Naphthalenesulfonic acid, butyl-, Me derivs., sodium salts

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No data is available on the product Naphthalenesulfonic acid, methyl, butyl, sodium salt (ANS B) (C7-alkyl naphthalene sulfonate) itself. Evaluation is based on read-across to the comparable C7-alkyl naphthalene sulfonate ANS IP and other substances in the Alkyl Naphthalene Sulfonates (ANS) category.

Naphthalenesulfonic acid, bis(1-methylethyl)-,methyl derivs., sodium salt (ANS IP; C7-alkyl naphthalene sulphonate) was found to be not toxic to reproduction in the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening study according to OECD 422 at highest dose tested of 500 mg/kg bw/day.

Link to relevant study records

Reference

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).

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

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.

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

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

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).

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: 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

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.

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.

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)

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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.

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

Key result

Reproductive effects observed:

no

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Valid and GLP compliant 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

No data is available on the product Naphthalenesulfonic acid, methyl, butyl, sodium salt (ANS B) (C7-alkyl naphthalene sulfonate) itself. Evaluation is based on read-across to the comparable C7-alkyl naphthalene sulfonate ANS IP and other substances in the Alkyl Naphthalene Sulfonates (ANS) category. See chapter 13 for support for read-across within the category of Alkyl Naphthalene Sulfonates (ANS).

A Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed withNaphthalenesulfonic acid, bis(1-methylethyl)-,methyl derivs., sodium salt (ANS IP; C7-alkyl naphthalene sulphonate) in 10 Wistar Han rats/dose/sex by oral gavage.

The test item was administered daily in 4 groups of test animals at dose levels of 0, 30, 200 and 700/500 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 due to overt toxicity. Females were treated for up to 54 days (until post-natal day 12) and males for 28 days. Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg bodyweight. Dose volumes were adjusted individually based on weekly body weight measurements. Dose levels were based on results from a RF involving evaluation of0, 50, 200, 700 mg/kg in 5 ml/kg in 3 animals/sex/group, according to the principles of OECD 421 guidance. Fertility and litter parameters were not affected in this range finder.

 

Parental effects:

In the first week of the main study, before lowering the HD dosing from 700 to 500 mg/kg bw /day, the HD groups showed overt toxicity.Three males and two females of the HD groups were found dead between day 3 and 6. No further deaths occurred after the dose was lowered from 700 to 500 mg/kg bw/day. Clinical symptoms were mainly confined to the HD groups, and specifically in the first one to two weeks consisted of moderate reduced spontaneous activity, prone position, and eye closed. Further in the study clinical signs included moving the bedding, piloerection and slight reduced spontaneous activity. In the females also the MD animals showed signs of moving the bedding during gestation and lactation. There were no effects on functional observations.

BW of the HD males showed an actual BW loss during the first week (12%), then recovery to MD level. The MD males showed decreased BW gain resulting to 6% lower BW compared to control, but this was not statistically significant. LD dose showed no effects. For the females, there were no significant effects observed on BW. Food consumption was in HD group lower during the first week, and showed a compensatory increase the second week.

During the first 14 days of the study (premating period) estrous cyclicity was evaluated. Most (6/8) females were shown to be completely acyclic, which is considered the result from severe toxicity observed at the start. Upon lowering the dose from 700 to 500 mg/kg bw/day, also recovery of the estrous cycle occurred as is evidenced by pregnancy of all but one of the HD females the week thereafter at mating. Haematology showed no remarkable findings besides a higher platelet number in HD males which was considered to be incidental. Clinical biochemistry and urinalysis were without findings. Pathology showed no relevant changes besidesgastric changes (ulceration / erosion) in the two females and slight to moderate gastric changes (hyperkeratosis, squamous hyperplasia, mixed cell infiltrates, epithelial vacuolization) in the three males that died in the first week.

The organ weights show an increased liver weight in the HD males and females which histologically correlated with slight hepatocellular hypertrophy in some animals from the HD group. In absence of effects on hepatic-derived enzyme profiling, the toxicological relevance of the liver weight increase cannot be established. In females there were also statistically significantly higher kidney weights observed in the HD group. These wereconsidered of no toxicological relevance in absence of correlating histological changes.

