Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 20 April 2012 to 5 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
N° 2011/40
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River Laboratories France, L’Arbresle, France. Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier Sustained-Virus Antibody Free, (COBS-VAF®).
- Age at study initiation: the males were approximately 10 weeks old, the females were approximately 9 weeks old
- Weight at study initiation: males: a mean body weight of 415 g (range 381 g to 440 g), and female a mean body weight of 243 g (range: 218 g to 288 g).
- Fasting period before study: no
- Housing: The animals were individually housed, except during pairing, in polycarbonate cages (Techniplast 2154.940 cm²) with stainless steel lids, and containing autoclaved sawdust (SICSA, Alfortville, France).
Toward the end of gestation and during lactation with their litter, autoclaved wood shavings (SICSA Alfortville, France) was provided as nesting material, a few days before delivery and during the lactation period.
half hut was given as enrichment of the environment of the rats.
The cages were placed in numerical order on the racks.
- Diet (e.g. ad libitum): The animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 5776559 and 2626975 (SSNIFF Spezialdiäten GmbH, Soest, Germany) which was distributed weekly.
- Water (e.g. ad libitum): The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: the animals were acclimated for a period of 5 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12h/12h (7:00 - 19:00)

IN-LIFE DATES: From:2012-05-02 To: 2012-06-08
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PPREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution in the vehicle. The test item was mixed with the required quantity of vehicle.
The dosage formulations were prepared on a daily basis.
The dosage forms were stored and delivered at room temperature in brown flasks.


VEHICLE
drinking water treated by reverse osmosis using an ELIX 5 apparatus
- Concentration in vehicle: 0, 30, 100 and 300 mg/kg of active ingredient
- Amount of vehicle (if gavage): 5 mL/kg
Details on mating procedure:
- M/F ratio per cage:1/1
- Length of cohabitation: until mating occurs or 14 days
- Proof of pregnancy: vaginal plug or sperm in a vaginal lavage. referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Alone
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The High Performance Liquid Chromatography with UV detection (HPLC/UV) analytical method for the determination of Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda in dosage form samples was validated at CIT prior to dosage form analysis.
The analytical method was validated for concentration ranging from 2 to 200 mg/mL.

The concentration of the test item in samples of each control and test item dosage form prepared for use in weeks 1, 3 and 5 was determined. In addition, the concentration of the test item in a sample of the high-dose test item form prepared for use in week 2 was determined.

Acceptance criterion: measured concentration = nominal concentration ± 10%

The validation of the analytical method was conducted in CIT/Study No. 38263 VAA and precise details concerning the checked parameters, acceptance criteria and obtained results were documented in the corresponding validation report.

Each analytical sequence consisted of at least:
+ A blank sample (diluent only),
+ Ten standard samples at nominal concentration, prepared from two independent standard solutions,
+ Study samples prepared from aliquots of the dosage forms.
The standard samples bracketed the dosage form samples.
The blank sample was checked for the absence of chromatographic interference.
Duration of treatment / exposure:
The dosage forms were administered daily according to the following schedule:
 in the males:
- 2 weeks before pairing (from study day 1 to 14),
- during the pairing period (from study day 15 until study day 16 to 25),
- until sacrifice (at least 5 weeks in total)(from study day 17 to 26 until study day 36).

 in the females:
- 2 weeks before pairing (from study day 1 to 14),
- during the pairing period (from study day 15 to 25),
- during gestation (from study day 16 to 26 until study day 36 to 46),
- during lactation until day 5 post-partum inclusive (from study day 37 to 47 until study day 42 to 52),

Study day 1 corresponds to the first day of the treatment period.
Frequency of treatment:
Once a day at approximately the same time
Details on study schedule:
Not appropriate
Remarks:
Doses / Concentrations:
30; 100; 300 mg/kg bw/d
Basis:
other: expressed as active material in mg/kg bw/d (purity 75.4% considering the three forms of sodium isopropylnaphtalenesulphonate: mono, di and tri)
Remarks:
Doses / Concentrations:
39.8; 133 and 398 mg/kg bw/d
Basis:
other: expressed as registered substance (UVCB 100%)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The dose-levels were selected in agreement with the Sponsor based on 2 week dose range finding study by oral route (gavage) in rat (CiToxLAB France/Study No. 38818 TSR). In this preliminary study, the following findings were recorded:
- mortality: at 1000 mg/kg/day, all animals were found dead or sacrificed moribund on study day 2. Severe clinical signs preceded the deaths or decision to sacrifice (i.e.: thin appearance, hunched posture, piloerection, loud breathing, soiled fur, cold to the touch and/or hypoactivity),
- clinical signs: all test item-treated animals had ptyalism (at the end of the treatment period at 100 or 300 mg/kg/day). Clinical signs (thin appearance, hunched posture, loud breathing, and/or dyspnea) were recorded at 100 or 300 mg/kg/day, but with an overall increased incidence in males when compared to females,
- body weight: there were no effects on mean body weight in all surviving animals at 100 or 300 mg/kg/day,
- food consumption: there were no effects on mean food consumption in all surviving animals at 100 or 300 mg/kg/day,
- pathology: in found dead or sacrificed moribund animals, there were red discoloration(s) and/or thickened wall in stomach, jejunum, ileum, cecum, forestomach and/or duodenum. The digestive organs were or not distended with gas. In surviving males, there were increased (≥ 10% vs. controls) absolute and/or relative liver, kidneys and testes weights at 300 mg/kg/day. In surviving females, there were increased (≥ 10% vs. controls) absolute and/or relative kidneys weight at 300 mg/kg/day and decreased (≤ -8 to -15% vs. controls) absolute and/or relative heart and ovaries weight from 100 mg/kg/day. At necropsy there were a few isolated findings (liver with yellow discoloration at 100 mg/kg/day in 1/3 males and lung(s) with dilatation in 1/3 females at 300 mg/kg/day).

