Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2017-04-21 to 2018-mm-dd
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
See chapter 13 for support for read-across within the category of Alkyl Naphthalene Sulfonates (ANS).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Based on stability data (Eurofins Munich study no. 166362) the test item and control formulations were prepared at least once every ten days and stored at room temperature and the repetition of homogeneity measurement in the main study was not necessary.
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Naphthalenesulfonic acid, bis(1-methylethyl)-, Me derivs., sodium salts
EC Number:
272-715-8
EC Name:
Naphthalenesulfonic acid, bis(1-methylethyl)-, Me derivs., sodium salts
Cas Number:
68909-82-0
Molecular formula:
UVCB substance
IUPAC Name:
Aromatic hydrocarbons, C10-13, reaction products with isopropylalcohol, sulphonated, sodium salts
Test material form:
solid: granular
Details on test material:
Name: Naphthalenesulfonic acid, bis(1-methylethyl)-, methyl derivs., sodium salt
Product: MORWET IP Powder
Chemical Name: Naphthalenesulfonic acid, bis(1-methylethyl)-, methyl derivs., sodium salt
CAS No.: 68909-82-0
Batch No.: 1452486
Physical State: solid, powder
Colour: tan
pH: 7.5 to 10 in 5 % solution
Active Components: UVCB substance with 100% purity
Average molecular weight: 346 g/mol (range from 213-474 g/mol)
Purity: 100 %
Storage Conditions: room temperature
Expiry Date: 07 June 2021
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test System
Species/strain: 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: approx. 14-15 weeks old
Body weight at the allocation of the animals to the experimental groups:
males: 338 - 380 g
(mean: 360.23 g, ± 20 % = 288.18 – 432.27 g)
females: 212 - 254 g
(mean: 234.20 g, ± 20 % = 187.36 – 281.04 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes.
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.

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
- 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 5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages during the premating period for both males and females and during post-mating period for 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 in type III H, polysulphone cages and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
- Nesting material were provided latest on GD 18 for all mated females
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. None of the animal showed pathological signs before the first administration.
Before dosing all females were screened for two weeks for regular estrous cyclicity and animals (10 females/ group) with regular estrous cycle (4-5 day cycle) were used in the study.
Before the first administration all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively. Randomisation was performed with validated IDBS Workbook 10.1.2 software.
Each animal was marked with its identification number by individual ear tattoo or tail marking.




Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: aqua ad iniectabilia (sterile water)
Remarks:
Manufacturer: AlleMan Pharma, Batch No.: 601101, 511535, 605070, 612118, Expiry Date: 12/2018 (601101), 10/2018 (511535), 04/2019 (605070), 11/2019 (612118)
Details on exposure:
The test item was weighed into a tared plastic vial on a precision balance.
The test item was dissolved in aqua ad iniectabilia (Sterile Water).
The dose formulations were prepared by adding the required volume of vehicle and further vortexing it for 2-3 minutes.
The vehicle was selected following solubility check and also in consultation with sponsor. The test item and control formulations were prepared at least once in 10 days based on the available stability results (Eurofins Munich Study No.166362). The prepared formulation was stored at room temperature.
The vehicle was also used as control item.


According to the results of the dose range finding study (BSL Munich study no. 163485) and in consultation with the sponsor the following doses were selected for the three dose groups (LD = low dose, MD = medium dose, HD = high dose) and one control group (C). The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Control: 0 mg/kg/d
Low Dose: 300 mg/kg/d
Medium Dose: 200 mg/kg/d
High Dose: 700 mg/kg/d
High Dose*: 500 mg/kg/d
* = the high dose level was reduced from 700 mg/kg bw to 500 mg/kg bw after 6 or 7 days of dose application initiation (staggered start of dosing) due to overt toxicity.

The highest dose level was chosen with the aim of inducing toxic effects, but no 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 animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the high dose group.


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






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 mating. 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
Remarks:
During the study samples were collected for the investigation of substance concentration.
Details on analytical verification of doses or concentrations:
Samples were taken from the middle of prepared formulations from all dose groups and from the middle of the control group in study week 1 (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).
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were analysed at Eurofins Munich and until then stored under appropriate conditions based on available stability data (Eurofins Munich Study No. 166362). The procedures followed for the study sample analysis were mentioned in a phase plan (study no. 166363) that was amended to the study plan. The B-samples were retained at -15 to -35 °C at BSL Munich (test facility) and discarded after completion of the final study report. The results were reported in the appendix 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 maximum period of 63 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.
Frequency of treatment:
daily
Details on study schedule:
Arrival of the Test Item: 01 September 2016
Study Initiation Date: 21 April 2017
Amendment to Study Plan: 19 May 2017
2nd Amendment to Study Plan: 23 May 2017
3rd Amendment to Study Plan: 29 May 2017
Delivery of Animals: 04 May 2017
Acclimatisation Period: 04 May 2017 to 09 May 2017
Experimental Starting Date: 09 May 2017
Treatment Period: 23 May 2017 to 15 July 2017
Necropsies: 20 June 2017, 21 June 2017, 02 July 2017, 05 July 2017, 10 July 2017 to 16 July 2017
Experimental Completion Date: 16 July 2017
Completion Date of Delegated Phase (Histopathology): xx
Completion Date of Delegated Phase (Formulation Analysis): 07 September 2017
Study Completion Date: xx




Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
30 mg/kg bw/day
Dose / conc.:
200 mg/kg bw/day
Dose / conc.:
700 mg/kg bw/day
Remarks:
the high dose level was reduced from 700 mg/kg bw to 500 mg/kg bw after 6 or 7 days of dose application initiation (staggered start of dosing) due to overt toxicity
Dose / conc.:
500 mg/kg bw/day
No. of animals per sex per dose:
100 animals (40 males and 60 females) were included in the study. All females were screened for regular estrous cycles for 14 days before the treatment initiation and only 40 females (10 females/group) showing regular estrous cycles were continued in the study.
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
Clinical Observations
General clinical observations were made at least once a day, preferably at the same time each 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.
Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. 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.
During the study all animals were visually monitored to see if there is condition of polydipsia.

Functional Observations
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 during the last week of lactation of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each group outside the home cage using a functional observational battery of tests.
Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

Body Weight and Food Consumption
The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter 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), on PND 4 and PND 13 along with pups. All animals were weighed directly before termination.
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.
Individual food consumption was calculated based on survival of animals on each day during two food measurement intervals. Factor was derived for that particular period and total cage consumption was divided by factor (e.g. for male cage 1 during premating day 1-7, factor was 4.67) to get food consumption per animal for that particular period.

Haematology
Haematological parameters were examined in 5 randomly selected males and females (only lactating females were evaluated) from each group at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in EDTA-coated tubes. The following haematological parameters were examined:
haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso) and large unstained cells (Luc).

