Sodium 4-vinylbenzenesulphonate

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:

1 August to 29 October 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 4-vinylbenzenesulphonate

EC Number:

220-266-3

EC Name:

Sodium 4-vinylbenzenesulphonate

Cas Number:

2695-37-6

Molecular formula:

C8H8O3S.Na

IUPAC Name:

.

Test animals

Species:

rat

Strain:

other: SPF Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were identified by making marks to the tails with an oily felt pen before allocation to groups, and after allocation to each group, the animals were identified by the numbers tattooed in the ear auricles.
For identification of a housing cage, before allocation to groups, the study number and animal numbers assigned at receiving were clearly described on the labels having the different color by sex, and these labels were displayed on the front of each housing cage. After allocation to groups, the study number, group identification, and animal numbers were clearly described on the labels having the different color by sex, and these labels were displayed on the front of each housing cage.

Animal husbandry
1) Housing environment
Animals were housed in an environmentally controlled animal room (Room No. 308), with a room temperatures of 22±3oC (a range of actual measured temperature: 21~23oC), relative humidity of 50±20% (a range of actual measured humidity: 47~68%), ventilation frequency of 10~15 times/hour, and 12-hour light (8:00~20:00, artificial light).
2) Housing equipment and methods
Bracket type metal cages with a metal mesh floor (260W x 380D x 180H, mm) were used for housing. Before allocation to groups, 3 animals each were housed in a cage, and after allocation, one animal was housed in a cage. Cages and feeders were changed at grouping, on 7 days, and once every 2 weeks thereafter during administration period. Sanitary plates under cages were exchanged with cleaned and sterilized plates twice a week. Draining of the automatic watering system was conducted once a week. Cleaning and wiping sanitization were conducted once every day. For the sanitization, chlorine disinfectant and iodine disinfectant were alternatively used every one week.
3) Food
Animals were allowed to free access to pellet form diet of CRF-1 irradiated with gamma rays (ORIENTAL YEAST CO., LTD.) served in steel feeders.
Used diet with the Lot No.07612 was analyzed for contaminants or microorganisms which may have harmful influences on the study. The analysis for contaminants was conducted by JAPAN FOOD RESEARCH LABORATORIES, and examination for microorganisms was conducted by the diet producer. Items of analysis and their allowance values were followed the standard operation procedure in Safety Research Institute for Chemical Compounds Co., Ltd. According to the results of analysis, no deviations from the allowance values were noted.
4) Drinking Water
Animals were provided free access to drinking water (supplied by Sapporo city Waterworks Bureau) with an automatic watering system. However, water bottles were used on the days of urinalysis.
The existences of contaminants which may have harmful influences on the study in the water were analyzed on the water samples obtained from this housing room and an end located room (Room No.306) on the same distributing pipe line. The analysis was conducted at Nihoneisei co., Ltd. Items of analysis and allowance values were followed the standard operation procedure in Safety Research Institute for Chemical Compounds Co., Ltd. According to the results of analysis, no deviations from the allowance values were noted.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

System of the high speed liquid chromatography (HPLC) and analysis conditions are given in the following.

High speed liquid chromatography (HPLC)
UV-VIS Detector: L-4200 Hitachi Ltd.
Intelligent Pump: L-6200 Hitachi Ltd.
Colum Oven: L-5025 and 655A-52 Hitachi Ltd.
Auto-sampler: AS-2000 Hitachi Ltd.
Degasser: ERC-3315α ERC Inc.
Data Processor: Empower 2 Nihon Waters K.K.
Analysis condition:
Column: L-column ODS, 4.6 mm I.D. x 250 mm, Chemical Examination and Research Institution Japan
Mobile phase: Acetonitrile/distilled water/tetrabutylammoniumbromide (400 ml : 600 ml : 1.612 g)
Auto-sampler cleaning solution: Acetonitrile / distilled water (400 : 600)
Injector cleaning solution: Acetonitrile / distilled water (400 : 600) SR06113 17
Wave length: 255 nm
Column temperature: 35oC
Flow rate: 1 ml/min
Injection volume: 10 μL
Temperature of auto-sampler: 10 oC
Analysis time: 15 minutes

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

6 males and 5 females per dose

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Clinical observations
The starting day of administration was set to be 1 day of administration, and the next day after 28 day of administration was set 1 day of recovery, and the days were counted up. All animals were examined for mortality, appearance, behavior, and others twice a day, once in the morning and once in the afternoon, from 1 day of administration to the days of necropsy (which were 28 day of administration and the day after 14 day of recovery). On the day of necropsy, the animals were observed once in the morning.