Overall, 1000 mg/kg/day was considered to be an excessive high dose-level for the further OECD 422 study while 300 mg/kg/day could be an appropriate high dose-level.

Therefore 300 mg/kg/day was selected as the high dose-level. The low-dose and mid dose have been selected using a ratio representing a 3-fold interval (i.e. 30 and 100 mg/kg/day).

Positive control:
Not included
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day before the treatment period and at least twice a day during the treatment period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations were performed on all animals outside the home cage, in a standard arena, once before the beginning of the treatment period and then once a week until the end of the study.

BODY WEIGHT: Yes
- Time schedule for examinations:
+ males: on the first day of treatment (day 1), then once a week until sacrifice.
+ femelles: on the first day of treatment (day 1), then once a week until mated and on days 0, 7, 14 and 20 p.c. and days 1 and 5 p.p..
The body weight of female sacrificed on day 25 p.c. for no delivery was recorded the day of sacrifice, presented in the report but not including in the statistical evaluation.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed by each male was measured once a week, over a 7 day period, from the first day of treatment until the start of the pairing period.
The quantity of food consumed by each female was measured once a week, over a 7 day period, from the first day of treatment until the start of pairing period, during pregnancy at the intervals days 0-7, 7-14 and 14-20 p.c. and during lactation for interval days 1 5 p.p..
During the pairing period, the food consumption was measured neither for males nor females.
Food intake per animal and per day was calculated by noting the difference between the food given and that in the food-hopper the next time.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes pupillary reflex during the FOB test
- Dose groups that were examined: 5 males and 5 female per group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of sacrifice
- Anaesthetic used for blood collection: Yes light isoflurane anesthesia
- Animals fasted: Yes for an overnight period of at least 14 hours
- How many animals: the first five males and the first five females to deliver from each group
- Parameters checked in table 7.8.1/1 were examined.

CLINICAL CHEMISTRY: Yes / No / No data
- Time schedule for collection of blood: on the day of sacrifice
- Anaesthetic used for blood collection: Yes light isoflurane anesthesia
- Animals fasted: Yes for an overnight period of at least 14 hours
- How many animals: the first five males and the first five females to deliver from each group
- Parameters checked in table 7.8.1/1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once at the end of the treatment period
- Dose groups that were examined: The first five males and the first five females to deliver
- Battery of functions tested:
+"touch escape" or ease of removal from the cage
+In the hand: fur appearance, salivation, lachrymation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis)
+ In the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia
+ The following parameter measurements, reflexes and responses were recorded:
 touch response,
 forelimb grip strength,
 pupillary reflex,
 visual stimulus response,
 auditory startle reflex,
 tail pinch response,
 righting reflex,
 landing foot splay,
 at the end of observation: rectal temperature.

OTHER:
+ at the end of observation: rectal temperature.
+Motor activity of all animals was measured once by automated infra-red sensor equipment over a 60-minute period
Oestrous cyclicity (parental animals):
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until the females are mated.
Sperm parameters (parental animals):
No data
Litter observations:
Each live pup was identified individually on day 1 p.p., by subcutaneous injection of Indian ink.

Litter size
The total litter size and numbers of pups of each sex were recorded as soon as possible after birth. Any gross malformations in pups were noted.
The litters were observed daily in order to note the number of live, dead and cannibalized pups.

Clinical signs
The pups were observed daily for clinical signs or abnormal behavior.

Body weight
The body weight of each pup was recorded on days 1 and 5 p.p..
Postmortem examinations (parental animals):
SACRIFICE
GROSS PATHOLOGY: Yes
Parent animals:
A complete macroscopic post-mortem examination was performed on all parent animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. The numbers of corpora lutea and implantation sites were also recorded for females sacrificed as scheduled on day 6 post partum and for female sacrificed on day 25 post-coitum due to no delivery. For apparently non-pregnant females the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.
Pup examinations:
A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all pups showing relevant external abnormalities. No tissues were preserved.