Blood Coagulation
Coagulation parameters from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in citrate tubes. The following coagulation parameters were examined: prothrombin time (PT) and activated partial thromboplastin time (aPTT).

Clinical Biochemistry
Parameters of clinical biochemistry from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in serum separator tubes. The following parameters of clinical biochemistry were examined:
alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP),
albumin (Alb), urea, total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na) and potassium (K).
From 2 female pups/litter on day 4 after birth from all dams and 2 pups/litter at termination on day 13 and from all adult males at termination, blood samples were collected from the defined site in serum separator tubes. All blood samples were stored under appropriate conditions. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4).
Further assessment of T4 in blood samples from the main study dams and day 4 pups was not deemed necessary. Additionally, assessment of TSH in day 4 pups, adult females, day 13 pups, and adult males were not considered necessary based on the fact that no histopathological findings were observed in thyroid/parathyroid gland of selected male and female adult animals and T4 hormone levels of males and day 13 pups. Pup blood was pooled by litter for thyroid hormone analysis.
Two pups per litter were sacrificed on day 4 after birth and blood samples were taken for possible serum hormone assessments. The two pups per litter were female pups to reserve male pups for nipple retention evaluations. No pups were eliminated where litter size dropped below 8 pups. When there was only one pup available above a litter size of 8, only one pup was sacrificed on PND 4.

Urinalysis
A urinalysis was performed with samples from 5 randomly selected males and females (only lactating females were evaluated) prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance was recorded. The following parameters were measured using qualitative indicators (Henry Schein Urine Stripes URI 10SL): specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood and leukocytes.












Oestrous cyclicity (parental animals):
Estrous cycles of all females were monitored before the treatment starts to select the females with regular cyclicity (using vaginal smears). Further on, vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.
Litter observations:
The duration of gestation was recorded and is calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after the 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 littering (PND 0) and on PND 4 and PND 13. Live pups were identified by tattooing. In addition to the observations of the parent animals, any abnormal behaviour of the offspring was recorded.
The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD/Cube root of pup weight). The number of nipples/areolae in male pups was counted on PND 12.


Postmortem examinations (parental animals):
Pathology
All surviving males were sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days) and females were sacrificed on the respective PND 13 by using anesthesia (ketamine/xylazine). All surviving pups were killed by cervical dislocation on PND 13.
Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle.
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), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved in 4 % neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70 % ethanol.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. The number of corpora lutea and implantation sites was recorded for any females sacrificed 26 days after the end of the mating period with no evidence of mating and for any females sacrificed on day 26 post-coitum due to non-delivery.

Organ Weights
The wet weight of the organs [testes (paired weight), epididymides (paired weight), prostate, seminal vesicles and coagulating glands (complete weight), uterus with cervix, ovaries (paired weight), thyroid/parathyroid glands (from 1 pup/sex/litter/group and from all adult males and females) – weighed after fixation (complete weight), thymus, liver, spleen, kidneys (paired weight), brain, adrenal (paired weight), heart and pituitary gland] of 5 randomly selected male and female animals (only lactating females were evaluated) from each group was recorded as soon as possible. Paired organs were weighed together. Organ weights of animals found dead were not recorded. Reproductive organs were weighed in all animals.
Thyroid/parathyroid glands from 1 pup/sex/litter/group (sacrificed on PND 13) and from all adult males and females were preserved. Weight of thyroid/parathyroid glands was measured after fixation.

Adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, eyes, Harderian glands, femur with knee joint, heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (mandibular), lymph nodes (mesenteric), lymph nodes (axillary), mammary gland area (male and female), oesophagus,optic nerves, ovaries, oviducts, pancreas, pituitary, prostate and seminal vesicles with coagulating glands as a whole, rectum, salivary glands (sublingual, submandibular), sciatic nerve, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar segments), spleen, sternum (with bone marrow), stomach, testes, thymus, thyroid/parathyroid glands, tongue, trachea, ureters, urinary bladder, uterus with cervix and vagina of the same selected animals from each group were preserved in 4% neutral-buffered formaldehyde except for eyes, testes and epididymides that were fixed in Modified Davidson’s Fixative for approximately 24 hours before they were transferred to 70% ethanol. All animals found dead and/or intercurrently euthanised for animal welfare reasons were subjected to a gross necropsy and the organs preserved for a histopathological examination.
Thyroid/parathyroid glands from 1 pup/sex/litter/group sacrificed on PND 13 and non-selected adult animals were preserved for potential histopathological examination. The thyroid gland weight was measured after fixation.


Histopathology
A full histopathology was carried out on the preserved organs and tissues of the selected animals of the control and high dose groups which were sacrificed at the end of the treatment period. Evaluation of thyroid/parathyroid glands from pups and from the remaining non-selected adult animals was not deemed necessary as no test item related histopathological findings were observed in thyroid/parathyroid gland of selected animals and there was also no test item related effect observed on T4 hormone level in males and pups sacrificed on PND 13. A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study.
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 examined in control and HD animals and in non-pregnant female animals of the LD and MD animals. Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were also examined in the mating partners of the non-pregnant female LD and MD animals.
These examinations were extended to animals of all other dosage groups for treatment-related changes observed in the high dose group for stomach, liver and kidney. Only organs and tissues of the other dosage groups showing changes in the high dose group were examined.
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.
The 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). The histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for histopathology). The study phases from test site 1 and 2 were 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):
Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.
Statistics:
A statistical assessment of the results of body weight, food consumption, parameters of haematology, blood coagulation, clinical biochemistry and litter data were performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights were statistically analyzed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see Details on results P0
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
see Details on results P0
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see Details on results P0
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see Details on results P0
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
see Details on results P0
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results P0
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results P0
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results P0
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see Details on results P0
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
see Details on results P0

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results P0
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
see Details on results P0

Details on results (P0)


Clinical Observations
In terminally sacrificed males, predominant clinical signs observed during the treatment period (premating day 1 to mating/post mating day 14) were moderately increased salivation in one animal of MD and moving the bedding in two animals of MD group during very few days of mating and postmating period. In HD group, major clinical signs observed were slight to moderately reduced spontaneous activity, half eyelid closure, moving the bedding, slight to moderate salivation and piloerection in all animals during majority of premating and mating/postmating period.
In terminally sacrificed females, major clinical signs observed during the treatment period (Premating day 1 to PND 12) were moving the bedding on one day during lactation period in one female of LD group, moving the bedding, slightly to moderately increased salivation in few animals on few days of MD group and moving the bedding, slightly to severely increased salivation, slightly to moderately increased piloerection, reduced spontaneous activity, half eyelid closure in all animals on majority of days during treatment period in HD group.
Isolated incidences of alopecia on various body parts, abnormal breathing and nasal discharge was observed on few occasions in one each female animal of MD group and considered to be incidental in nature.

