Naphthalenesulfonic acid, di-C9-rich C8-10-branched alkyl derivs., calcium salts

Endpoint summary

Description of key information

In the original submission of this dossier, a testing proposal was included for conducting a 90 -day oral toxicity study on the substance. In a decision finalized on November 25, 2015, ECHA instructed the registrant to conduct an OECD 408 oral gavage study on rats. This updated submission includs the results of the study.

Rats (10/sex/dose) were exposed to the substance by gavage for a period of 90 days (doses 0, 100, 300 and 1000 mg/kg bw). Detailed examinations related to mortality, clinical signs, body weight, food consumption, ophthalmoscopy, behavioural effects, haematology, clinical chemistry, macroscopy, histopathology and organ weights were performed in a study that is performed according to OECD 408. In the highest dose group 6/10 females died showing alterations in the gastro-intestinal tract, a small thymus and bone marrow atrophy. The surviving females at 1000 mg/kg bw showed similar effects and a reduced body weight (gain). The effects on the gastro-intestinal tract also became apparent in males at 300 and 1000 mg/kg bw. These animals also had a reduced body weight (gain). Other effects included changes in numbers of white blood cells, lymphocytes, platelets as well as effects on several biochemical parameters. Macroscopy and histopathology indicated that next to the GI-tract mainly the thymus and bone marrow could be considered as potentially affected in males at 300 mg/kg bw and above and in females at 1000 mg/kg bw.

The NOAEL as derived from this study is 100 mg/kg bw.

In addition to this study as study according to DNNSA (di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid) provides reliable read-across. The NOAEL for DNNSA in this study is 95 mg/kg/day based on local effects to the gastro-intestinal tract.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 May 2016 to 23 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Qualifier:

according to guideline

Guideline:

other: Japanese Chemical Substances Control Law 1973, Notification of Mar. 31 2011 by MHLW (0331 No.7), METI (H23.03.29 SeiKyoku No. 5) and MOE (No. 110331009).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Recognized by international guidelines as the recommended test system

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:WI(Han) (outbred, SPF-Quality)
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: ca 6 weeks
- Weight at study initiation: Males 119-157 g; females 115-135 g
- Fasting period before study: NA
- Housing: 5/sex/cage (Macrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material and paper as cage-enrichment
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH, Soest, Germany): ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40-70%
- Air changes (per hr): at least 10/hr
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: prepared daily within 6 hours prior to dosing, and homogenized to visually acceptable levels. Adjustment was made for specific gravity of the vehicle (0.92). No correction was made for purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: based on pre-test
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

yes

Remarks:

many measurements are outside the validity criteria as set in the report

Details on analytical verification of doses or concentrations:

Analyses By UPLC-MS: accuracy of formulation in all doses in week 2, 6 and 13, stability in week 2 and homogeneity at 100 and 1000 mg/kg bw in week 2 and 13

Instrument Acquity UPLC system (Waters, Milford, MA, USA)
Detector Xevo TQ-S mass spectrometer (Waters)
Column Acquity UPLC BEH C18, 100 mm × 2.1 mm i.d., dp =1.7 µm (Waters)
Column temperature 40°C ± 1°C
Injection volume 5 µL
Mobile phase 75/25 (v/v) A/B ( A - 5 mM ammonium formate in 95/5 (v/v) acetonitrile/water; B - 5 mM ammonium formate in 5/95 (v/v) acetonitrile/water)
Flow 0.6 mL/min
MS detection
Ionisation source ESI-
Cone voltage 40 V
Collision energy 46 eV
Quantitation m/z 459.1 --> m/z 373.1

Stock and spiking solution: standard solution of 1000 and 2000 mg/L in methanol
Linearity: proven over 1.00-200 mg/g (r>0.99)
QC: 73-166% of nominal at 20 mg/g; 82-116% of nominal at 220 mg/g
accuracy of preparation : at 100 mg/kg bw 90-120%; at 300 mg/kg bw 94-109%; at 1000 mg/kg bw 96-109% (all corrected for the results of the QC samples)
stability over 6 hours :98-102% of initial
homogeneity: CV 4.2-9.7 with the exception of the 100 mg/kg bw sample in week 13 --> CV 19%

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/sex

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on results of a 14-day oral range finding study (Study No. 500723, 2013).