HISTOPATHOLOGY: Yes (see table 7.8.1/2)
Postmortem examinations (offspring):
A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all found dead pups. Special attention was paid to whether the pup had fed (e.g. presence of milk in the stomach). No tissues were preserved.

GROSS NECROPSY
A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all pups showing relevant external abnormalities. No tissues were preserved.
Statistics:
see attached documents statistique.doc
Reproductive indices:
 pre-implantation loss:
Number of corpora lutea - Number of implantation sites
_____________________________________________ x 100
Number of corpora lutea

 post-implantation loss:
Number of implantation sites - Number of live pups
_____________________________________________ x 100
Number of implantations

 mating index:
Number of mated animals
_____________________ x 100
Number of paired animals

 fertility index:
Number of pregnant female partners
_______________________________ x 100
Number of mated pairs

 gestation index:
Number of females with live born pups
________________________________ x 100
Number of pregnant females
Offspring viability indices:
 live birth index:
Number of live born pups
_____________________ x 100
Number of delivered pups

 viability index on day 4 post-partum:
Number of surviving pups on day 4 post-partum
_______________________________________ x 100
Number of live born pups
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see below
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths in control or test item-treated groups.
Emaciated appearance was observed from 100 mg/kg/day in a few males only. Hypoactivity and loud/abdominal breathing were observed at 300 mg/kg/day in some females only. All these clinical signs were considered to be related to the treatment with the test item. Ptyalism, observed in most high dose animals, was considered to be related to the test item but of minor toxicological importance.

BODY WEIGHT AND WEIGHT GAIN
In males, there were a dose related decrease in mean body weight gain for the period of days 1 to 8 only. When compared with control the differences were statistically significant from 100 mg/kg/day (+11 g vs. +25 g, p<0.05) and resulted in a body weight loss at 300 mg/kg/day (-2 g vs. +25 g, p<0.001). These findings were considered to be related to treatment with the test item. In females, there were no effects on mean body weight or mean body weight gain.

FOOD CONSUMPTION
When compared with control, there was a statistically significant decrease in mean food consumption in males given 300 mg/kg/day for the period of days 1 to 8 only (29 g/day vs. 34 g/day, p<0.001). This finding was considered to be related to treatment with the test item. In females, there were no effects on mean food consumption during the premating, mating, gestation or lactation periods.

HAEMATOLOGY
There were no toxicologically significant findings.

CLINICAL CHEMISTRY
There were no toxicologically significant findings.

NEUROBEHAVIOUR
There were no relevant differences in treated groups when compared with control group.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no treatment related effects on mating and fertility data. All animals mated within comparable mean number of days taken to mate.

ORGAN WEIGHTS
Minimal increases in adrenal weights were seen in females given the test item at 300 mg/kg/day. In the absence of microscopic correlates, this variation was considered to be of equivocal significance.

GROSS PATHOLOGY
There were no macroscopic findings after administration of the test item at 30, 100 or 300 mg/kg/day.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic findings after administration of the test item at 30, 100 or 300 mg/kg/day.
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
100 mg/kg bw/day (nominal)
Based on:
other: expressed as active material in mg/kg bw/d (purity 75.4% considering the three forms of sodium isopropylnaphtalenesulphonate: mono, di and tri)
Sex:
male/female
Basis for effect level:
other: Clinical signs in males at 100 mg/kg bw/d: emaciated appearance, decrease in body weight change
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
133 mg/kg bw/day (nominal)
Based on:
other: expressed as registered substance (UVCB 100%)
Sex:
male/female
Basis for effect level:
other: Clinical signs in males at 100 mg/kg bw/d: emaciated appearance, decrease in body weight change
Dose descriptor:
NOEL
Remarks:
reproductive performance
Effect level:
300 mg/kg bw/day (nominal)
Based on:
other: expressed as active material in mg/kg bw/d (purity 75.4% considering the three forms of sodium isopropylnaphtalenesulphonate: mono, di and tri)
Sex:
male/female
Basis for effect level:
other: absence of any treatment-related effect on mating and fertility
Dose descriptor:
NOEL
Remarks:
reproductive performance
Effect level:
398 mg/kg bw/day (nominal)
Based on:
other: expressed as registered substance (UVCB 100%)
Sex:
male/female
Basis for effect level:
other: absence of any treatment-related effect on mating and fertility
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
see below
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
see below
VIABILITY (OFFSPRING)
There were no obvious treatment-related effects on live birth, viability and lactation indexes with the exception of a trend towards decreased viability and lactation indexes at 300 mg/kg/day.
In the 300 mg/kg/day group, there were increases in the number of found dead and cannibalized pups. One litter had four found dead pups and three cannibalized pups. While not dose related, a test item treatment-related effect cannot be excluded.