The clinical signs salivation and moving the bedding were observed immediately after the dose administration and therefore were considered to be a sign of discomfort due to a local reaction to the test item rather than a systemic adverse effect and has no toxicological relevance. However, clinical signs like slightly to moderately increased piloerection, reduced spontaneous activity and half eyelid closure in HD group could be attributed to treatment with the test item.
None of the females showed signs of abortion or premature delivery.
During the weekly detailed clinical observation, no relevant differences between the groups were found.

Mortality
During the treatment period of this study, few mortalities were observed as follows:
- Male no. 31 (HD) was found dead on premating day (PMD) 6. Clinical signs observed before death were spontaneous activity reduced (moderate), piloerection (moderate), half eyelid closure during PMD 2-6 and moving the bedding during PMD 5-6.
- Male no. 35 (HD) was found dead on premating day (PMD) 6. Clinical signs observed before death were slight increased salivation on PMD 1, moving the bedding from PMD 1-6 and moderately reduced spontaneous activity, moderate piloerection and half eyelid closure during PMD 2-6.
- Male no. 38 (HD) was found dead on premating day (PMD) 5. No specific clinical signs were observed before death.
- Female no. 75 (HD) was found dead on premating day (PMD) 3. Clinical signs observed before death were moderately reduced spontaneous activity, moderate piloerection and half eyelid closure during PMD 2-3.
- Female no. 77 (HD) was found dead on premating day (PMD) 5. Clinical signs observed before death were moderately increased salivation on PMD 3, moving the bedding during PMD 3-5 and slightly increased salivation during PMD 4-5.
Histopathologically, in females, gastric changes (ulceration in female No. 75 and erosion in female No. 77) contributed to both morbidity and mortality. In males, based on the histopathology investigation, the cause of death was not evident although, there were slight to moderate gastric changes were observed which might have contributed to the morbidity.

Body Weight Development
In males, there was no statistically significant difference observed on body weight between the LD and the control group during the entire study period. However, lower group mean body weights from premating day 14 to terminal sacrifice were observed in MD group without achieving statistical significance when compared with the controls. A statistically significantly lower group mean body weight was also observed in HD group from day 7 during entire study period although statistical significance was achieved only on premating day 7, 14 and mating/postmating day 7 when compared with the controls.
In correlation to body weight, group mean body weight gain was statistically significantly lower during mating/post mating day 7-14, premating day 1-mating/postmating day 14 and premating day 1 to terminal sacrifice in MD group males when compared with the controls. There was also statistically significantly lower group mean body weight gain observed during premating day 1-7, premating day 1 to mating/postmating day 14, premating day 1 to terminal sacrifice and statistically significantly higher group mean body weight gain during premating day 7-14 and mating/postmating day 7-14 in HD group when compared with the controls.
In females, no statistically significant difference in group mean body weight was observed in treatment groups during entire study period when compared with controls. There was statistically significantly higher group mean body weight gain observed during premating day 7-14 in HD group when compared with the controls.
This statistically and biologically significant effect on body weight and body weight gain in MD and HD group male was considered as test item related and toxicologically relevant. However, statistically significant effect on female group mean body weight gain in HD group was attributed to possible compensatory recovery after the reduction of dose from second week of the study.

Food Consumption
In males, the food consumption during treatment period tended to increase with the progress of the study in the control, the LD, the MD and the HD group. However, In HD group, during premating day 1-7, food consumption was lower without achieving statistical significance compared to the control group and this effect on food consumption in males was considered to be test item related.
In females, no statistically significant effect on food consumption was observed during premating period, gestation and lactation period in treatment groups when compared with the controls. However, in correlation to body weight development, marginally lower food consumption was observed in HD females during premating day 1-7 when compared with the controls. As this effect on female food consumption was marginal and in the light of no significant effect on body weight development, this effect on food consumption in females was not considered to be adverse.

Haematology and Coagulation
In males sacrificed at the end of treatment period, no test item related adverse effects were observed for haematological parameters. However, there was a statistically significantly higher platelets (PLT) count observed in HD group compared to the control group. All group mean and most of the individual values were within the historical control data range. As group mean value was within historical control data limit, statistically significant effect on PLT in HD group was considered to be incidental and not related to the treatment with test item. No test item related effect was observed on coagulation parameters when compared with the controls.
In females sacrificed at the end of treatment period, no statistically significant or test item effect observed on any of the haematology or blood coagulation parameters when compared with the controls. All group mean and most of the individual values were within the historical control data range.

Clinical Biochemistry
In males sacrificed at the end of treatment period, significantly lower total bile acids (TBA) was observed in MD and HD group although statistical significance In males and females sacrificed at the end of treatment period, no test item related or statistically significant effect on any clinical biochemistry parameter in treatment groups was observed when compared with the control. There was higher TBA group mean value observed in female LD group without achieving statistical significance. Due to lack of dose dependency and consistency, this effect on TBA in LD group was not considered to be test item related and toxicologically relevant.

Urinanalysis
The urinalysis performed in selected male and female animals sacrificed at the end of treatment period revealed no test item treatment related effect and all urinary parameters were in the normal range of variation. High protein levels were found in the urine of few male and females of all groups including control group. Therefore, this effect on urine parameters was not considered to be test item related.

Functional Observations
In males, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period except incidental statistically significantly higher defecation count before the initiation of treatment in LD group when compared with the controls. There were no biologically relevant differences observed in body temperature between the groups.
In females, statistically significantly higher supported rearing count in MD, lower supported rearing count in LD, MD and HD and statistically significantly lower not supported rearing count in all treatment groups was observed before initiation of the treatment which was not considered to be toxicologically relevant. There was also statistically significantly higher supported rearing count in LD and HD group, statistically significantly lower urination count in all treatment groups and statistically significantly lower defecation count in LD group observed during last week of treatment. As this type of difference was marginal and without dose dependency/consistency, it has no toxicological relevance and could be considered as biological variation. There were no biologically relevant differences in body temperature between the groups except statistically significantly lower body temperature was observed before initiation of the treatment in LD group which was considered to be toxicologically irrelevant.

Organ Weights
In males sacrificed at the end of treatment period, there were statistically significantly higher absolute liver weights in all treatment groups (except MD group), higher relative (to body weight) liver weights in MD and HD group and higher kidney weights in LD and HD group although statistical significance only achieved in LD group when compared with the controls. Increased liver absolute weights were histologically correlated with slight hepatocellular hypertrophy only in one animal from the HD group, whereas in the MD and LD group neither the absolute nor the relative increase in liver weight correlated with underlying histological changes. Therefore, without a dose dependent correlation between liver weight increase and the observed histopathological hepatic changes and in absence of hepatic-derived enzyme profiling, the toxicological relevance of the liver weight increase could not be established.
In females sacrificed at the end of treatment period, significantly higher absolute liver weights were observed in HD group when compared with the controls although statistical significance was not achieved. There were also statistically significantly higher relative (to body weight) kidney weights observed in HD group when compared with the controls. In females, statistically significantly higher relative kidney weights in HD group were considered of no toxicological relevance in absence of correlating histological changes.

