Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

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:

other: GLP guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

N° 2011/40

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda

EC Number:

939-368-0

Cas Number:

1322-93-6

Molecular formula:

Not applicable (a generic molecular formula can not be provided for this specific UVCB substance)

IUPAC Name:

Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): SUPRAGIL WP
- Storage condition of test material: at room temperature and protected from light

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

treated by reverse osmosis using an ELIX 5 apparatus

Details on oral 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

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 CITixLAB 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:

daily

Doses / concentrations

Doses / concentrations 1

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)

Doses / concentrations 2

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 a 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 applicable

Examinations

Observations and examinations performed and frequency:

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 7.5.1/1 were examined.

CLINICAL CHEMISTRY: 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 7.5.1/1 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 period: rectal temperature.
+Motor activity of all animals was measured once by automated infra-red sensor equipment over a 60-minute period

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 7.5.1/2)
HISTOPATHOLOGY: Yes (see table 7.5.1/2)

Other examinations:

no other examination

Statistics:

Citox software (version D.06) was used for the statistical analyses of hematology and blood biochemistry data according to the sequence, described in attached document.
Other data was compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions).
PathData software (version 6.2d2) was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01) according to the sequence described in the attached document.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no unscheduled deaths in controls or test item-treated groups. Emaciated appearance from 100 mg/kg bw/d in few males. Hypoactivity and loud/abdominal breathing at 300 mg/kg bw/d in some females. Ptyalism, observed in most high dose animals, was considered to be related to the test item but of minor toxicological importance.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were no unscheduled deaths in controls or test item-treated groups. Emaciated appearance from 100 mg/kg bw/d in few males. Hypoactivity and loud/abdominal breathing at 300 mg/kg bw/d in some females. 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 changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decreases in body weight changes from 100 mg/kg bw/d, resulting in body weight loss at 300 mg/kg bw/d in males. In females, no effect on body weight was observed.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decrease in mean food consumption in males given 300 mg/kg bw/d for the period of days 1 to 8. No effect on food consumption in females.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Minimal increase in adrenal weights in females given the test item at 300 mg/kg bw/d. In absence of microscopic correlates, this variation was considered to be of equivocal significance.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

no further details

Effect levels

1

Dose descriptor:

NOAEL

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

Basis for effect level:

other: Clinical signs in males at 100 mg/kg bw/d: emaciated appearance, decrease in body weight change

2

Dose descriptor:

NOAEL

Effect level:

133 mg/kg bw/day (nominal)

Based on:

other: expressed as registered substance (UVCB 100%)

Sex:

male

Basis for effect level:

other: Clinical signs in males at 100 mg/kg bw/d: emaciated appearance, decrease in body weight change

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

no other information

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the test item Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda induced slight clinical signs in males only from the dose of 100 mg/kg bw/d, but no adverse systemic effects were observed after a repeated dose exposure to the test item. The NOAEL was determined to be 100 mg/kg bw/d when expressed as the active material and 133 mg/kg bw/d when expressed as the registered substance (UVCB at 100%) based on clinical signs observed in male rats. Therefore no classification as STOT-RE is required according to the Regulation (EC) 1272/2008 (CLP) and as harmful after repeated dose exposure according to the Directive 67/548/EEC.

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).

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. A Functional Observation Battery was performed on five males and females per group at the end of the study. Prior to sacrifice, blood samples were also taken from these animals for analysis of hematology and blood biochemistry parameters. The males were sacrificed after completion of the mating period. Body weights and selected organs weights were recorded and a complete macroscopic post-mortem examination performed. A microscopic examination was also conducted on selected organs from the first five males in the control group and the high-dose group. Microscopic examination was conducted on all macroscopic lesions from all groups. Dams were sacrificed on day 6 p.p.. Body weights and selected organs weights were recorded and a complete macroscopic examination was performed. A microscopic examination was then conducted on selected organs from the first five females to deliver in the control group and the high-dose group and on any macroscopic lesions from all groups.

 

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 repeated-dose toxicity parameters, the following observations were made:

There were no unscheduled deaths in control or test item-treated groups. Emaciated appearance was observed in few males from 100 mg/kg bw/d. Hypoactivity and loud/abdominal breathing was observed in some females exposed to 300 mg/kg bw/d. Statistically significant decrease in body weight changes were observed in males from 100 mg/kg bw/d for the period of days 1 to 8 only, resulting in body weight loss at 300 mg/kg bw/d. This was associated with a statistically significant decrease in mean food consumption at 300 mg/kg bw/d for the period of days 1 to 8. In females, there was neither effect on body weight nor on food consumption. A minimal increase in adrenal weights was observed in females given the test item at 300 mg/kg bw/d. But in absence of microscopic correlates, this variation was considered to be of equivocal significance.

 

Based on the results of this study, 133 mg/kg bw/d of test item (as registered) was established as the No Observed Adverse Effect Level (NOAEL) considering the clinical signs observed in males.

Therefore, the registered substance is not classified for repeated dose toxicity 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.