CLINICAL SIGNS (OFFSPRING)
There were no obvious treatment-related findings.

BODY WEIGHT (OFFSPRING)
There were no effects on mean body weight neither in male nor in female pups during the lactation period (day 1 or day 5 p.p.).

GROSS PATHOLOGY (OFFSPRING)
There were no obvious treatment-related findings at necropsy of found dead pups or scheduled sacrifice pups.



Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (nominal)
Based on:
other: expressed as active material in mg/kg bw/d (purity 75.4% considering the three forms of sodium isopropylnaphtalenesulphonate: mono, di and tri)
Sex:
male/female
Basis for effect level:
other: equivocal reduced pup survival at 300 mg/kg/day which can be considered as secondary effect to general toxicity in parents.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
398 mg/kg bw/day (nominal)
Based on:
other: expressed as registered substance (UVCB 100%)
Sex:
male/female
Basis for effect level:
other: equivocal reduced pup survival at 300 mg/kg/day which can be considered as secondary effect to general toxicity in parents.
Reproductive effects observed:
not specified

no other information

Conclusions:
The test item, Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda, was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and (for females) throughout gestation and until day 5 post-partum, at dose-levels of 30, 100 or 300 mg/kg/day.

This study provides evaluations of reproduction / developmental toxicity associated with administration of repeated doses. In particular, the study allows the discrimination between reproduction / developmental effects occurring in the absence of general toxicity and those which are only expressed at levels that are also toxic to parent animals: Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda elicited no reproductive/developmental toxicity potential in the absence of parental toxicity. Therefore, the reduced pup survival observed at 300 mg/kg/day can be considered as a secondary effect to general findings observed in both sexes at this dose-level.

Based on the experimental conditions of this study:
- the No Observed Adverse Effect Level (NOAEL) for general toxicity in parents was considered to be 100 mg/kg/day (i.e. 133 mg/kg/day in terms of test item as registered) based on clinical findings in males,
- the No Observed Effect Level (NOEL) for reproductive performance was considered to be 300 mg/kg/day (i.e. 398 mg/kg/day as registered substance) in the absence of any treatment-related effect on mating and fertility at this dose-level,
- the No Observed Adverse Effect Level (NOAEL) for toxic effects on progeny was considered to be 300 mg/kg/day (i.e. 398 mg/kg/day as registered substance) based on equivocal reduced pup survival at this dose-level which can be considered as secondary effect to the general toxicity in parents.


Executive summary:

In a combined repeated dose toxicity study with the reproductive/developmental toxicity screening test by oral route performed in compliance with the GLP and in accordance with the OECD 422 test guideline, Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda (purity of 75.4%) diluted in water was administered daily by gavage to Sprague Dawley rats (10 animals/sex/dose) at doses of 0; 30; 100 and 300 mg/kg bw/d (based on the three forms of sodium isopropylnaphtalenesulphonate (mono, di and tri)content i.e. 0; 39.8; 133 and 398 mg/kg bw/d in terms of registered substance).Males were exposed 2 weeks before pairing, during pairing and until sacrifice (at least 5 weeks in total) whereas females were exposed 2 weeks before pairing, during pairing, during gestation and during lactation until day 5 post-partum (6-7 weeks in total). The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5 p.p..

Animals were checked daily for clinical signs, mortality, and detailed clinical observations were conducted weekly. Body weight was recorded weekly until sacrifice and then at designated intervals throughout gestation and lactation for the females. Female estrous cycle stage was determined each morning during the pairing period, until the females are mated. Prior to sacrifice, blood samples were also taken from these animals for analysis of hematology and blood biochemistry parameters. At sacrifice body weights and selected organs weights were recorded and a complete macroscopic post-mortem examination performed with particular attention paid to the reproductive organs. A microscopic examination was also conducted on selected organs from the first five animals in the control group and the high-dose group. Microscopic examination was conducted on all macroscopic lesions from all groups.

The total litter sizes and numbers of pups of each sex were recorded after birth. The pups were observed daily for clinical signs of toxicity and pup body weights were recorded on days 1 and 5 p.p..

Pups showing relevant external abormalities at scheduled sacrifice and those found dead before study termination, were submitted for a macroscopic post-mortem examination.

The test item concentrations in the administered dose formulations analyzed in weeks 1, 3 and 5 remained within an acceptable range of -3.7% to +4.4 when compared to the nominal values. SUPRAGIL WP was not detected in control samples.