Pathology
Few specific macroscopic changes were recorded for the male and female animals, which based on microscopic examination were not considered to be of test item treatment relevance.
The macroscopic changes observed were lung- dark red discolouration (male no. 35 and 38 of HD group), thymus - red discolouration (male no. 35 and 38 of HD group), stomach - gas filled (male no. 31 if HD group, female no. 77 of HD group), Intestinal tract- gas filled (male no. 31, 35, 38 of HD group, female no. 77 of HD group), left testes- absent (male no. 21 of MD group), testes- small (male no. 14 of LD group), left epididymides- small (male no. 14 of LD group and male no. 21 of MD group), right kidney- dilatation (male no. 30 of MD group), lung- dark abnormal colour (female no. 75 of HD group) and liver- diaphragmal herniation (female no. 59 of LD group)
Macroscopic findings correlating with histopathological observations were observed in following animals: 
- In decedents, red discoloration observed in the thymus of males no. 35 and No. 38 of HD group was correlated microscopically with congestion and hemorrhage in male no. 35 and with hemorrhage in male no. 38. The above mentioned changes were considered incidental agonal changes which are occasionally found in animals being subjected to necropsy. All other changes recorded at necropsy in decedents were considered incidental and most likely associated with autolytic processes.
- In survivors, small testes and epididymides recorded in male no. 14 (LD group) correlated microscopically with testis and epididymis atrophy and aspermia. This changes were considered to be most likely incidental.
In male No. 21 (MD group) small epididymis on the left side was correlated microscopically with unilateral epididymis atrophy and aspermia. The above mentioned changes were considered to be most likely incidental. In addition, in the male no. 30, a dilated right kidney was correlated microscopically with pelvic dilatation. All observed changes in male no. 30 were considered to be most likely incidental.
In female No. 59 (LD group), the observed diaphragmal herniation corresponded histologically to a hepatic nodule. This liver change was to be considered incidental.
All other changes recorded at necropsy in surviving animals were considered incidental and most likely associated with autolytic processes. In absence of corresponding histopathological findings, these necropsy findings were considered to be of no toxicological relevance.


Histopathology
Under the conditions of this study, there were a number of early decedents. In females, gastric changes (ulceration in female No. 75 and erosion in female No. 77) contributed to both morbidity and mortality. In males (31, 35 and 38) based on the histopathology investigation, the cause of death was not evident although, there were slight to moderate gastric changes were observed which might have contributed to the morbidity.
In decedents, test item related histopathology changes observed in stomach of females were ulceration (animal No. 75) and multifocal erosions (animal No. 77). The above mentioned changes were associated with vacuolization of forestomach epithelial cells (epithelial vacuolization), hyperkeratosis, mixed cell infiltrates and squamous hyperplasia. In male stomach, hyperkeratosis and squamous hyperplasia were observed. In liver minor degree of hepatocytes vacuolation (fatty change) and centrilobular hypertrophy was observed in few males and females.
In survivors of LD, MD and HD groups, test item related histopathology changes were observed in liver, kidney and stomach.
In liver, minor degree of centrilobular hepatocytes hypertrophy was observed only in few males from the HD group and was considered test item related. Further, minimal to moderate glycogen accumulation was observed in some males and females form the HD group only, whereas in control animals moderate glycogen accumulation was also observed in one female. The above mentioned changes were considered to be most likely incidental (related to the feeding schedule), whereas a test item contribution cannot be fully excluded. In addition, in one female from the low dose group, a slight bile duct hyperplasia, slight fibrosis and a hepatic nodule (hepatodiaphragmatic herniation) was observed and correlated with the liver herniation through the diaphragm recorded at necropsy. These findings were considered incidental and deemed not test item related.
In kidney, minimal to slight hyaline droplets accumulation in tubular epithelial cells were observed in the majority of males from all dose groups and the control group. This renal change is a male rat specific phenomenon of no toxicological relevance in humans. Minimal to slight tubular basophilia was observed in few animals from the high dose group only. The tubular basophilia was considered most likely related to the test item administration. Minimal to slight tubular simple dilatation was observed in some male and females from all dose groups and in one female from the control group. The tubular simple dilatation was considered most likely incidental. Furthermore, pelvic dilatation was observed in one female from the low dose group and two males from the mid dose group. These changes were deemed incidental and not treatment related.
In stomach, moderate forestomach ulceration accompanied by severe multifocal mixed cell infiltrates, moderate submucosal edema wall and epithelial vacuolization was observed in one male from the MD group. Minimal to moderate mixed cell infiltrates mainly located at the limiting ridge and sometimes extending in the adjacent forestomach and glandular stomach submucosa of males and females from the LD and MD groups and only in males from the HD group. Minimal to moderate epithelial vacuolization was noticed in few males from LD, MD and HD dose groups only. Furthermore, a minor degree of hyperkeratosis was observed in some males from the LD and HD dose groups, whereas slight to moderate squamous hyperplasia was present only in few males from the HD group. These changes in stomach were considered to be most likely related to the test item administration.
The test item did not produced any histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, oviducts, uterus and cervix, and vagina. Further, there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure.
In one male from the low dose group (male no. 14) atrophy of the testis and epididymis was observed, while in one male from the mid dose group (male no. 21) epididymis atrophy was noticed. In absence of any toxicologically significant changes in the male reproductive organs/tissues of survivors, the above mentioned changes were considered incidental.
Further, the treatment with test item did not induced histomorphological effects in the reproductive organs of the non-pregnant female from the low dose group (Animal No. 54). The infertility was caused by the testicular atrophy and subsequent aspermia observed in its mating partner male no. 14.
In conclusion, due to the early mortality observed in males and females from the high dose group and the presence of several histopathological adverse changes in different organs, a histomorphological NOAEL (no observed adverse level) could be established at 30 mg/kg bw/day for the male rats and 200 mg/kg bw/day for female rats.

Precoital Interval and Duration of Gestation
There was no effects on the duration of gestation. However, precoital interval was statistically significantly higher in HD group females compared to the controls. As this difference in precoital interval was marginal (1 day) and therefore considered as biological variation and not related to treatment with test item.

Estrous Cyclicity
Test item had no biologically significant effect on the estrous cycle analysed during 2 weeks premating period after the first administration in LD and MD group when compared to the controls. There were no considerable differences in the length or sequence of cycle stages between the LD and MD dose groups and the control group. However, statistically significantly higher mean cycle length and lower number of normal cycles were observed in HD group females when compared with controls. Furthermore, 6/8 females were observed with no single estrus cycles (acyclicity) and exhibited persistent diestrus stage. In the light of no effect on pregnancy rate and various reproductive indices in HD group, this effect on estrus cyclicity in HD group was considered as a secondary effect due the treatment with the test item.