At histopathology, a minor degree of centrilobular hepatocytes hypertrophy was observed is some animals in the HD group. Also minimal to moderate glycogen accumulation was seen in HD males and females. In kidney, minimalto slight tubular basophilia was observed in few animals from the high dose group only (See attached graph on kidney effects). In stomach,ulceration was observed in one MD male. Other effects as moderate hyperkeratosis, moderate squamous hyperplasia, minimal to moderate epithelial vacuolization, minimal to marked mixed cell infiltrates were observed over all three dose groups. Overall, effects on the stomach show a clear dose related response (See attached graph on stomach effects).The test item did not produce any histological evidence of toxicity in the male and female reproductive organs and tissues.

 

Effects on reproduction:

There were no differences in the length or sequence of cycle stages between the LD and MD dose groups and the control group. In the HD group, 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. Precoital interval was statistically significantly higher in HD group females compared to the controls. This difference in precoital interval was marginal (1 day) and considered as biological variation. In view of the observed acyclicity in the period immediately preceding mating, it is more a sign of rapid recovery from a too high toxicity from 700 mg/kg bw.

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. Also no effects were observed on the number of corpora lutea, implantation sites, live pups on PND 0, 4 and 13, percent preimplantation loss and post implantation.

 

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. There was no statistically significant effect on pup mean weight, total litter weight, female litter weight on PND 0, PND 4 and PND 13.

In males, statistically significant lower pup weight in LD and HD group (not dose dependent) compared with the controls. There was also statistically significantly higher absolute (not in HD group) and relative AGD in MD and HD group observed when compared to the controls. In females, statistically significantly higher absolute and relative AGD was observed in all treatment groups when compared to the controls. (See attached graphs on pup weight and AGD). 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 to 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.

Pup thyroid weight, male and PND 13 pup thyroxine hormone (T4) in the treatment groups were not affected. 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.

 

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 during first week of the study (mortality and effects on oestrous cycle) and 500 mg/kg (Bw males, slight effects food consumption, clinical signs, slight effects on liver and kidney weights, and increased platelets in males)

As no adverse effects on the reproduction were observed, the NOAEL for reproductive effects is 500 mg/kg bw.

 

Lack of effects on reproduction issupported from testing ofSodium (C10-13-)alkylnaphthalene sulfonate:

In an OECD 422 study, oral treatment with Sodium(C10-13-)alkylnaphthalene sulfonate resulted in deaths at 1000 mg/kg which were attributed to tracheal/pulmonary lesions associated with aspiration of test article (possibly due to the gavage procedure) and/or acute inflammation/ulceration in the stomach. The primary effects at 1000 mg/kg resulting from treatment in surviving animals and early deaths/sacrifices were confined to local effects in the stomach (being most pronounced in those animals sacrificed of found dead during the study), and an increased incidence and/or severity of hemorrhage in the mesenteric lymph nodes. Clinical signs, clinical biochemistry, haematology parameters and motor activity were unremarkable.

Specifically, histopathological examinations showed no remarkable findings in reproductive organs: cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles,testes, uterus and vagina). No toxicologically relevant effects on mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were noted.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

-        Parental NOAEL: 300 mg/kg/day (local and systemic)

-        Reproduction NOAEL: at least 1000 mg/kg/day

-        Developmental NOAEL: at least 1000 mg/kg/day

Effects on developmental toxicity

Description of key information

No data is available on the product Naphthalenesulfonic acid, methyl, butyl, sodium salt (ANS B) (C7-alkyl naphthalene sulfonate) itself. Evaluation is based on read-across to the comparable C7-alkyl naphthalene sulfonate ANS IP and other substances in the Alkyl Naphthalene Sulfonates (ANS) category. See chapter 13 for support for read-across within the category of Alkyl Naphthalene Sulfonates (ANS).Naphthalenesulfonic acid, bis(1-methylethyl)-,methyl derivs., sodium salt(ANS IP; C7-alkyl naphthalene sulphonate) was found to be not toxic to reproduction in the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening study according to OECD 422 at highest dose tested of 500 mg/kg bw/day.An OECD 422 study with oral treatment withANS N (‘High nonene’)resulted to a NOAEL for reproduction and development of at least 1000 mg/kg bw/day. An additional OECD 414 developmental toxicity study in rats on ANS N (‘high nonene’) showed no developmental toxicity, and resulted to a NOAEL of at least 600 mg/kg bw/day, being the highest dose tested.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

04 December 2011 - 20 January 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Justification for type of information:

See chapter 13 for support for read-across within the category of Alkyl Naphthalene Sulfonates (ANS).

