Magnesium bis(dinonylnaphthalenesulphonate)

Endpoint summary

Description of key information

OECD 422 studies were conducted with 2 analogues: DNNSA (di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid) and Barium DNNSA (Barium bis( di C8-C10, branched, C9 rich, alkylnaphthalene sulphonate). The NOAEL for DNNSA is 95 mg/kg/day and the NOAEL for Barium DNNSA is 55 mg/kg/day. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

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 for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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 conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

95 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

Quality of whole database:

For dermal exposure route-to-route extrapolation from the available oral study will be applied

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

DNNSA provides the best read-across for MgDNNSA because DNNSA is the major component of MgDNNSA and the presence of the magnesium moiety, which is known to have low systemic toxicity,would not contribute significantly to the toxicity. The NOAEL for BaDNNSA is lower than the NOAEL for DNNSA most likely due to the presence of the barium ion. The NOAEL for BaDNNSA is based on effects on the kidney which is known as the target organ for barium toxicity.

Based on these results, the No Observed Adverse Effect Levels (NOAEL) used as starting point for the derivation of the DNELs was established as 95 mg/kg, the NOAEL for DNNSA

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
DNNSA provides the best read-across for MgDNNSA because DNNSA is the major component of MgDNNSA and the presence of the magnesium moiety would not contribute significantly to the toxicity. The NOAEL for BaDNNSA is lower than the NOAEL for DNNSA most likely due to the presence of the barium iom. The NOAEL for BaDNNSA is based on effects on the kidney which is known as the target organ for barium toxicity.
Based on these results, the No Observed Adverse Effect Levels (NOAEL) used as starting point for the derivation of the DNELs was established as 95 mg/kg, the NOAEL for DNNSA.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
based on the use of the substance (no aerosol formation with respirable droplets expected) and the very low vapour pressure, the inhalation route is considered not relevant for the exposure assessment.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
based on the use of the substance (no aerosol formation with respirable droplets expected) and the very low vapour pressure, the inhalation route is considered not relevant for the exposure assessment.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
No local effects on the skin are anticipated.

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. As the single mortality of one female at 298 mg/kg bw/d occurred at day 27 post coitum and thus approximately after 45 days of exposure, the effects are not considered leading to a classification for specific target organ systemic toxicity, repeat exposure according to CLP (Regulation EC No 1272/2008) or according to DSD.