxicity in males and females.

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

Quality of whole database:

High quality GLP study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A combined repeated dose oral toxicity and reproduction/developmental toxicity screening test with FAT 20003/K TE in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg bw/d was carried out. There were no adverse toxicological effects in the adult males and females or significant reproductive effects at any administered dose. Thus, the no observed adverse effect level (NOAEL) of FAT 20033/K TE is considered to be 1000 mg/kg bw/d for systemic toxicity and also for reproduction toxicity in males and females.

Effects on developmental toxicity

Description of key information

The NOAEL of FAT 20003/K for developmental toxicity was considered to be 1000 mg/kg bw/d.

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:

High 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

Additional information

In the combined repeated dose oral toxicity and reproduction/developmental toxicity screening test with FAT 20003/K TE in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg/d, there were no developmental effects in pups at any administered dose. Thus, the no observed adverse effect level (NOAEL) of FAT 20033/K TE is considered to be 1000 mg/kg bw/d for developmental toxicity in males and females.

Justification for classification or non-classification

Based on the above stated assessment of the reprotoxic potential of sodium 6-amino-5-[[2-[(cyclohexylmethylamino)sulphonyl]phenyl]azo]-4-hydroxynaphthalene-2-sulphonate in a combined repeated dose reproductive and developmental screening test (OECD 422), the substance does not need to be classified as a reproductive or developmental toxicant according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and according to CLP (Regulation (EC) No 1272/2008 Of The European Parliament And Of The Council) as implementation of UN-GHS in the EU.