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: Organisation of Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, March 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

no

Principles of method if other than guideline:

In addition, the procedures described in the report essentially conform to the following guidelines:
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany..
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: mean weight at start of treatment was 297 gr (males) or 201 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle.

IN-LIFE DATES: From: 04 December 2011 - 20 January 2012

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

- Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for density (1.2 g/mL) and purity (52.95 w/w%) of the test substance.
- Storage conditions of formulations: At room temperature in the dark under nitrogen
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Some animals were not dosed according to the latest body weight. For animals on Day 1 of lactation, the amount of test substance was maximally10% less than intended. For animals on Day 4 of lactation, the amount of test substance wasmaximally 34% more than intended. Given the incidental nature of this deviation, and since inlife data did not indicate an effect of this deviation, interpretation of the study results was considered to have been unaffected.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (22 December 2011), according to a validated method (Project 497219). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature under nitrogen was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Details on mating procedure:

- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).
Detection of mating was not confirmed for one animal at 100 mg/kg which had total litter loss. One female at 1000 mg/kg was mated with a proven male of the same dose level, since the male with which the female should originally be mated with was sacrificed during the premating period.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 40-47 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Main females were not dosed during littering.

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

Frequency of treatment:

Once daily for 7 d/w.

Duration of test:

Males: 29 days
Females: 40-47 days

No. of animals per sex per dose:

10, plus 5 males/group in Groups 1 and 4 (Recovery).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of the dose range finding study (Project 497221), and in consultation with the sponsor.
- Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.
- Identification of pups: On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.
- Selection of animals for selected measurements: 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The
circumstance of any death was recorded in detail.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually. These clinical observations were at least conducted between 0 and 1 hour after dosing (i.e. based on the peak period of anticipated effects after dosing in the dose range finding study (Project 497221)). The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.
Clinical signs for one animal at 1000mg/kg on 10 January 2012 were recorded off-line and were not identified with severity grades. Based on clinical signs recorded, an adequate evaluation of the study results could still be made. Clinical signs were entered in the computer system later, and a grade 1 was entered for all symptoms, including those for which a maximum grade 3 or 4 applied (the software requires a grade to be entered).

BODY WEIGHT:
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION:
- Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY: yes

WATER CONSUMPTION : No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling).
- Dose groups that were examined: all
- Battery of functions tested: According to test guidelines
Motor activity of males at the end of the recovery period was not determined, although a (potentially) treatment-related effect was noted at the end of treatment. No clinical signs were noted to support the lower motor activity at the end of the treatment period, nor were any clinical signs noted at the end of the recovery period. The mean motor activity did not show a clear dose-related trend over the dose groups. This suggests that an effect (if any) on motor activity at the end of the recovery phase would have been small, and not have any significant impact on the overall conclusion of the study.

GENERAL REPRODUCTION DATA
- Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.
- Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.

- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.

SACRIFICE
Pups surviving to planned termination were killed by decapitation on lactation Days 5-7.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.

HISTOPATHOLOGY / ORGAN WEIGHTS
No.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data.
- The following additional methods of statistical analysis were used: Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. No statistical analysis was performed on histopathology findings.

Indices:

Reproductive indices; For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring indices:
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic and microscopic abnormalities were observed in the 1000 mg/kg group that were considered to be related to treatment in stomach (thickened limiting ridge in 2/5 females and irregular surface in 1/8 males and 1/5 females) and mesenteric lymph nodes (haemorrhagic appearance in 1/8 males and 3/5 females). Such effects were also observed in the animals found dead or sacrificed, but not in animals at the end of the recovery period.