Detailed clinical observations
Detailed clinical observations were conducted on all animals before administration, on 7, 14, 21, and 28 day of administration, and on 7 and 14 day of recovery. When observing from the outside of a cage, observation items were posture, palpebral closure, respiration, tremor or convulsion, stereotype movement / rotation or circling, and bizarre behavior / self-mutilation. When taking out an animal forma a cage, observation items were degree of easiness of taking out, degree of easiness of handling, muscle tonus, piloerection, abnormal fur, skin, exophthalmos, pupil size, visible mucosa, lacrimation, salivation, and body temperature. In the open field, convulsion, gait, arousal condition, urination, defecation, stereotype movement / grooming•sniffing, bizarre behavior / retropulsion or vocalization, and respiration were observed. The observation results were recorded using the scoring system established previously in each observation item.

Sacrifice and pathology:

Necropsy
All animals were necropsied on the next day of 28 day of administration, and on the next day of 14 day of recovery. After the observation of external appearance, animals were anesthetized with ether to sample blood specimens, and then euthanized by exsanguinations. In the necropsy, all organs and tissue were observed macroscopically. In addition, the following organs and tissue were fixed in phosphate buffered 10 % formalin, and then preserved. The eyes and Harderian glands were fixed in Davidson’s solution and preserved. The testes and epididymides were fixed in Bouin’s solution, and preserved in 70 % ethanol. The lung was injected with fixative and then immersed in the fixative. Both sides of paired organs were fixed and preserved as a rule.
Organs and tissue: Brain (cerebrum, cerebellum, and medulla), pituitary, spinal cord, thymus, thyroid, parathyroid, adrenal, spleen, heart, tongue, esophagus, stomach, liver, pancreas, duodenum, jejunum, ileum (including Payer’s patch), cecum, colon, rectum, mesenteric lymph node, submandibular lymph node, trachea, lung, kidneys, urinary bladder, testes, epididymides, prostate gland, seminal vesicle (including coagulating gland), ovaries, uterus, vagina, eyes, Haderian glands, femur(including bone marrow, right side), and sciatic nerve.
Besides, as an abnormal site, skin of the left neck including the border of crust and normal tissue of one animal (No. 304).

Other examinations:

Organ weight
The following organs from all animals were weighed with an electronic even balance, (ER-180A, A&D Company Limited) at the necropsy. Paired organs were weighed right and left together.
Organs: Brain, pituitary, thyroid, adrenals, spleen, heart, liver, kidneys, thymus, testes, epididymides, prostate, seminal vesicle (including coagulation glands), ovaries, uterus.

Statistics:

Statistical analysis
Statistical data on all the administration groups and the recovery group obtained during the administration period were calculated together.
Mean values and standard deviations were calculated for grip strength, locomotion activity, body weights, body weight increase and its rate, food consumption, urine volume, hematology, blood biochemistry, absolute organ weights, and relative organ weights, and then the homogeneity of variances was analyzed by Bartlett’s test. If the variances were shown to be homogenous (p>0.05), one-way analysis of variance was conducted, meanwhile if the unequal variances (p≦0.05 )were shown, data were analyzed by Kruskal-Wallis test. If the statistical significance (p>0.10) was found in the one-analysis, a pair wise comparison with the control group was conducted by Dunnett’s test. If the statistical significance (p>0.10) was found by Kruskal-Wallis test, a comparison with the control group was conducted by Mann-Whitney U test.
Detailed clinical observations and observation items in the functional observation, the qualitative parameters in urinalysis, and urine specific gravity were analysed with Kruskal-Wallis test. If the significant difference (p>0.10) was found, a comparison with the control group was conducted by Mann-Whitney U test.
For the comparison test with the control group, a significance level of 5% was applied. Indication for the methods of statistical analysis is given at the top of INDIVIDUAL DATA.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the 1000 mg/kg group, soft feces were sporadically observed in 6 males.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In females of the 1000 mg/kg group, a significant decrease in body weight gain. No significant difference was observed in male.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Slight increase in females at 300mg/kg, no effect with males at any dose or females at any other dose.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Slight decrease in leukocyte count in males at 300 mg/kg and females at 1000 mg/kg. No effects at other dose levels in either sex