Positive control:

NA

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality/viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least daily (immediately after dosing), once weekly in a standard arena

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION : yes
- Time schedule for examinations: weekly

WATER CONSUMPTION: no (not quantitatively)

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-test and in week 13
- Dose groups that were examined: pre-test all; week 13 control and 1000 mg/kg bw

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes (< 24 hours)
- How many animals: all surviving animals
- Parameters examined: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, PT and APTT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of treatment
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes (< 24 hours)
- How many animals: all surviving animals
- Parameters examined: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Bile acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 12
- Dose groups that were examined: all groups first five animals/sex
- Battery of functions tested: hearing ability, pupillary reflex ,static righting reflex (STATIC R), fore- and hind-limb grip strength, locomotor activity (movements and ambulations over 1 hour)

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Ovaries, Adrenal glands, Pancreas, Aorta, Peyer's patches [jejunum, ileum] if detectable, Brain [cerebellum, mid-brain, cortex], Pituitary gland, Caecum, Ileum, Jejunum, Duodenum, Colon, Rectum, Preputial gland, Cervix, Prostate gland, Clitoral gland, Salivary glands - mandibular, sublingual, Sciatic nerve, Skeletal muscle, Epididymides, Seminal vesicles including coagulating gland, Eyes with optic nerve and Harderian gland, Skin, Female mammary gland area, Spinal cord -cervical, midthoracic, lumbar, Femur including joint, Spleen, Heart, Sternum with bone marrow, Stomach, Testes, Kidneys, Thymus, Larynx, Thyroid including parathyroid, Lacrimal gland, exorbital, Tongue, Liver, Trachea, Lung, infused with formalin, Urinary bladder, Lymph nodes - mandibular, mesenteric, Uterus, Nasopharynx, Vagina, Oesophagus, All gross lesions

ORGAN WEIGHTS: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate, Liver, Seminal vesicles including coagulating glands, Ovaries, Thyroid including parathyroid

HISTOPATHOLOGY: Ovaries, Adrenal glands, Pancreas, Aorta, Peyer's patches [jejunum, ileum], Brain [cerebellum, mid-brain, cortex], Pituitary gland, Caecum, Ileum, Jejunum, Duodenum, Colon, Rectum, Cervix, Prostate gland, Salivary glands - mandibular, sublingual, Sciatic nerve, Epididymides, Seminal vesicles including coagulating gland, Eyes with optic nerve and Harderian gland, Skin, Female mammary gland area, Spinal cord -cervical, midthoracic, lumbar, Spleen, Heart, Sternum with bone marrow, Stomach, Testes, Kidneys, Thymus, Larynx, Thyroid including parathyroid [if detectable], Liver, Trachea, Lung, infused with formalin, Urinary bladder, Lymph nodes - mandibular, mesenteric, Uterus, Vagina, Oesophagus, All gross lesions

Statistics:

Dunnett-test, Steel-test, The Fisher Exact-test, Kruskal-Wallis nonparametric ANOVA test

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw: males + females rales, salivation
100 and 300 mg/kg bw: males + females salivation

non survivors: lethargy, flat and/or hunched posture, shallow/laboured respiration, piloerection, salivation, chromodacryorrhoea, diarrhea, red discolouration of the mouth and snout, ptosis and/or lean appearance

Mortality:

mortality observed, treatment-related

Description (incidence):

1000 mg/kg bw: 1 male + 6 females found dead in week 7 to 13; 2 females killed in extremis in week 10 and 11
control: 1 female killed in extremis in week 13

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

males: 300 and 1000 mg/kg bw: dose dependent significant decrease (onset week 10 onwards for 300 mg/kg bw; week 6-7 onwards 1000 mg/kg bw)

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

1000 mg/kg bw: males + females increased

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

within normal ranges for rats of this strain and age

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw: females significantly decreased number of lymphocytes and platelets, significantly increased number of neutrophils; decreased number of reticulocytes (rel); significantly decreased APTT;
males significantly decreased APTT
300 mg/kg bw: females significantly decreased APTT; significantly increased number of neutrophils

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw: males significantly decreased ALAT, cholesterol and bile acids, sign. increased organic phophate;
females significantly increased ALAT, calcium, sign decreased protein (albumin), cholesterol and potassium

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Results were either in normal ranges or comparable to those of controls

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw: males sign. decreased liver and thymus (abs), sign increased kidneys and adrenals (rel);
females: sign decreased thymus (abs+rel), sign increased liver and adrenals (abs+rel)
300 mg/kg bw: females decreased thymus (abs+rel)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

males: focus/foci on the glandular mucosa in 3/10, 3/10, 1/10 and 5/10 animals at 0, 100, 300 and 1000 mg/kg bw resp.
Thickening and/or irregular surface of the forestomach: in 1/10 and 5/10 animals at 300 and 1000 mg/kg bw
females: Thickening and/or irregular surface of the forestomach: 4/4 females at 1000 mg/kg bw
intestines distended with gas and/or gelatinous content:4/4 females at 1000 mg/kg bw
Small thymus: 2/4 females at 1000 mg/kg bw

decendents: stomach distended with gas and/or irregular surface and/or reddish foci, intestines distended with gas, reduced size of thymus and spleen