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality only occurred in the high dose group of 1000 mg/kg (2/15 males and 4/10 females) which were attributed to tracheal/pulmonary lesions associated with aspiration of test article (possibly due to the gavage procedure) and/or acute inflammation/ulceration in the stomach.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY
Two males at 1000 mg/kg were sacrificed in extremis on Days 4 and 5 of the premating period, respectively. Three females at 1000 mg/kg were found dead on Days 20, 39 and 23 of treatment, respectively (i.e. Days 1, 22 and 5 of the post-coitum phase, respectively). One other female at 1000 mg/kg was found dead on Day 25 of treatment (i.e. Day 11 of the mating period). One female at 1000 mg/kg and one female at 100 mg/kg were sacrificed due to total litter loss. The incidence of total litter loss showed no relation to the dose, and the incidence was low. No toxicological relevance was ascribed to these occurrences.

CLINICAL SIGNS
Hunched posture was shown by all males and most females at 1000 mg/kg (except the two males sacrificed in extremis during the first week of treatment), generally from the first week of treatment onwards. Surviving males and females at 1000 mg/kg showed sporadic incidences of lethargy, breathing difficulties (rales and/or shallow respiration), piloerection and pale appearance. Reduced faeces production was noted for males at 1000 mg/kg during the last week of the mating period. For males, all these findings had resolved at commencement of/during the recovery phase. Clinical signs noted among the four females at 1000 mg/kg found dead included lethargy, flat/hunched posture, uncoordinated movements, ptosis, rales, piloerection, lean or pale appearance, abdominal swelling and/or ptosis. The incidence of these findings was not clearly related to the day of death. Two males at 1000 mg/kg were sacrificed in extremis during the first week of treatment based on their significantly deteriorated health condition (clinical signs were not recorded on the day of sacrifice). Salivation seen among all animals at 1000 mg/kg, and at lower incidence at 100 and 300 mg/kg, was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance. Incidental findings that were noted included rales, a dark left eye, alopecia, scabs, hypersensitivity to touch (single day for a single animal), and chromodacryorrhoea. These findings occurred within the
range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

FUNCTIONAL OBSERVATIONS
Motor activity (total movements and/or ambulations) was reduced for males at 100, 300 and 1000 mg/kg and for females at 100 and 300 mg/kg with statistical significance at the end of the treatment period, but no clear dose-related trend was apparent and control values were considered to be high (motor activity at the end of the recovery period for males was not determined). All groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period. Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.

BODY WEIGHTS
No toxicologically relevant changes in body weights and body weight gain were noted. The slightly lower body weight gain of males at 1000 mg/kg at the end of the premating period did not prevail or became more pronounced as treatment progressed. This change was therefore considered to be of no toxicological significance. One male at 1000 mg/kg and one female at 100 mg/kg showed weight loss of up to 9% over Days 1-8 of the premating period. As these animals again gained weight during the mating period, and similar changes in body weight were absent among other animals of these dose groups, this was considered to be of no toxicological relevance.

FOOD CONSUMPTION
No toxicologically relevant changes in food consumption before or after correction for body weight were noted. Absolute food intake of males and food intake corrected for body weight for both sexes at 1000 mg/kg was lower during the first week of the premating period. Since this change did not prevail or became more pronounced as treatment progressed, it was considered to be of no toxicological significance.

HAEMATOLOGY
The following (statistically significant) changes in haematology parameters distinguished animals at 1000 mg/kg from control animals at the end of the treatment phase:
- Lower red blood cell counts in males and females,
- Higher red cell distribution width (RDW) in two males and one female at 1000 mg/kg
- Lower haemoglobin level in males and females,
- Lower haematocrit level in males and females.
At the end of the recovery phase, males at 1000 mg/kg showed lower red blood cell counts, haemoglobin and haematocrit levels, and a higher mean corpuscular volume (MCV).

CLINICAL BIOCHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher alkaline phosphatase activity (ALP) in two females at 1000 mg/kg (mean not statistically significant).
- Higher glucose levels in females at 1000 mg/kg (mean not statistically significant).
- Lower total protein levels in males at 300 and 1000 mg/kg,
- Lower total bilirubin levels in males at 100, 300 and 1000 mg/kg, and in females at 300 and 1000 mg/kg.
- Higher cholesterol level in females at 1000 mg/kg.
These findings had resolved during the recovery phase in males at 1000 mg/kg.
The lower bile acid level of males at 100, 300 and 1000 mg/kg was considered to be of no toxicological significance, since the opposite effect would be expected in case of target organ toxicity. This change in bile acid level, as well as the higher chloride level of males at 1000 mg/kg and the lower potassium levels in females at 300 mg/kg at the end of treatment showed no dose-related trend. The apparent higher glucose level of females at 1000 mg/kg was due to a high variation in individual values, the range of which was comparable to the variation encountered among control animals. No
toxicological relevance was therefore ascribed to these changes.