 

With regards to reproductive and developmental parameters, the following observations were made:

There were no treatment related effects on mating and fertility data. No relevant differences between control and test item-treated groups were observed regarding the mean duration of gestation, the mean number of corpora lutea, the mean number of implantations, the mean pre-implantation loss, the mean number of pups delivered and the mean post-implantation loss. In the 300 mg/kg bw/d group, there were increases in the number of found dead and cannibalized pups: one litter had four found dead pups and three cannibalized pups. While not dose-related, a test item treatment-related effect cannot be excluded. There were no obvious treatment-related effects on live birth, viability and lactation indexes with the exception of a trend towards decreased viability and lactation indexes at 300 mg/kg bw/d. There were no effects on the mean body weight of pup during the lactation period (day 1 or day 5p.p.). While not statistically significant, there was a dose-related decrease in sex-ratio (% of male pups) both on day 1 and 5 p.p.. At 300 mg/kg bw/d, a test item treatment-related effect cannot be excluded. There were no obvious treatment-related findings at necropsy of found dead pups or scheduled sacrifice pups.

In conclusion, based on the experimental conditions of this study:

- the No Observed Effect Level (NOEL) for reproductive performance was considered to be 398 mg/kg/day (highest dose tested) in the absence of any treatment-related effect on mating and fertility at this dose-level,

- the No Observed Adverse Effect Level (NOAEL) for toxic effects on progeny was considered to be 398 mg/kg/day based on equivocal reduced pup survival at this dose-level which can be considered as secondary effect to the general toxicity in parents.

 

Therefore, the registered substance is not classified for reprotoxicity according to the classification criteria of the Regulation (EC) 1272/2008 (CLP) and of the Directive 67/548/EEC. This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 422.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
398 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and OECD 422 compliant study (read-accross, Klimisch 2)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The toxicity to reproduction of Reaction product of naphthalene, butanol, sulfonated and neutralized by caustic soda was determined by a read-across approach using the analogue substance Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda.

The toxicity study to reproduction of Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda is summarized below:

In a combined repeated dose toxicity study with the reproductive/developmental toxicity screening test by oral route performed in compliance with the GLP and in accordance with the OECD 422 test guideline, Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda (purity of 75.4%) diluted in water was administered daily by gavage to Sprague Dawley rats (10 animals/sex/dose) at doses of 0; 30; 100 and 300 mg/kg bw/d (based on the three forms of sodium isopropylnaphtalenesulphonate (mono, di and tri)content i.e. 0; 39.8; 133 and 398 mg/kg bw/d in terms of registered substance).Males were exposed 2 weeks before pairing, during pairing and until sacrifice (at least 5 weeks in total) whereas females were exposed 2 weeks before pairing, during pairing, during gestation and during lactation until day 5 post-partum (6-7 weeks in total). The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5 p.p.. Particular attention was paid to the reproductive organs during the macroscopic examination. Female estrous cycle stage was determined each morning during the pairing period, until the females are mated.

 With regards to reproductive and developmental parameters, the following observations were made:

There were no treatment related effects on mating and fertility data. In the 300 mg/kg bw/d group, there were increases in the number of found dead and cannibalizedpups: one litter had four found dead pups and three cannibalized pups. While not dose-related, a test item treatment-related effect cannot be excluded. There were no obvious treatment-related effects on live birth, viability and lactation indexes with the exception of a trend towards decreased viability and lactation indexes at 300 mg/kg bw/d. There were no effects on the mean body weight of pup during the lactation period (day 1 or day 5p.p.). While not statistically significant, there was a dose-related decrease in sex-ratio (% of male pups) both on day 1 and 5p.p.. At 300 mg/kg bw/d, a test item treatment-related effect cannot be excluded. There were no obvious treatment-related findings at necropsy of found dead pups or scheduled sacrifice pups.

 

In conclusion, the No Observed Effect Level (NOEL) for reproductive performance was considered to be 300 mg/kg/day (i.e. 398 mg/kg/day as registered substance) in the absence of any treatment-related effect on mating and fertility at this dose-level and

the No Observed Adverse Effect Level (NOAEL) for toxic effects on progeny was considered to be 300 mg/kg/day (i.e. 398 mg/kg/day as registered substance) based on equivocal reduced pup survival at this dose-level which can be considered as secondary effect to the general toxicity in parents.

Short description of key information:
Following daily oral administration (by gavage) of the Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda (purity of 75.4%) at dose levels of 0; 30; 100 and 300 mg/kg bw/d (based on the three forms of sodium isopropylnaphtalenesulphonate (mono, di and tri)content i.e. 0; 39.8; 133 and 398 mg/kg bw/d in terms of registered substance) to Sprague-Dawley rats (10/gender/dose) for at least 5 weeks of dosing in a GLP OECD 422-compliant study, the NOEL for reproductive performance and the NOAEL for toxic effects on progeny were both set at 398 mg/kg/day in the absence of any treatment-related adverse effects at this dose-level.