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In the 300 mg/kg group increase in chloride level was noted in male. and on both male and females at 1000 mg/kg.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

In the 1000 mg/kg group, a significant increase in the amount of protein excretion in male, and a significant decrease in pH value in female were noted.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No significant difference was observed in male or female

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In the 1000 mg/kg groups, a significant increase in the relative weight of kidneys was noted in female. No significant difference was noted in male

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the 1000 mg/kg groups, thickening of the limiting ridge of the stomach was observed in all of males and females

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the 1000 mg/kg groups, slight hyperplasia of squamous epithelium was observed in all of males and females (6 animals in each group), and slight hyperkeratosis was observed in 3 males and 4 females.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

2

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

3

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

The changes related to the test material administration were the significant increase in chloride level in males of 300 mg/kg and of higher dose group, sporadic soft feces in males and females of 1000 mg/kg, the suppression of body weight gain in females of 1000 mg/kg during the last half of the administration period, the increase in protein excretion and decrease in urinary pH, the macroscopic thickening and histopathology findings of squamous epithelium hyperplasia and hyperkeratosis in the limiting ridge of the stomach in 1000 mg/kg group.

In blood biochemistry, a significant increase in chloride was noted in males of 300 mg/kg and in both males and females of 1000 mg/kg group, but the mechanism of this change was not clear. In organ weights, a significant increase in relative weight of the kidneys was noted in females of 1000 mg/kg group at the end of administration. This kidney weight change was thought to be related to the test material administration because of the relevance to urinary protein, pH, and chloride level in serum.

The observation of increased chloride level in males at 300 mg/kg are associated with no other effects to organs, whilst the effects in males and females at 1000 mg/kg were associated also with an increase in relative kideney weights in females. The increased chloride levels are therefore considered significant adverse effects at 1000 mg/kg.

Applicant's summary and conclusion

Conclusions:

The NOAEL is determined to be 300 mg/kg. The NOEL in females is also 300 mg/kg and the NOEL in males is 100 mg/kg

Executive summary:

The 28 -day repeated dose oral toxicity has been assessed according to the OECD 407 test guideline in accordance with GLP with doses levels set at 1000, 300, 100 and 0 mg/kg/ day with water as the solvent.

The changes related to the test material administration were the significant increase in chloride level in males of 300 mg/kg and of higher dose group, sporadic soft feces in males and females of 1000 mg/kg, the suppression of body weight gain in females of 1000 mg/kg during the last half of the administration period, the increase in protein excretion and decrease in urinary pH, the macroscopic thickening and histopathology findings of squamous epithelium hyperplasia and hyperkeratosis in the limiting ridge of the stomach in 1000 mg/kg group.

In blood biochemistry, a significant increase in chloride was noted in males of 300 mg/kg and in both males and females of 1000 mg/kg group, but the mechanism of this change was not clear. In organ weights, a significant increase in relative weight of the kidneys was noted in females of 1000 mg/kg group at the end of administration. This kidney weight change was thought to be related to the test material administration because of the relevance to urinary protein, pH, and chloride level in serum.

The NOAEL is determined to be 300 mg/kg. The NOEL in females is also 300 mg/kg and the NOEL in males is 100 mg/kg.

The observation of increased chloride level in males at 300 mg/kg are associated with no other effects to organs, whilst the effects in males and females at 1000 mg/kg were associated also with an increase in relative kideney weights in females. The increased chloride levels are therefore considered significant adverse effects at 1000 mg/kg.