MACROSCOPIC EXAMINATION
The following macroscopic abnormalities at the end of the treatment period in surviving animals at 1000 mg/kg were considered to be directly related to treatment with the test substance:
- Thickened limiting ridge of the stomach in 2/5 females.
- Irregular surface of the stomach in 1/8 males, and irregular surface of the forestomach in 1/5 females,
- Reddish/dark red/black discolouration of the mesenteric lymph nodes in 1/8 males, and 3/5 females.
Findings in sacrificed animals (except for the female with total litter loss) or animals found dead at 1000 mg/kg considered to be directly related to treatment included:
- Dark red/black discolouration of the mesenteric lymph nodes in 3/4 females.
- Thickened limiting ridge of the stomach in 3/4 females.
- Irregular surface of the forestomach in 1/4 females,
- Dark red discolouration or dark red foci of the glandular mucosa of the stomach in 2/2 males.
- Dark red discolouration of the gastro-intestinal tract in 1/2 males.
No treatment-related macroscopic abnormalities were noted at the end of the recovery period in males at 1000 mg/kg. Only a background level of hemorrhage/congestion in the mesenteric lymph node was recorded in 1/4 surviving Recovery males at 1000 mg/kg (minimal). Dark red foci/reddish discolouration of the thymus noted in several surviving animals and intercurrent sacrifices/deaths was considered to be related to necropsy procedures (congestion), and the incidence of microscopic correlates did not indicate a relationship to treatment. Other findings among animals sacrificed in extremis or found dead consisted of a reduced size of the thymus and spleen, enlarged liver, an advanced stage of autolysis, incomplete exsanguination, dilation of the gastrointestinal tract and mucous contents of the gastro-intestinal tract. These were considered to be related to the necropsy procedures and/or to be secondary to the autolytic process. One female at 100 mg/kg and one female at 1000 mg/kg showed total litter loss. One female at 1000 mg/kg showed dead foetuses in the uterus (five in each horn). The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, did not show a dose-related incidence trend and/or had no treatment-related microscopic correlates. These necropsy findings were therefore considered to be of no toxicological relevance, and included red foci on the lungs, pelvic dilation of the kidneys, a yellowish or reddish soft or hard nodule on the epididymides, a tan focus on the preputial glands, irregular surface of the liver, a dark red hard nodule on the spleen, uterus containing fluid, tan discolouration of the clitoral glands, and alopecia.

ORGAN WEIGHTS
No toxicologically relevant changes in organ weights and organ to body weight ratios were noted. The (statistically significant) lower absolute thymus weights and thymus to body weight ratio and higher liver to body weight ratio in males at 1000 mg/kg at the end of the treatment phase were slight
in nature, had no morphological correlates, and these organ weights were similar to control levels at the end of the recovery period. The statistically significant lower absolute thyroid weight of males at 100 and 1000 mg/kg and lower thyroid to body weight ratio of males at 100 mg/kg occurred in the absence of a dose-related trend, and means remained within the range considered normal for rats of this age and strain. The higher thyroid weight, thyroid to body weight ratio, and seminal vesicle weight of males at 1000 mg/kg at the end of the recovery period was absent at the end of the treatment period, and the change was slight. No toxicological relevance was therefore ascribed to these variations.