Justification for selection of Effect on fertility via oral route:
Only one reproductive and developmental screening toxicity study available on a similar substance (key study; GLP and OECD 422 compliant)

Effects on developmental toxicity

Description of key information
Groups of 27 inseminated Wistar rats each were treated daily orally by gavage with Reaction product of naphthalene, butanol, sulfonated and neutralized by caustic soda formulated in demineralized water from day 6 to day 15 p.c. at the doses of 0, 10, 60, or 360 mg/kg body weight, respectively.
NOAEL for maternal toxicity : 10 mg/kg b.w./day
NOAEL for developmental toxicity : 60 mg/kg b.w./day
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from August 1993 to 11 September 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant study conducted according to OECD guideline.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan-Winkelmann, Borchen
- Age at study initiation: not documented
- Weight at study initiation: 187 to 236 g
- Fasting period before study: no
- Housing: During the adaptation period, the females were kept in groups in Type III Makrolon cages, and starting from gestation day 0 they were iindividually accomodated in Type II Makrolon cages on low-dust wood shavings supplied by ssniff GmbH in Soest.
- Diet (e.g. ad libitum): standard diet (Altromin 1324) given ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 7 days before mating.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30-70%
- Air changes (per hr): at least 10 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial lighting from 6 a.m. to 6 p.m.

IN-LIFE DATES: From: 24 Aug 1993 To: 27 Oct 1993
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Administration formulations (solutions) were prepared with demineralised water. The stability (over 8 days) of the active ingredient in the formulations were analytically verified before initiation of the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A content check of the formulations in all dose levels was carried out. The results revealed no significant devioations of the active ingredient content from the nominal value in the formulations.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 2 females with one male
- Length of cohabitation: one night
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
from day 6 to day 15 p.c.
Frequency of treatment:
daily
Duration of test:
see above
No. of animals per sex per dose:
27 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected according to a preceding range-finding study in 5 rats per group with doses of 200, 300, 400 and 500 mg/kg bw. From the dose of 300 mg/kg and above breathing sounds, decreased feed intakes and body weight gains were observed (300 mg/kg marginally). One dam of the 400 mg/kg group died and all dam of the 500 mg/kg group revealed bloody snouts and decreased motility. The resorption rate and the external examination of the fetuses revealed no embryotoxic potential up to and including 500 mg/kg.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: from day 0 to 20 p.c. all animals were inspected twice daily on the basis of their appearance and behavior and not was given to any alterations in the excretory products.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule:see above

BODY WEIGHT: Yes
- Time schedule for examinations: on day 0 p.c., daily from day 6 to 15 p.c and on day 20 pc.

FOOD CONSUMPTION: Yes
The quantity of food consumed for each female was recorded for the following intervals: days 0-6, 6-11, 11-16 and 16-20 p.c.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: gross pathological evaluation (no further details)
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
Fetal examinations:
- External examinations: Yes, all fetuses
- Soft tissue examinations: Yes, evaluation of about half of the fetuses.
- Skeletal examinations: Yes, evaluation of about half of the fetuses.
- Head examinations: No data


- External examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data
- Soft tissue examinations: Yes: [all per litter / / #? per litter ] / No / No data
- Skeletal examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data
- Head examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data
Statistics:
Animals that had no implantation sites were not used for statistical evaluation. For calculation of body weight gain, feed intakes and fetal parameters only dams with living fetuses were used.

Statistical significance was tested using the following methods:

a. Fisher's exact significance test at significance levels of alpha = 5% and alpha = 1% (two-tailed) was used for the:
- fertility rate
-gestation rate

b. The F-test, and t-test or Welch t-test were used for the:
- feed intakes
- Body weight gains
- Corrected body weight gains
- Number of corpora lutea per dam
- Number of implantations per dam
- Number of live fetuses per dam
- Percentage of live fetuses in proportion to the number of implantations per dam
- Number of male or female fetuses per dam
- Fetal weights per group and per dam
- Placental weights per group and per dam

c. The Yates-corrected Chi2 test was used for the:
- Number of fetuses or litters exhibiting malformations
- Pre-implantation loss per dam
- Number of male or female fetuses per group
- Number of resorptions per dam

d. 2 by N CHI2-test; in case of signficant differences Fisher's exact test with Bonferonni correction for:
- Number of fetuses or litters with skeletal findings.