Pre- and Postnatal Data
There were no test item treatment related effects observed on the number of corpora lutea, implantation sites, live pups on PND 0, 4 and 13, percent preimplantation loss and post implantation loss in treatment groups when compared with the control group.

Reproductive Indices
There were no test item related effects on the reproductive indices (copulation, viability and delivery indices) in the dose groups when compared to the control group. However, a slightly reduced fertility index (number of females pregnant/ No. of females copulated X 100) of 87.5 % was observed in the HD group as compared to 90 % in control group. Although there was reduction in fertility index in HD group, it was within the standard pregnancy rate of rat i.e. ≥ 80 % and therefore this effect on fertility index was considered as biological variation and not related to treatment with the test item administration.






Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Remarks:
UVCB substance with 100% purity
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
LOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Remarks:
UVCB substance with 100% purity
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other:
Remarks:
Close examination shows that the only effects observed at MD (200 mg/kg) are a slight lower BW compared to control (-6%) in males, and a limited increase in the combined effects in stomach upon histopathological examinations in males and females.

Target system / organ toxicity (P0)

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
other: Additionally, slight effect on BW in males
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
liver
other: At higher dose levels the local effects in stomach are most prominent, possibly causing mortaility to occur (at 700 mg/kg bw/day)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
see Details on results F1
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
see Details on results F1
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results F1
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results F1
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on results F1
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
see Details on results F1

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Litter Data
There were no test item treatment related or statistically significant effects observed in treatment groups on litter data parameters like group mean total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, still birth, runt on PND 0 as well as number of live pups, male pups, number of female pups and sex ratio on PND 4 and PND 13 when compared with the controls.

Litter Weight Data
There was no statistically significant effect on pup mean weight, total litter weight, female litter weight on PND 0, PND 4 and PND 13 observed in treatment groups when compared with the controls.
However, in correlation to low number of male pups and sex ratio, lower group mean male litter weight values were observed on PND 0, 4 and 13 although statistical significance was achieved only on PND 0 and 4. This decrease in male litter weight in HD group was attributed to low male pups in few females (71, 73 and 78) of the HD group. Therefore this effect on male litter weight was not considered to be test item related and assumed to be biological variation.

Pup Survival Data
No effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in treatment groups when compared to the control group and all pups survived until terminal sacrifice on PND 13.

Anogenital Distance and Nipple Retention
In males, statistically significant lower pup weight and cube root of pup weight on the day of anogenital measurement was observed in male LD and HD group when compared with the controls. There was also statistically significantly higher absolute (not in HD group) and relative anogenital distance in MD and HD group observed when compared to the controls.
In females, statistically significantly higher absolute and relative anogenital distance was observed in all treatment groups when compared to the controls.
In male and females, parameters like pup body weight, litter size and sex ratio were not affected in LD and MD groups and these parameters are correlated with anogenital distance (AGD) although statistically significant group mean AGD value in MD group males and LD and MD group females were observed. AGD is always correlated with crown rump length - CRL (not part of this study) or Cube root of pup body weight, litter size and sex ratio (for data normalization to simulate linear measurement). Therefore effect on AGD in LD and MD group males and females cannot be considered as test item related.
In HD males, effect on absolute and relative AGD was not consistent and dose dependent although statistically significantly higher relative AGD in HD group was observed when compared to the controls. In females also, as no effect on body weight, litter size and sex ratio was observed, marginal higher but statistically significant effect on female absolute and relative AGD was not considered to be adverse.
All AGD values in male and female pups were within historical control data range. Furthermore, the statistically significant mean values in male and female AGD could also be attributed to variation in individual data values for absolute and relative AGD from few females from each group as AGD varies based on litter size, pup weight and timings of littering on PND 0 (between 0-24 hours) before AGD was measured on PND 0. If AGD is permanently changed (at birth and adulthood) then only it would establish a permanent structural change with possible impact on sexual function but in most of the times this finding disappear during sexual maturation and can therefore not be considered as adverse.
Moreover, according to the recent draft OECD guidance document No. 150 (2 July 2017) for many assays, individual endpoints may not in themselves be diagnostic of an endocrine disruption modality. Such diagnosis often relies on a combination of endpoints or assays in a weight of evidence assessment. Specifically, with regard to changes in pup AGD, a positive result for apical endpoints could be statistically significant changes in pup AGD, accompanied by treatment-related histopathologic changes in parental reproductive organs.
In this guidance document, as per the table B.1 (page 76-78), in the case that a change in AGD is observed, it has to be always accompanied by treatment-related histopathologic changes in parental reproductive organs and other endpoints. In this study, this means that the increased AGD in female pups could indeed indicate an androgen-mediated activity (agonistic) if this finding would be accompanied by consistent increased AGD in male pups (Absolute AGD not dose dependent and consistent but only relative AGD which is a calculated value), Genital abnormalities in male pups, changes in weights of uterus, ovaries (decrease), Increase in weights of epididymides, prostate, seminal vesicles and coagulating glands, decreased testes weight, histopathologic changes in ovary and uterus and histopathologic changes in testes, epididymides, male accessory sex organs. However, in neither of these endpoints listed, a test item related effect of toxicological relevance was observed in the present study. Besides, there was no effect observed on nipple retention in the pups of any of the groups. There was no accompanied effect on parameters like litter size, sex ratio, estrus cyclicity (just secondary effect in HD females without affecting pregnancy rate), weights of uterus, ovaries, testes, epididymides, prostate, seminal vesicles and coagulating glands). There were no accompanied histopathologic changes in parental reproductive organs. There was no effect observed on pup thyroid weight, male and PND 13 pup thyroxine hormone (T4) in the treatment groups.
Since no additional finding is supporting a possible androgen-mediated activity (agonistic) or any other endocrine disruption modality of the test item as specified in the guidance document. Thus, there is no conclusive evidence of an endocrine disrupting effect of the test item and as a result of that, the findings on AGD in this study cannot be considered as adverse.
No statistically significant effect of toxicological relevance was observed on nipple retention in the pups of any of the groups when compared with the controls. However, group mean number of nipple retention in HD group was higher compared to the controls although statistical significance was not achieved. This increase in group mean pups with nipple retention was attributed to just few pups from 3 females of the HD group (73, 79 and 80) and other 4 females from HD group were not observed with any pup with nipple retention and therefore this effect on nipple retention was not considered to be adverse. Furthermore areolae/nipples observed in early postnatal rats are temporary.


Thyroid Hormone (T4) Analysis and Pup Thyroid Weight on PND 13
No test item related effect of toxicological relevance or statistical significance was observed on pup thyroid weight, male and PND 13 pup thyroxine hormone (T4) in the treatment groups when compared to the controls.