MICROSCOPIC EXAMINATION
The following treatment-related findings were noted in surviving main study animals at 1000 mg/kg:
- Lymphogranulocytic inflammation of the forestomach in 3/4 males.
- Hyperkeratosis of the stomach in 1/4 males (minimal degree).
- Hyperplasia of the squamous epithelium of the forestomach in 1/4 males (minimal) and 2/4 females (minimal-slight).
- Increased incidence and severity of hemorrhage/congestion in the mesenteric lymph nodes of 4/4 males and 4/4 females.
No treatment-related histopathological findings were noted at the end of the Recovery phase. The major histologic findings for the animals found dead or sacrificed in extremis were present in the respiratory tract (trachea/lungs) and/or stomach, and primarily consisted of tracheal/bronchiolar necrosis with inflammation, ulceration/erosion/inflammation of the glandular stomach and subcapsular necrosis of the liver. The presence of lesions in the trachea/lungs suggests a local reaction to aspiration of test article and the possibility of refluxive inhalation or gavage procedure rather than
systemic toxicity. Stomach lesions (most pronounced in animal nos. 40, 48, and 84) were considered to be test substance related. Findings of atrophy and increased apoptosis in the thymus and lymphoid depletion in the spleen of early death animals were considered secondary to stress. Reticuloendothelial hyperplasia in the spleen and histiocytic infiltrates in the mesenteric lymph nodes were possibly treatment related.
Reproductive performance:
No test article related abnormalities were seen in the reproductive organs. Staging of spermatogenesis in testes did not provide any evidence of test article related impairment to the spermatogenetic cycle. Other microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar Han rats of this age and strain.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.

Details on embryotoxic / teratogenic effects:
There was no evidence of teratogenic effects based on the absence of relevant clinical signs and external macroscopic findings.

EARLY POSTNATAL PUP DEVELOPMENT
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings. The higher sex ratio at 100 mg/kg occurred in the absence of a dose-related trend, and was therefore considered to be of no toxicological relevance.

MORTALITY PUPS
Three pups of the control group, four pups at 100 mg/kg, and eight pups at 1000 mg/kg (due to total litter loss of a single female (no. 90)) were found dead or missing during lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS PUPS
Clinical symptoms of pups were confined to a scab on the nose of one pup. The nature and incidence of this clinical sign remained within the range considered normal for pups of this age, and was therefore considered to be of no toxicological relevance. BODY WEIGHT PUPS Body weights of pups were considered to have been unaffected by treatment.

MACROSCOPY PUPS
Incidental macroscopic findings of pups that were found dead included absence of milk in the stomach, a stage of beginning autolysis and partial cannibalism. A hard nodule on the lower lip was noted for one of the surviving pups. The nature and incidence of this finding remained within the range considered normal for pups of this age, and was therefore considered to be of no toxicological relevance.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Abnormalities:

not specified

Developmental effects observed:

not specified

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored under nitrogen at room temperature under normal laboratory light conditions for at least 6 hours.

REPRODUCTIVE DATA No toxicologically relevant effects on mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were noted. Fertility and conception indices at 1000 mg/kg were lower than controls. This was ascribed to death of three females, and not related to a primary effect of the test substance on reproductive performance. Three females were recorded as non-pregnant, two of which at 1000 mg/kg were found dead early in the post-coitum phase, on respectively Days 1 and 5). Hence, potential pregnancy of these two females could not be confirmed by eg. counting of implantation sites. One female at 1000 mg/kg was found dead on Day 11 of the mating phase, and necropsy of this female showed implantation sites confirming pregnancy. All females which completed the full study duration were found to be pregnant, either based on delivery of pups or based on presence of implantation sites. Two females (one at 100 mg/kg and one at 1000 mg/kg) had total litter loss. Given the low incidence of this finding, this was considered to be of no toxicological relevance. The variation in the number of corpora lutea over the dose groups (achieving a level of statistical significance at 1000 mg/kg) occurred in the absence of a clear dose related trend, and the variation in individual values remained essentially within the range encountered among control animals. Hence, no toxicological relevance was ascribed to these variations. GESTATION No toxicologically relevant effects on gestation index and duration were noted. The lower gestation index at 1000 mg/kg was due to total litter loss of one female, and spontaneous death of two females out of the seven pregnant females. This lower gestation index was therefore considered to be of no toxicological relevance. PARTURITION/MATERNAL CARE No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Conclusions:

In an OECD 422 study, oral treatment with Sodium alkylnaphthalene sulfonate resulted in deaths at 1000 mg/kg which were attributed to tracheal/pulmonary lesions associated with aspiration of test article (possibly due to the gavage procedure) and/or acute inflammation/ulceration in the stomach. The primary effects at 1000 mg/kg resulting from treatment in surviving animals and early deaths/sacrifices were confined to local effects in the stomach (being most pronounced in those animals sacrificed of found dead during the study), and an increased incidence and/or severity of hemorrhage in the mesenteric lymph nodes. Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: 300 mg/kg/day (local and systemic)
Reproduction NOAEL: at least 1000 mg/kg/day
Developmental NOAEL: at least 1000 mg/kg/day

Reference 2

Endpoint:

developmental toxicity

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).