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Stomach: males at 1000 mg/kg bw slightly increased incidence of ulceration, hyperkeratosis, edema, hyperplasia; hemorrhages in 1-2 males in all groups
females at 1000 mg/kg bw: increased incidence of hyperkeratosis in 4/4 survivors
Thymus: males 1000 mg/kg bwlymphocytolysis (5/10)
females 1000 mg/kg bw: lymphoid depletion (2/4)
females 300 mg/kg bw: lymphocytolysis (5/10)
Thyroid: males: moderate follicular cell hypertrophy in 0/10, 2/10, 5/10 and 4/10 at 0, 100, 300 and 1000 mg/kg bw resp.
Lung: males increased presence of alveolar macrophages in 0/10, 0/10, 4/10 and 5/10 at 0, 100, 300 and 1000 mg/kg bw resp
Intestines (duodenum, jejunum, ileum, cecum, rectum): females at 1000 mg/kg bw mucosal basophilia
Adrenals: females at 300 mg/kg bw vacuolation, zona glomerulosa (4/10), no effects in survivors at 1000 mg/kg bw
Bone marrow: females at 1000 mg/kg bw atrophy (2/4)

decendents: erosion/ulceration, squamous hyperplasia and/or hyperkeratosis of the forestomach or hemorrhages, basophilic mucosa with decreased Goblet cells and some animals with mucosal atrophy or mucosal erosion/ulceration, lymphoid depletion in the thymus and diffuse atropy of the bone marrow, tubular vacuolation in the kidneys, lymphoid atrophy in the spleen, lymphocytolysis in the mesenteric lymph nodes, acinar degranulation in the pancreas, acinar atrophy in the salivary glands and inactive uterus and vaginal atrophy

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

The high mortality in females at 1000 mg/kg bw was attributed to alterations in the gastro-intestinal tract (erosions/ulcerations in stomach and mucosal atrophy/basophilia with decreased Goblet cells in the intestines). Additional test substance–related effects in the early sacrificed females were observed in the hematopoietic system (lymphoid depletion in thymus and diffuse atrophy in the bone marrow).
The alterations in the gastro-intestinal tract in males from 300 mg/kg onwards and females at 1000 mg/kg are considered to be related to the substance. The surviving animals showed a reduced body weight gain or body weight loss in males from 300 mg/kg onwards and in females at 1000 mg/kg.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

mortality

other: alterations in the GI-tract

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

gross pathology

histopathology: non-neoplastic

other: alteration in the GI-tract

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

intestine

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

other: bone marrow

Treatment related:

yes

Dose response relationship:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

immune system

Organ:

thymus

Treatment related:

yes

Dose response relationship:

yes

correlations in findings

Necropsy	Organ Weight	Clinical Pathology	Histopathology
Stomach: red foci, glandular	-	-	Hemorrhage(s)
Stomach: thickening/irregular surface	-	-	Erosions/ulcerations/hyperkeratosis/edema
Intestines: distended with gas/ gelatinous content	-	-	Mucosal basophilia together with decreased Goblet cells
Thymus: reduced in size	↓	Lymphocytes ↓	Lymphoid depletion
Bone marrow	-	Platelets ↓	Atrophy

Conclusions:

The NOAEL as derived from this 90-day study is 100 mg/kg bw

Executive summary:

Rats (10/sex/dose) were exposed to the substance by gavage for a period of 90 days (doses 0, 100, 300 and 1000 mg/kg bw). Detailed examinations related to mortality, clinical signs, body weight, food consumption, ophthalmoscopy, behavioural effects, haematology, clinical chemistry, macroscopy, histopathology and organ weights were performed in a study that is performed according to OECD 408. In the highest dose group 6/10 females died showing alterations in the gastro-intestinal tract, a small thymus and bone marrow atrophy. The surviving females at 1000 mg/kg bw showed similar effects and a reduced body weight (gain). The effects on the gastro-intestinal tract also became apparent in males at 300 and 1000 mg/kg bw. These animals also had a reduced body weight (gain). Other effects included changes in numbers of white blood cells, lymphocytes, platelets as well as effects on several biochemical parameters. Macroscopy and histopathology indicated that next to the GI-tract mainly the thymus and bone marrow could be considered as potentially affected in males at 300 mg/kg bw and above and in females at 1000 mg/kg bw.

The NOAEL as derived from this study is 100 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

guideline study

System:

other: GI tract, heamatopoietic and immune system

Organ:

intestine

stomach

thymus

other: bone marrow

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The effects as reported in the 90 -day toxicity study are not considered to lead to a classification for specific target organ systemic toxicity, repeat exposure according to CLP (Regulation EC No 1272/2008).