Registration Dossier

Administrative data

Description of key information

A GLP OECD 422 study indicated clinical biochemistry effects at 298 mg/kg/day and established a NOAEL at 95 mg/kg/day. The effects seen on the GI tract (hyperkeratosis of the forestomach epithelium, mucosal hyperplasia and increased severity of lymphogranulocytic inflammation in the caecum and increased amounts of mucus in the large intestines) are most probably related to acidic nature of DNNSA. In addition effects on spleen. liver and bone marrow were reported.

In a study that is performed according to OECD 408 with the calcium salt of the substance, 6/10 females in the highest dose group 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.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 April 2012 to 25 June 2012 (end of in-life phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study with no significant deficiencies
Reason / purpose:
reference to other 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)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Source F0: Charles River Deutschland, Sulzfeld, Germany.
Age at start F0-treatment: Approximately 11 weeks.
Number of F0-animals: 40 females and 40 males.
Acclimatization F0: At least 5 days prior to start of treatment.
Health inspection F0: Upon receipt of the animals.
Identification F0: Earmark and tattoo.

Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
Method: Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Frequency: Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted according to a validated method (Project 498817). These analyses were conducted after the in-life phase as no suitable analytical method was available at an earlier stage. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
Males were exposed for 31 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41-52 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Female nos. 44 (Group 1), 65, (Group 3) and 77 (Group 4) were not dosed during littering.
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
Oral gavage in propylene gylcol
Basis:
actual ingested
No. of animals per sex per dose:
10
Details on study design:

After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 80, 250 and 750 mg/kg/day. Males were exposed for 31 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-52 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

Positive control:
No
Observations and examinations performed and frequency:
The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (Week 4 (males); end of lactation (females)), body weight and food consumption (at least at weekly intervals), clinical pathology (Week 4 (males); end of lactation (females)), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). Formulations were analyzed once during the study to assess accuracy, homogeneity and stability.
Sacrifice and pathology:
All males and the selected 5 females/group (see Allocation) were deprived of food overnight (with a maximum of approximately 24.5 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food.
Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 893 mg/kg, four males were sacrificed in extremis during the first week of treatment, and one female was sacrificed in extremis on Day 23 of the post-coitum phase. At 250 mg/kg, one female was sacrificed in extremis on Day 27 of the post-coitum phase. This single death at 298 mg/kg was considered to be related to treatment considering the mortality incidence at 893 mg/kg.
Mortality:
mortality observed, treatment-related
Description (incidence):
At 893 mg/kg, four males were sacrificed in extremis during the first week of treatment, and one female was sacrificed in extremis on Day 23 of the post-coitum phase. At 250 mg/kg, one female was sacrificed in extremis on Day 27 of the post-coitum phase. This single death at 298 mg/kg was considered to be related to treatment considering the mortality incidence at 893 mg/kg.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 893 mg/kg, males showed lower mean body weights and body weight gain throughout the mating period. At 95 mg/kg, body weight and body weight gain was similar to control animals.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Animals sacrificed in extremis showed weight loss, and food intake was reduced among these sacrificed animals. Food intake of surviving animals was similar to control levels.
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):
At 893 mg/kg, males showed a statistically significant higher mean white blood cell count (due to high counts for animal nos. 36 and 37). Other differences were considered to be of no toxicological relevance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Changes in clinical biochemistry were higher ALT activity, higher AST activity, higher APT activity at 893 mg/kg, higher albumin and bilirubin level (females), and higher urea levels (males). At 298 mg/kg, changes were higher ALP and lower cholesterol.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No behaviour or locomotor effects. See attached data table.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Effects at 893 mg/kg in males in heart and thymus. Effects in females at 893 mg/kg in thymus.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Scattered effects but not apparent dose trend. See attached data tables.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Effects at 893 mg/kg/day. See details section.
Histopathological findings: neoplastic:
no effects observed
Details on results:

Histopathology Findings: Treatment-related microscopic findings in surviving animals at 893 mg/kg (correlating to necropsy findings) were noted in the gastro-intestinal tract, and included hyperkeratosis of the forestomach epithelium, mucosal hyperplasia and increased severity of lymphogranulocytic inflammation in the caecum and increased amounts of mucus in the large intestines. Other microscopic findings at this dose included lymphoid atrophy of the thymus, decreased severity of hemopoietic foci in the spleen, increased severity of alveolar foamy macrophages in the lungs and scattered hepatocellular vacuolation in the liver. Reduced contents in the prostate gland, seminal vesicles and preputial gland among some males at 893 mg/kg were considered to have occurred secondary to lower body weights. Microscopic findings observed in early sacrifices were generally similar in nature and severity as those recorded for surviving animals. The female at 893 mg/kg additionally showed mucosal hyperplasia of the small intestines, reduced red pulpa of the spleen and cortical necrosis in the adrenals.
Dose descriptor:
NOAEL
Effect level:
> 95 - < 298 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: At 298 mg/kg, changes in clinical biochemistry were confined to a higher alkaline phosphatase activity in females, and lower cholesterol level in males.
Critical effects observed:
not specified

 Analysis of dose preparations

The concentrations analysed in the formulations of Group 2 (95mg/kg), Group 3 (298mg/kg) and Group 4 (893mg/kg) were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). 

No test substance was detected in the Group 1 (control) formulation. 

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation≤ 10%). 

Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Conclusions:
In conclusion, treatment with di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) by oral gavage in male and female Wistar Han rats at dose levels of 95, 298 and 893 mg/kg body weight/day revealed parental toxicity at298 and 893 mg/kg body weight/day. Based on these results, the No Observed Adverse Effect Levels (NOAEL) for repeat dose exposure to adult parental animals was 95 mg/kg/day.
Executive summary:

A Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test ofdi C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) was conducted in rats by oral gavage (OECD 422).

 

Based on the results of a 10-day dose range finding study, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 95, 298 and 893 mg/kg.

 

Study outline

After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 95, 298, and 893 mg/kg/day. Males were exposed for 31 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-52 days, i.e. during 2 weeks prior to mating, during mating, duringpost-coitum, and during at least 4 days of lactation.

 

Evaluated parameters

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (Week 4 (males); end of lactation (females)),  body weight and food consumption (at least at weekly intervals), clinical pathology (Week 4 (males); end of lactation (females)), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy).Formulations were analyzed once during the study to assess accuracy, homogeneity and stability.

Results/discussion

Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

 

At 893 mg/kg, four males were sacrificed in extremis during the first week of treatment, and one female was sacrificed in extremis on Day 23 of the post-coitum phase. At 298 mg/kg, one female was sacrificed in extremis on Day 27 of the post-coitum phase. This single death at 298 mg/kg was considered to be related to treatment considering the mortality incidence at 893 mg/kg.

 

Animals sacrificed in extremis showed clinical signs including lethargy, hunched posture, piloerection, diarrhoea and/or a lean appearance. All these animals showed weight loss, and food intake was reduced among these sacrificed animals. Food intake of surviving animals was similar to control levels. Some of these clinical signs noted for sacrificed animals were also noted among surviving animals at 298 and 893 mg/kg, albeit at much lower incidence.

 

At 893 mg/kg, surviving males showed a lower mean body weight (gain) throughout the mating period, with occasional weight loss among two surviving animals towards the end of their scheduled treatment period. Changes in clinical biochemistry parameters consisted of higher alanine aminotransferase activity in males and females, higher aspartate aminotransferase activity in males, higher alkaline phosphataseactivity in males and females at 893 mg/kg, higher albumin and total bilirubin level in females, higher urea level in males, lower cholesterol level in males and females, and higher calcium level in females.

 

At 298 mg/kg, changes in clinical biochemistry were confined to a higher alkaline phosphatase activity in females, and lower cholesterol level in males.

 

Treatment-related microscopic findings in surviving animals at 893 mg/kg (correlating to necropsy findings) were noted in the gastro-intestinal tract, and included hyperkeratosis of the forestomach epithelium, mucosal hyperplasia and increased severity of lymphogranulocytic inflammation in the caecum and increased amounts of mucus in the large intestines. Other microscopic findings at this dose included lymphoid atrophy of the thymus, decreased severity of hemopoietic foci in the spleen, increased severity of alveolar foamy macrophages in the lungs and scattered hepatocellular vacuolation in the liver. Reduced contents in the prostate gland, seminal vesicles and preputial gland among some males at 893 mg/kg were considered to have occurred secondary to lower body weights. Microscopic findings observed in early sacrifices were generally similar in nature and severity as those recorded for surviving animals. The female at 893 mg/kg additionally showed mucosal hyperplasia of the small intestines, reduced red pulpa of the spleen and cortical necrosis in the adrenals.