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Develomental 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

Species:

rat

Strain:

Wistar

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.

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: 30 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.

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.

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.

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

Duration of test:

Litter and lactating females were observed until PND 13.

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

No. of animals per sex per dose:

00 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

Maternal examinations:

Estrous Cyclicity
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.

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.

Ovaries and uterine content:

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.

Fetal examinations:

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.

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).

Indices:

The Copulation Index (%), Fertility Index (%), Delivery Index (%), Viability Index PND 0 - 4 (%) and Viability Index PND 4 - 13 (%) were calculated.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Urinalysis findings:

effects observed, non-treatment-related

Behaviour (functional findings):

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Gross pathological findings:

effects observed, non-treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

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.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Total litter losses by resorption:

effects observed, non-treatment-related

Early or late resorptions:

effects observed, non-treatment-related

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

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.

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Remarks:

UVCB substance with 100% purity

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

LOAEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Remarks:

UVCB substance with 100% purity

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

Basically clear effects are observed in females at 700 mg/kg bw (mortality and effects on oestrous cycle during first week of study) and 500 mg/kg (slight effects food consumption, clinical signs, slight effects on liver and kidney weights)

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: Local effects stomach.

Description (incidence and severity):

Effects are observed at 700 mg/kg bw during first week of study (mortality possibly contributed by local effects stomach, and effects on oestrous cycle) and 500 mg/kg (slight effects food consumption, clinical signs, slight effects on liver and kidney weights)

Fetal body weight changes:

effects observed, non-treatment-related

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, non-treatment-related

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Skeletal malformations:

not examined

Visceral malformations:

not examined

Details on embryotoxic / teratogenic effects:

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.

Key result

Dose descriptor:

NOAEL

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

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

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:       200       mg/kg bw/day

High Dose:       700       mg/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 during 14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 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 body weight.

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 females of 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, few mortalities observed were male nos. 31, 35 (HD) found dead on premating day (PMD) 6, male no. 38 (HD) was found dead on 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 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.

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.

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 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 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 period as 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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Valid and GLP compliant 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

No data is available on the product Naphthalenesulfonic acid, methyl, butyl, sodium salt (ANS B) (C7-alkyl naphthalene sulfonate) itself. Evaluation is based on read-across to the comparable C7-alkyl naphthalene sulfonate ANS IP and other substances in the Alkyl Naphthalene Sulfonates (ANS) category. See chapter 13 for support for read-across within the category of Alkyl Naphthalene Sulfonates (ANS).

A Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed with Naphthalenesulfonic acid, bis(1-methylethyl)-,methyl derivs., sodium salt (ANS IP; C7-alkyl naphthalene sulfonate)in 10 Wistar Han rats/dose/sex by oral gavage.

The test item was administered daily in 4 groups of test animals at dose levels of 0, 30, 200 and 700/500 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 due to overt toxicity. Females were treated for up to 54 days (until post-natal day 12) and males for 28 days. Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg bodyweight. Dose volumes were adjusted individually based on weekly body weight measurements. Dose levels were based on results from a RF involving evaluation of0, 50, 200, 700 mg/kg in 5 ml/kg in 3 animals/sex/group, according to the principles of OECD 421 guidance. Fertility and litter parameters were not affected in this range finder.

 

Parental toxicity:

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 during first week of the study (mortality and effects on oestrous cycle) and 500 mg/kg (Bw males, slight effects food consumption, clinical signs, slight effects on liver and kidney weights, and increased platelets in males)

 

Effects on reproduction:

There were no differences in the length or sequence of cycle stages between the LD and MD dose groups and the control group. In the HD group, 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. Precoital interval was statistically significantly higher in HD group females compared to the controls. This difference in precoital interval was marginal (1 day) and considered as biological variation. In view of the observed acyclicity in the period immediately preceding mating, it is more a sign of rapid recovery from a too high toxicity from 700 mg/kg bw.