Pup External Findings
No test item related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups. Few specific findings like dark snout (pup no. 7 from dam 45 of control group on PND 0) and dark necrotic tag (all pups from dam 76 of HD group between PND 0-12) were observed in control and HD group.
The external finding like absent hair coat (pup no. 1-9 from dam no. 52 of LD group and pup no. 1-7, 9 from dam 70 of MD group) and partial necrotic tag/spot (pup no. 2, 8, 9, 12 and 13 of dam no. 76 of HD group) at death was considered to be spontaneous and not related to test item treatment.


Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Remarks:
UVCB substance with 100% purity
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Naphthalenesulfonic acid, bis(1-methylethyl)-,methyl derivs., sodium salt showed no adverse effects to reproduction and development in the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening study according to OECD 422 up to the highest dose tested of 500 mg/kg bw/day.
Executive summary:

The aim of this study was to assess the possible effects of Naphthalenesulfonic acid, bis(1-methylethyl)-,methyl derivs., sodium salt on male and female fertility and embryofoetal development after repeated dose administration in Wistar rats.

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 63 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 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. Animals of an additional control group were handled identically as the dose groups but received aqua ad iniectabilia (sterile water), the vehicle used in this study.The 4 groups comprised 10 male and 10 female Wistar rats. Before dosing all females were screened for two weeks for regular estrous cyclicity and animals (10 females/ group) with regular estrous cycle (4-5 day cycle) were used in the study.

The following doses were evaluated:

Control:          0   mg/kg bw/day

Low Dose:      30  mg/kg bw/day

Medium Dose:200mg/kg bw/day

High Dose:     700mg/kg bw day / 500 mg/kg bw/day*

* = the high dose level was reduced from 700 mg/kg bw to 500 mg/kg bw after 6 or 7 days of dose application (staggered start of dosing) due to overt toxicity.

The test item formulation was prepared once in 10 days based on stability data. The test item was dissolved in aqua ad iniectabilia and administered daily during14 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 12 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 bodyweight.

During the period of administration, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

Haematological and clinical biochemistry evaluations were performed on blood samples collected at terminal sacrifice from five randomly selected males and females from each group. Urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males 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 from all animals and in the last week of treatment in five randomly selected males and femalesof each group.

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, on day 4 and day 13 post-partum. The anogenital distance (AGD) of each pup was measured on PND 0. The number of nipples/areolae in male pups was counted on PND 12.

From 2 pups/litter on day 4 after birth; from all dams and 2 pups /litter at termination on day 13 and from all adults males at termination, blood samples were collected from the defined site. All blood samples were stored under appropriate conditions. Blood samples from the day 13 pups and from the adult males were assessed for serum levels for thyroid hormones (T4). Further assessment of T4 in blood samples from adult females and day 4 pups were not deemed necessary. Pup blood was pooled by litter for thyroid hormone analysis.

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 13. 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 or 13 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the preserved tissues was performed on high dose and control animals, in non pregnant female animals and male mating partners of the LD and MD animals. These examinations were extended to animals of all other dosage groups as treatment-related changes were observed in the high dose group for stomach, liver and kidney. 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. All gross lesions macroscopically identified were examined microscopically in all animals.

Summary Results

Mortality:

During the treatment period of this study,fewmortalities observed were male nos. 31, 35 (HD) found dead on premating day (PMD) 6, male no. 38 (HD) was found deadon premating day (PMD) 5, female no. 75 (HD) was found dead on premating day (PMD) 3 and female no. 77 (HD) was found dead on premating day (PMD) 5.Histopathologically,in females, gastric changes (ulceration in female No. 75 and erosion in female No. 77) contributed to both morbidity and mortality. In males, based on the histopathology investigation, the cause of death was not evident although, there were slight to moderate gastric changes were observed which might have contributed to the morbidity.

Clinical Observations:

In terminally sacrificed males, predominant clinical signs observed during the treatment period (premating day 1 to mating/post mating day 14) were moderately increased salivation in one animal of MD and moving the bedding in two animals of MD group during very few days of mating and postmating period. In HD group, major clinical signs observed were slight to moderately reduced spontaneous activity, half eyelid closure, moving the bedding, slight to moderate salivation and piloerection in all animals during majority of premating and mating/postmating period.

In terminally sacrificed females, major clinical signs observed during the treatment period (Premating day 1 to PND 12) were moving the bedding on one day during lactation period in one female of LD group, moving the bedding, slightly to moderately increased salivation in few animals on few days of MD group and moving the bedding, slightly to severely increased salivation, slightly to moderately increased piloerection, reduced spontaneous activity, half eyelid closure in all animals on majority of days during treatment period in HD group.

The clinical signs salivation and moving the bedding were observed immediately after the dose administration and therefore were considered to be a sign of discomfort due to a local reaction to the test item rather than a systemic adverse effect and has no toxicological relevance. However, clinical signs likeslightly to moderately increased piloerection, reduced spontaneous activity and half eyelid closure in HD group could be attributed to treatment with the test item.

None of the females showed signs of abortion or premature delivery.

During the weekly detailed clinical observation, no relevant differences between the groups were found.

Functional Observations:

In males and females, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period except incidental statistically significantly higher/lower count for few parameters before the initiation of treatment or at the end of treatment when compared with the controls. There were no biologically relevant differences observed in body temperature between the groups.

Body Weight Development:

In males, there was no statistically significant difference observed on body weight between the LD and the control group during the entire study period. However, lower group mean body weights from premating day 14 to terminal sacrifice were observed in MD group without achieving statistical significance when compared with the controls. A statistically significantly lower group mean body weight was also observed in HD group from day 7 during entire study period although statistical significance was achieved only on premating day 7, 14 and mating/postmating day 7 when compared with the controls.

In correlation to body weight, group mean body weight gain was statistically significantly lower during mating/post mating day 7-14, premating day 1-mating/postmating day 14 and premating day 1 to terminal sacrifice in MD group males when compared with the controls. There was also statistically significantly lower group mean body weight gain observed during premating day 1-7, premating day 1 to mating/postmating day 14, premating day 1 to terminal sacrifice and statistically significantly higher group mean body weight gain during premating day 7-14 and mating/postmating day 7-14 in HD group when compared with the controls.

In females, no statistically significant difference in group mean body weight was observed in treatment groups during entire study period when compared with controls. There was statistically significantly higher group mean body weight gain observed during premating day 7-14 in HD group when compared with the controls.

This statistically and biologically significant effect on body weight and body weight gain in MD and HD group male was considered as test item related and toxicologically relevant. However, statistically significant effect on female group mean body weight gain in HD group was attributed to possible compensatory recovery after the reduction of dose from second week of the study.