Indices:
see Statistics section
Historical control data:
Historical control data were provided to allow comparison with concurrent controls. These included data for clinical signs, pathological and anatomical findings, food consumption during pregnancy, placenta findings, fertility and pregnancy index, caesarian section data, summary of reproduction data, number of malformations, spontaneous malformations, external and visceral deviations of fetuses, skeletal retardations and 14th rib, skeletal findings of fetuses/litters.
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: Reduced body weight gain and feed intake at 60 and 360 mg/kg

Details on maternal toxic effects:
The following parameters of maternal toxicity were impaired: The body weight gain in the 60 and 360 mg/kg group dams and the feed intake in the 360 mg/kg group (60 mg/kg marginally) were reduced during the treatment period (details in Tables 1 and 2). Decreased water consumption and reduced feces excretion were observed in some dams of the 360 mg/kg group.
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: based on maternal toxicity
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: based on developmental toxicity
Remarks on result:
other: observed with maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: Increased numbers of fetuses with skeletal variations and retardations were observed at 360 mg/kg

Details on embryotoxic / teratogenic effects:
The following parameters of intrauterine development were affected: In the 360 mg/kg group increased numbers of placentas with necrotic border and slightly reduced placental weights were observed (details below and in Table 4). In the 360 mg/kg group increased numbers of fetuses with skeletal variations and retardations were observed (details below and in Table 5).
At the dose of 360 mg/kg b.w. increased incidences of vertebral and multiple malformations that may occur spontaneously were evident, while the overall incidence of malformations was comparable between control and 360 mg/kg group. A treatment related effect for this slightly different distribution cannot be excluded.
However, primary developmental toxicity was not evident, since all embryotoxic findings observed at the dose of 360 mg/kg appeared in a maternally toxic dose range.
Remarks on result:
other: not specified
Abnormalities:
not specified
Developmental effects observed:
not specified

Appearance, behavior and mortality:

Appearance, behavior and mortality of the dams were unaffected up to and including the dose of 360 mg/kg b.w.

Gross pathological findings in dams:

No treatment related findings were evident at necropsy up to and including the dose of 360 mg/kg b.w.

General Reproduction data:

The number of corpora lutea, preimplantation losses and implantations was comparable in all experimental groups, with exception of the significantly lower preimplantation losses in the 360 mg/kg group (details in Table 3).

Gestation rate:

The gestation rate was unaffected by the treatment with doses up to and including 360 mg/kg.

Weight and appearance of placentas:

The placental weight was statistically significantly decreased at the dose of 360 mg/kg when calculated on litter basis and from 60 mg/kg and above when calculated in individual basis.Since the toxicologically more relevant calculation on litter basis was not affected at the dose of 60 mg/kg and as the values even of the 360 mg/kg group lay within the range of historical control data, the statistical significance at 60 mg/kg is not regarded as toxicologically relevant and even the values obtained at the dose of 360 mg/kg as equivoqual. Nevertheless, together with the increase number of placentas with necrotic placental borders at 360 mg/kg, this dose is considered to be the effect level for placental weight and appearance (details in Table 4).

Resorption rate, number of fetuses:

The resorption rate and the mean number of fetuses were not affected by the treatment up to and including the dose of 360 mg/kg.

Sex and fetuses:

The number of male and female fetuses did not diffe from those in the controls to any extent at levels up to and including 360 mg/kg.

Fetal weight:

The fetal weight was unaffected by the treatment up to and including 360 mg/kg.

Skeletal system deviations (variations, retardations):

At the highest dose of 360 mg/kg, slightly reduced ossification of the parietal and interparietal bones and the 7th cervical vertebra were observed as well as an increase in the incidence of fetuses with wavy ribs. These findings are considered to be treatment related.

Malformations (details in Table 5):

Neither the type nor the incidence of malformations were affected by treatment with doses up to and including 60 mg/kg.

The number of abnormal fetuses at 360 mg/kg was comparable to the control group. All findings in the control and the 360 mg/kg were also observed in previous controls. Nevertheless, there was a slight difference concerning the distribution of these findings. Microphthalmia mainly observed in the control group of this study occured in comparable incidences in previous controls, while the incidence of vertebral and multiple malformations mainly obtained in the 360 mg/kg group was rather low in previous controls. Therefore a treatment related effect on the incidence of the verteral and multiple malformations cannot be excluded. However, incresed incidences of spontaneoulsy occuring malformations are not regarded as indication of a specific teratogenic potential.

The remaing individual fetal findings (deviations) observed at visceral examinations revealed no evidence of tes substance-related effects in any of the dose troups.

Conclusions:
The test item, Reaction product of naphthalene, butanol, sulfonated and neutralized by caustic soda was administered by gavage, once daily from days 6 to 15 p.c. to inseminated Wistar rats at doses of 0, 10, 60, or 360 mg/kg/day in a GLP-compliant study in accordance with OECD Guideline 414.
On the basis of the results obtained in this study:
- The No Observed Adverse Effect Levels (NOAEL) for maternal toxicity was 10 mg/kg b.w./day.
- The NOAEL for developmental toxicity was 60 mg/kg b.w./day.

At the dose of 360 mg/kg b.w. increased incidences of vertebral and multiple malformations that may occur spontaneously were evident, while the overall incidence of malformations was comparable between control and 360 mg/kg group. A treatment related effect for this slightly different distribution cannot be excluded. However, primary developmental toxicity was not evident, since all embryotoxic findings observed at the dose of 360 mg/kg appeared in a maternally toxic dose range.