 

The increased severity of myeloid hyperplasia with increased granulopoiesis in the sternal bone marrow at 893 mg/kg (all sacrificed males and one surviving male) was in line with the higher neutrophil count (and resulting higher white blood cell count) for males at this dose level.

 

At 95 mg/kg, no toxicologically relevant effects were noted in any of the parameters examined.

 

In conclusion, treatment with di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) by oral gavage in male and female Wistar Han rats at dose levels of 95, 298, and 893 mg/kg body weight/day revealed parental toxicity at 298 and 893 mg/kg body weight/day. Based on these results, the No Observed Adverse Effect Levels (NOAEL) for repeat dose exposure to adult parental animals was 95 mg/kg/day.

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:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Qualifier:
according to
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 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:
mortality
gross pathology
histopathology: non-neoplastic
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:
High Quality GLP study following current guidelines.
System:
gastrointestinal tract
Organ:
bone
thymus

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

Additional information

Treatment of male and female Wistar rats with DNNSA by oral gavage in a study according to OECD 422 at dose levels of 95, 298 and 893 mg/kg bw revealed parental toxicity at 298 and 893 mg/kg bw. At 95 mg/kg, no toxicologically relevant effects were noted in any of the parameters examined. In a 90 -day study on the calcium salt of the substance 6/10 females at 1000 mg/kg bwdied, 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.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
A well-conducted GLP OECD 408 study without significant deficiencies that established a dose-response relationship and a NOAEL.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Due to low vapor pressure (3.3E-5 Pa at 20 °C) and high boiling point (> 50 °C), the inhalation route is not considered to be a relevant exposure route to human. In addition, the data of acute toxicity by oral is available and sufficient for assessment of acute toxicity of test substance. Thus, the acute inhalation toxicity study can be waived.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Due to low vapor pressure (3.3E-5 Pa at 20 °C) and high boiling point (> 50 °C), the inhalation route is not considered to be a relevant exposure route to human. In addition, the data of acute toxicity by oral is available and sufficient for assessment of acute toxicity of test substance. Thus, the acute inhalation toxicity study can be waived.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
It is considered that exposure via the oral route is considered a worst case exposure whereby the systemic availability of the substance is considered to be maximised. Exposure via the dermal route is expected to be considerably less than via the oral route due to lack of absorption under expected exposure conditions. In addition, the acute dermal study shows that no adverse effects were observed. The data of acute toxicity by oral is available and sufficient for assessment of acute toxicity of test substance. Thus, the acute dermal toxicity study can be waived.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
It is considered that exposure via the oral route is considered a worst case exposure whereby the systemic availability of the substance is considered to be maximised. Exposure via the dermal route is expected to be considerably less than via the oral route due to lack of absorption under expected exposure conditions. In addition, the acute dermal study shows that no adverse effects were observed. The data of acute toxicity by oral is available and sufficient for assessment of acute toxicity of test substance. Thus, the acute dermal toxicity study can be waived.

Justification for classification or non-classification

In the OECD 422 study, side effects were seen at 298 mg/kg bw/d (mid dose group) whereas no effects occurred at 95 mg/kg bw/d (low dose group). The most prominent effects were the mortalities at 893 mg/kg/day (4 males and 1 female) and at 298 mg/kg/day (1 female). Surviving animals in both treatments showed some of the same clinical signs as those in the animals sacrificed. Thus, the NOAEL was set to 95 mg/kg bw/d. In the 90 day oral repeated dose toxicity study with the calcium salt mortality was observed at 1000 mg/kg bw in females. At 300 and 1000 mg/kg in males and at 1000 mg/kg bw in females effects on body weight, GI-tract alterations and histopathlogical changes in the thymus and bonemarrow became apparent. These effects are not considered leading to a classification for specific target organ systemic toxicity, repeat exposure according to CLP (Regulation EC No 1272/2008).

.