Food Consumption:

In males, the food consumption during treatment period tended to increase with the progress of the study in the control, the LD, the MD and the HD group. However, In HD group, during premating day 1-7, food consumption was lower without achieving statistical significance compared to the control group and this effect on food consumption in males was considered to be test item related.

In females, no statistically significant effect on food consumption was observed during premating period, gestation and lactation period in treatment groups when compared with the controls. However, in correlation to body weight development, marginally lower food consumption was observed in HD females during premating day 1-7 when compared with the controls. As this effect on female food consumption was marginal and in the light of no significant effect on body weight development, this effect on food consumption in females was not considered to be adverse.

Estrus Cyclicity:

There were no considerable differences in the length or sequence of cycle stages between the LD and MD dose groups and the control group. However, statistically significantly higher mean cycle length and lower number of normal cycles were observed in HD group females when compared with controls. Furthermore, 6/8 females were observed with no single estrus cycles (acyclicity) and exhibited persistent diestrus stage. In the light of no effect on pregnancy rate and various reproductive indices in HD group, this effect on estrus cyclicity in HD group was considered as a secondary effect due the treatment with the test item.

Litter Data:

There were no test item treatment related or statistically significant effects observed in treatment groups on litter data parameters like group mean total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, still birth, runt on PND 0 as well as number of live pups, male pups, number of female pups and sex ratio on PND 4 and PND 13 when compared with the controls.

Litter Weight Data:

There was no statistically significant effect on pup mean weight, total litter weight, female litter weight on PND 0, PND 4 and PND 13 observed in treatment groups when compared with the controls. However, in correlation to low number of male pups and sex ratio, lower group mean male litter weight values were observed on PND 0, 4 and 13 although statistical significance was achieved only on PND 0 and 4. This decrease in male litter weight in HD group was attributed to low male pups in few females (71, 73 and 78) of the HD group. Therefore this effect on male litter weight was not considered to be test item related and assumed to be biological variation.

Precoital Interval and Duration of Gestation:

There was no effects on the duration of gestation. However, precoital interval was statistically significantly higher in HD group females compared to the controls. As this difference in precoital interval was marginal (1 day) and therefore considered as biological variation and not related to treatment with test item.

Pre and Post-Natal Data:

There were no test item treatment related effects observed on the number of corpora lutea, implantation sites, live pups on PND 0, 4 and 13, percent preimplantation loss and post implantation loss in treatment groups when compared with the control group.


Reproductive Indices:

There were no test item related effects on the reproductive indices (copulation, fertility, viability and delivery indices) in the dose groups when compared to the control group.

Pup Survival Data:

No effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in treatment groups when compared to the control group and all pups survived until terminal sacrifice on PND 13.

Anogenital Distance and Nipple Retention:

In males, statistically significant lower pup weight and cube root of pup weight on the day of anogenital measurement was observed in male LD and HD group (not dose dependent) when compared with the controls. There was also statistically significantly higher absolute (not in HD group) and relative anogenital distance in MD and HD group observed when compared to the controls.

In females, statistically significantly higher absolute and relative anogenital distance was observed in all treatment groups when compared to the controls.

In male and females, parameters like pup body weight, litter size and sex ratio were not affected in LD and MD groups and these parameters are correlated with anogenital distance (AGD) although statistically significant group mean AGD value in MD group males and LD and MD group females were observed. Therefore effect on AGD in LD and MD group males and females cannot be considered as test item related.

In HD males effect on absolute and relative AGD was not consistent and dose dependent although statistically significantly higher relative AGD in HD group was observed when compared to the controls. In females also, as no effect on body weight, litter size and sex ratio was observed, marginal higher but statistically significant effect on female absolute and relative AGD was not considered to be adverse.

All anogenital values in male and female pups were within historical control data range. AGD is not the only parameter to confirm the endocrine disruption and AGD itself needs to be correlated with lot of other parameters in the study. No additional finding in the study supporting a possible androgen-mediated activity (agonistic) or any other endocrine disruption modality of the test item. Thus, there is no conclusive evidence of an endocrine disrupting effect of the test item and as a result of that, the findings on AGD cannot be considered as adverse.

Additional comments indicating that statistical relevenat efefcts on AGD are of no biological consequence: The AGD of the females are still below the average historical control for all does groups, and additionally the variability (SD) is exceptionally low when compared variability in historical control data. Besides, there is hardly any dose related increase visible. Also the indicated AGD increase in males is not biologically relevant. Also here, the increase is only minimal and only slightly above the average historical control, also for the control group. Besides, studies with testosterone indicated that specifically AGD did not increase: “masculinization in males  appears to operate at  maximum capacity with normal endogenous  testosterone concentrations.” (Welsh M. et al., 2008, Identification in rats of a programming window for reproductive tract masculinization, disruption of which leads to hypospadias and cryptorchidism). This suggests that an observed increase in AGD in males is of no biological significance.

No statistically significant effect of toxicological relevance was observed on nipple retention in the pups of any of the groups when compared with the controls.

Thyroid Hormone (T4) Analysis:

No test item related effect of toxicological relevance or statistical significance was observed on pup thyroid weight, male and PND 13 pup thyroxine hormone (T4) in the treatment groups when compared to the controls.

Pup External Findings:

No test item related gross external abnormalities of toxicological relevance on PND 0-12 and death were observed in the pups of any of the groups.

Haematology and Coagulation:

In males sacrificed at the end of treatment period, no test item related adverse effects were observed for haematological parameters. However, there was a statistically significantly higher platelets (PLT) count observed in HD group compared to the control group. All group mean and most of the individual values were within the historical control data range. As group mean value was within historical control data limit, statistically significant effect on PLT in HD group was considered to be incidental and not related to the treatment with test item. No test item related effect was observed on coagulation parameters when compared with the controls.

In females sacrificed at the end of treatment period, no statistically significant or test item effect observed on any of the haematology or blood coagulation parameters when compared with the controls. All group mean and most of the individual values were within the historical control data range.

Clinical Biochemistry:

In males and females sacrificed at the end of treatment period, no test item related or statistically significant effect on any clinical biochemistry parameter in treatment groups was observed when compared with the control.

Urinalysis:

The urinalysis performed in selected male and female animals sacrificed at the end of treatment period revealed no test item treatment related effect and all urinary parameters were in the normal range of variation. High protein levels were found in the urine of few male and females of all groups including control group. Therefore, this effect on urine parameters was not considered to be test item related.