Therefore, in the absence of evident direct embryotoxic effect, the registered substance is not classified for reprotoxicity according to the classification criteria of the Regulation (EC) 1272/2008 (CLP) and of the Directive 67/548/EEC.
Executive summary:

Groups of 27 inseminated Wistar rats each were treated daily orally by gavage with Reaction product of naphthalene, butanol, sulfonated and neutralized by caustic soda formulated in demineralized water from day 6 to day 15 p.c. in doses of 0, 10, 60, or 360 mg/kg body weight, respectively. The fetuses were delivered by cesarian section on the 20th day of gestation. Investigations were performed to determine the general tolerance of the test substance by the dams, as well as its effect on intrauterine development.

Mortality, behavior and appearance of the dams were not affected by the treatment up to and including 360 mg/kg. The body weight gain in the 60 and 360 mg/kg group dams and the feed intake in the 360 mg/kg group (60 mg/kg marginally) were reduced during the treatment period. Decreased water consumption and reduced feces excretion were observed in some dams of the 360 mg/kg group. There were no test substance-related findings at necropsy in any of the experimental groups.

With respect to intrauterine development, the gestation rate, the resorption rate and accordingly the number of fetuses, and the fetal sex as well as fetal weight were unaffected by the treatment up to and incuding 360 mg/kg.

In the 360 mg/kg group increased numbers of placentas with necrotic border and slightly reduced placental weights were observed.

External, visceral and skeletal examination of the fetuses revealed no treatment related effects on intrauterine development up to and including the dose of 60 mg/kg. At the dose of 360 mg/kg b.w. increased incidences of vertebral and multiple malformations that may occur spontaneously were evident, while the overall incidence of malformations was comparable between control and 360 mg/kg group. A treatment related effect for this slightly different distribution cannot be excluded.

However, primary developmental toxicity was not evident, since all embryotoxic findings observed at the dose of 360 mg/kg appeared in a maternally toxic dose range.

The No Observed Adverse Effect Levels (NOAEL) were thus:

Maternal toxicity : 10 mg/kg b.w./day

Developmental toxicity : 60 mg/kg b.w./day

Therefore, in the absence of evident direct embryotoxic effect, the registered substance is not classified for reprotoxicity according to the classification criteria of the Regulation (EC) 1272/2008 (CLP) and of the Directive 67/548/EEC.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
60 mg/kg bw/day
Species:
rat
Quality of whole database:
Klimisch 1
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

Groups of 27 inseminated Wistar rats each were treated daily orally by gavage with Reaction product of naphthalene, butanol, sulfonated and neutralized by caustic soda formulated in demineralized water from day 6 to day 15 p.c. in doses of 0, 10, 60, or 360 mg/kg body weight, respectively. The fetuses were delivered by cesarian section on the 20th day of gestation.

The following parameters of maternal toxicity were impaired: The body weight gain in the 60 and 360 mg/kg group dams and the feed intake in the 360 mg/kg group (60 mg/kg marginally) were reduced during the treatment period. Decreased water consumption and reduced feces excretion were observed in some dams of the 360 mg/kg group.

The following parameters of intrauterine development were affected: In the 360 mg/kg group increased numbers of placentas with necrotic border and slightly reduced placental weights were observed. In the 360 mg/kg group increased numbers of fetuses with skeletal variations and retardations were observed.

At the dose of 360 mg/kg b.w. increased incidences of vertebral and multiple malformations that may occur spontaneously were evident, while the overall incidence of malformations was comparable between control and 360 mg/kg group. A treatment related effect for this slightly different distribution cannot be excluded.

However, primary developmental toxicity was not evident, since all embryotoxic findings observed at the dose of 360 mg/kg appeared in a maternally toxic dose range.

The No Observed Adverse Effect Levels (NOAEL) were thus:

Maternal toxicity : 10 mg/kg b.w./day

Developmental toxicity : 60 mg/kg b.w./day

Based on these results, the registered substance is not classified for reprotoxicity according to the classification criteria of the Regulation (EC) 1272/2008 (CLP) and of the Directive 67/548/EEC.


Justification for selection of Effect on developmental toxicity: via oral route:
Only one developmental toxicity study available on the substance (key study)

Justification for classification or non-classification

Harmonized classification:

No harmonized classification is available according to the Regulation (EC) No 1272/2008 including ATP3.

Self-classification:

In the absence of any treatment-related adverse effects on reproductive and developmental parameters in available studies conducted on the registered substance itself or a close analogue, Reaction product of naphthalene, butanol, sulfonated and neutralized by caustic soda is considered as non reprotoxic according to the Regulation (EC) No 1272/2008 (CLP) and the Directive 67/548/EEC.