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. Also no effects were observed on the number of corpora lutea, implantation sites, live pups on PND 0, 4 and 13, percent preimplantation loss and post implantation.

 

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. There was no statistically significant effect on pup mean weight, total litter weight, female litter weight on PND 0, PND 4 and PND 13.

In males, statistically significant lower pup weight in LD and HD group (not dose dependent) compared with the controls. There was also statistically significantly higher absolute (not in HD group) and relative AGD in MD and HD group observed when compared to the controls. In females, statistically significantly higher absolute and relative AGD was observed in all treatment groups when compared to the controls. (See attached graphs on pup weight and AGD). 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 to 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.

 

The study therefore concluded to a NOAEL for developmental toxicity of 500 mg/kg bw/day.

Lack of effects on reproduction and development is furthersupported from studies on Sodium (C10-13)-alkylnaphthalene sulfonate (ANS N (‘High nonene’)). An OECD 422 study with oral treatment withANS N (‘High nonene’)resulted to a NOAEL for reproduction and development of at least 1000 mg/kg bw/day.

Further, developmental toxicity/teratogenicity for ANS N (‘High nonene’) was studied in rats according to OECD 414 guideline.Eighty-eight mated female Wistar Han rats were assigned to four dose groups. Sodium alkylnaphthalene sulfonate was administered once daily by oral gavage from Days 6 to 20 post-coitum at doses of 100, 300 and 600 mg/kg bw/day. The rats of the control group received the vehicle, water, alone. All animals surviving to Day 21 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. The stomach was collected and fixed from all animals at necropsy. Histopathological examination was performed on the stomach from 10 selected females per group. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placenta and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative; these fetuses were dissected and examined for visceral anomalies. The other one-half of the fetuses were processed and stained with Alizarin Red S for skeletal examinations.

Results: Test item-related morphological alterations were present in the stomach at 600 mg/kg bw/day. A slightly increased incidence of minimal inflammation of the glandular stomach was recorded at 600 mg/kg bw/day (5/10 females), compared to 1/10 females of the control group. In one female at this dose a moderate erosion/ulceration with a moderate lymphogranulocytic inflammation of the forestomach was recorded, which was considered to be related to the treatment with the test item. No maternal toxicity was observed in the 100 and 300 mg/kg bw/day groups. No developmental toxicity was observed in the 100, 300 and 600 mg/kg bw/day groups.

In conclusion, based on the results in this prenatal developmental toxicity study the following NOAELs were established:

- NOAEL maternal, local = 300 mg/kg bw/day based on morphological alterations of the stomach.

- NOAEL maternal, systemic toxicity = 600 mg/kg bw/day.

- NOAEL developmental = 600 mg/kg bw/day.

Mode of Action Analysis / Human Relevance Framework

Based on its surfactant properties, the structure is not expected to easily pass membrane structures, but cytotoxicity through disruption of cell membrane is expected. This is supported by study results: All available data indicates that the NOAEL is driven by local effects of the substance on gastro-intestinal tract (stomach). Consequently,Sodium alkylnaphthalene sulfonate is not likely to reach reproductive organs or exert effects on the developing foetus.

Justification for classification or non-classification

Sodium C7-alkylnaphthalene sulfonate (ANS IP) was found to be not toxic to reproduction and development in the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening study according to OECD 422 at highest dose of 500 mg/kg bw/day.

 

Lack of effects on reproduction and development is furthersupported from studies on Sodium (C10-13)-alkylnaphthalene sulfonate (ANS N (‘High nonene’)) where testing in OECD 422 resulted to NOAEL for reproduction/development of 1000 mg/kg bw, and developmental testing in OECD 414 resulted to a NOAEL of 600 mg/kg bw/day, the highest dose tested.

 

Based on mechanistic considerations,Sodium alkylnaphthalene sulfonate is not likely to reach reproductive organs or exert effects on the developing foetus.

For the same reasons, also effects via lactation are not to be expected.

 

Consequently, no classification for reproduction toxicity is needed.