Pathology:

Few specific macroscopic changes were recorded for the male and female animals, which based on microscopic examination were not considered to be of test item treatment relevance. However,In decedents, red discoloration observed in the thymus of males no. 35 and No. 38 of HD group was correlated microscopically with congestion and hemorrhage in male no. 35 and with hemorrhage in male no. 38. The above mentioned changes were considered incidental agonal changes which are occasionally found in animals being subjected to necropsy. In survivors, small testes and epididymides recorded in male no. 14 (LD group) correlated microscopically with testis and epididymis atrophy and aspermia. In male No. 21 (MD group) small epididymis on the left side was correlated microscopically with unilateral epididymis atrophy and aspermia. In addition, in the male no. 30, a dilated right kidney was correlated microscopically with pelvic dilatation.In female No. 59 (LD group), the observed diaphragmal herniation corresponded histologically to a hepatic nodule.

The above mentioned changes were considered to be most likely incidental in nature.

Organ Weight:

In males sacrificed at the end of treatment period, there were statistically significantly higher absolute liver weights in all treatment groups (except MD group), higher relative (to body weight) liver weights in MD and HD group and higher kidney weights in LD and HD group although statistical significance only achieved in LD group when compared with the controls. Increased liver absolute weights were histologically correlated with slight hepatocellular hypertrophy only in one animal from the HD group, whereas in the MD and LD group neither the absolute nor the relative increase in liver weight correlated with underlying histological changes. Therefore, without a dose dependent correlation between liver weight increase and the observed histopathological hepatic changes and in absence of hepatic-derived enzyme profiling,the toxicological relevance of the liver weight increase could not be established.

In females sacrificed at the end of treatment period, significantly higher absolute liver weights were observed in HD group when compared with the controls although statistical significance was not achieved. There were also statistically significantly higher relative (to body weight) kidney weights observed in HD group when compared with the controls.In females, statistically significantly higher relative kidney weights in HD group were considered of no toxicological relevance in absence of correlating histological changes.

 

Histopathology:

There were a number of early decedents.in females, gastric changes (ulceration in female No. 75 and erosion in female No. 77) contributed to both morbidity and mortality. In males (31, 35 and 38) based on the histopathology investigation, the cause of death was not evident although, there were slight to moderate gastric changes were observed which might have contributed to the morbidity.The above mentioned changes were associated with vacuolization of forestomach epithelial cells (epithelial vacuolization), hyperkeratosis, mixed cell infiltrates and squamous hyperplasia. In male stomach, hyperkeratosis and squamous hyperplasia were observed.In liver minor degree of hepatocytes vacuolation (fatty change) and centrilobular hypertrophy was observed in few males and females.

In Survivors of LD, MD and HD groups, test item related histopathology changes were observed in liver, kidney and stomach.

In liver, minor degree of centrilobular hepatocytes hypertrophy was observed only in few males from the HD group and was considered test item related. In kidney, minimal to slight hyaline droplets accumulation in tubular epithelial cells were observed in the majority of males from all dose groups and the control group.This renal change is a male rat specific phenomenon of no toxicological relevance in humans. Minimal to slight tubular basophilia was observed in few animals from the high dose group only. The tubular basophilia was considered most likely related to the test item administration. In stomach, moderate forestomach ulcerationaccompanied by severe multifocal mixed cell infiltrates, moderate submucosal edema wall and epithelial vacuolization was observed in one male from the MD group. Minimal to moderate mixed cell infiltrates mainly located at the limiting ridge and sometimes extending in the adjacent forestomach and glandular stomach submucosa of males and females from the LD and MD groups and only in males from the HD group. Minimal to moderate epithelial vacuolization was noticed in few males from LD, MD and HD dose groups only. Furthermore, a minor degree of hyperkeratosis was observed in some males from the LD and HD dose groups, whereas slight to moderate squamous hyperplasia was present only in few males from the HD group. These changes in stomach were considered to be most likely related to the test item administration.

The test item did not produced any histological evidence of toxicity in the male and female reproductive organs and tissues.

In conclusion, due to the early mortality observed in males and females from the high dose group and the presence of several histopathological adverse changes in different organs, a histomorphological NOAEL (no observed adverse level) could be established at 30 mg/kg bw/day for the male rats and 200 mg/kg bw/day for female rats.

Dose Formulation Analysis:

Dose formulation analysis for nominal concentration revealed that nominal concentrations for all formulations were confirmed throughout the study periodas measured concentrations were within acceptance criterion of 10%.

Conclusion:

On the basis of this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with Naphthalenesulfonic acid, bis(1-methylethyl)-,methyl derivs., sodium salt in male and female Wistar rats with dose levels of 30, 200, and 500/700 mg/kg body weight day the following conclusions can be made:

- There were few mortalities observed in the study (3 males and 2 females) during the early days of the study. Histopathologically cause of the death could not be established for all of them. In females, gastric changes (ulceration in female No. 75 and erosion in female No. 77) contributed to both morbidity and mortality.  

- No adverse effects of test item were found on male and female clinical observations in LD and MD group, clinical signs like slightly to moderately increased piloerection, reduced spontaneous activity and half eyelid closure in HD group could be attributed to treatment with the test item.

- No adverse effects of test item were found on female body weight development and food consumption in any treatment group. However, test item related effect on male body weight development observed in MD and HD group and on food consumption in HD group.

- Functional observations, haematology and coagulation, clinical biochemistry, urinalysis, gross pathological findings at necropsy and organ weight remained unaffected in male and females up to dose levels of 500/700 mg/kg bw/day.

- There were also no effects on litter data, litter weight data, nipple retention, precoital interval and duration of gestation, reproductive indices, pup thyroid weight and parental male and pup thyroxine hormone, pre and post-natal data, pup survival and pup external findings on PND 0 and at death observed up to dose levels of 500/700 mg/kg bw/day.

- There were test item related effects observed on estrous cyclicity in female HD group (secondary effect without effect on pregnancy rate) and anogenital distance in male and female HD group when compared with the controls. However, all anogenital values in male and female pups were within historical control data range and no additional finding is supporting a possible androgen-mediated activity (agonistic) or any other endocrine disruption modality of the test item.

- Histopathologically, in survivors of LD, MD and HD groups, test item related histopathology changes were observed in liver, kidney and stomach. However, the test item did not produced any histological evidence of toxicity in the male and female reproductive organs and tissues.

The report concluded that, due to the early mortality observed in males and females from the high dose group (at 700 mg/kg bw/day in first week) and the presence of several histopathological adverse changes in different organs, a histomorphological NOAEL could be established at 30 mg/kg bw/day for the male rats and 200 mg/kg bw/day for female rats.

Close examination shows that the only effects observed at MD (200 mg/kg) are a slight lower BW compared to control (-6%) in males, and an increased combined effects in stomach upon histopathological examinations in males and females. (See attached graphs).

All other effects are observed at HD at 700 mg/kg bw (mortality and effects on oestrous cycle first week study) and 500 mg/kg (Bw males, slight effects food consumption, clinical signs, slight effects on liver and kidney weights, and increased platelets in males)

No adverse effects were observed on reproduction an developmenatl parameters, and the NOAEL for reproduction and development in this study established at 500 mg/kg bw/day.