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

Toxicity to reproduction

Administrative data

Endpoint:

toxicity to reproduction

Remarks:

other: OECD 422 Reproductive Screening Study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 March 2012 to 11 June 2012 (in-life phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Study without significant deficiencies

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

barium bis(di C8-C10, branched, C9 rich, alkylnaphthalenesulphonate)

EC Number:

939-718-2

Cas Number:

1474044-80-8

Molecular formula:

C28H44O3S.1/2Ba

IUPAC Name:

barium bis(di C8-C10, branched, C9 rich, alkylnaphthalenesulphonate)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

UVCB

Test animals

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.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on 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.

Initial doses (0, 15, 50, 150 mg/kg respectively) were based on an estimated purity of 90%. After completion of the in-life phase the substance was classified as UVCB with a purity of 100%. Therefore, actual doses were 10% higher, i.e. 17, 55 and 165 mg/kg respectively.

Details on mating procedure:

Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted according to a validated method. 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%.
The concentrations analysed in the formulations of Group 2 (15 mg/kg), Group 3 (50 mg/kg) and Group 4 (150 mg/kg) were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). For the formulation of Group 4 (150 mg/kg) prepared for use during treatment, the mean accuracy was below the target concentration (i.e. 89% of target).
No test substance was detected in the Group 1 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.

Duration of treatment / exposure:

Exposure period: 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).

Frequency of treatment:

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.

Details on study schedule:

Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males. Female nos. 74 and 75 were re-mated with a proven male of the same dose group since male nos. 34 and 35 were sacrificed in extremis on Day 5 and 2 of the mating period respectively. For parturition, the females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.

Doses / concentrations

Remarks:

Doses / Concentrations:
15, 50, 150 mg/kg/day
Basis:
other: nominal doses

No. of animals per sex per dose:

10 (see table)

Control animals:

yes, concurrent vehicle

Positive control:

No

Examinations

Parental animals: Observations and examinations:

Daily detailed clinical observations were made in all animals immediately (0-15 min) after dosing. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

Oestrous cyclicity (parental animals):

Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Sperm parameters (parental animals):

From the selected 5 males of the control and high dose group, and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

Litter observations:

Each litter was examined to determine the following, if practically possible:

Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.

Clinical signs: At least once daily, detailed clinical observations were made for all animals.

Body weights: Live pups were weighed on Days 1 and 4 of lactation.

Postmortem examinations (parental animals):

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.

Postmortem examinations (offspring):

Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.

All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 3; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 4) was applied to frequency data.

Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test (Ref. 5) to determine intergroup differences followed by the Wilcoxon test (Ref. 6) to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

See tables below

Offspring viability indices:

See tables below

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Tubular crystals (polarizing) in the kidneys in 1/5 male (minimal) and 1/5 female (slight) of the 150 mg/kg treated rats. In the female, this was accompanied by minimal or slight degrees of tubular dilatation, epithelial hypertrophy and granular cast(s)

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

spermatogenic staging profiles were normal for all treated males evaluated

Reproductive performance:

no effects observed

Details on results (P0)

The lower motor activity of females at 150 mg/kg was temporary in nature since at commencement
and the end of the measurement interval motor activity appeared similar to control levels. Moreover,
the lower motor activity was not associated with concurrent changes in clinical appearance or changes
during functional observation tests. Therefore, this change in motor activity was considered to be of no
toxicological relevance.
Tubular crystals were noted in the kidneys of one male and one female at 150 mg/kg (up to slight
degree). Crystal deposition can lead to obstructive nephropathy. In this study, this finding was
accompanied in the female by tubular dilatation, epithelial hypertrophy and granular cast(s) (up to
slight degree) in the immediate vicinity as tubular crystals were noted, and is suggestive for findings
secondary to obstruction. Therefore, the presence of tubular crystals is considered to be adverse.
The increased severity of congestion/erythrophagocytosis in the mesenteric lymph nodes of females
treated at 15 mg/kg, 50 mg/kg and 150 mg/kg (up to moderate degree, and corresponding to dark red
foci/reddish discolouration of the mesenteric lymph node in some females at 15 and 150 mg/kg) was
considered to be non-adverse given the absence of any other indicators of cellular damage, e.g.
inflammation or necrosis. The increased incidence and severity of hyperplasia/hypertrophy of the
follicular epithelium of the thyroid gland in females treated at 150 mg/kg was subtle in nature and
occurred in the absence of a dose-related trend, and was therefore considered to be non-adverse.
There were no morphological findings in the reproductive organs of either sex which could be
attributed to the test item.
No toxicologically significant changes were noted in any of the remaining parental parameters
investigated in this study (i.e. clinical appearance, functional observations, body weight, food
consumption, clinical laboratory investigations, macroscopic examination and organ weights).

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

The nature and incidence of clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Mortality / viability:

no mortality observed

Description (incidence and severity):

No toxicological relevance was attributed to these missing/dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

Reproductive results:
No reproduction toxicity was observed up to the highest dose level tested (150 mg/kg).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites).

No abnormalities were seen in the reproductive organs of the rats who failed to sire or deliver healthy pups, which could account for their infertility. Furthermore, the spermatogenic staging profiles were normal for all selected control and high dose males, and for all males suspected to be infertile.

Developmental results:

At 893 mg/kg, pups showed a lower mean body weight on Day 4 of lactation. This effect on pup body weight was considered to be a primary developmental effect since surviving females did not show a treatment-related change in body weights or food intake, and there was no evidence of deficiencies in maternal care.

No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs and macroscopy).

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: 55 mg/kg/day
Reproduction NOAEL: at least 150 mg/kg/day

Executive summary:

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted with Barium bis( di c8-c10, branched, c9 rich, alkylnaphthalene sulphonate) in rats by oral gavage. This GLP study followed the OECD 422 guideline. Dose levels were 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 15, 50 and 150 mg/kg. This was based on an estimated purity of 90%. After completion of the testing, the substance was determined to be UVCB with a purity of 100%. Based on the UVCB purity, the doses are considered to be 17, 55 and 165 mg/kg/day.

 

After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 42-55 days, i.e. during 2 weeks prior to mating, during mating, duringpost-coitum, and during at least 4 days of lactation.

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, 15, 50, and 150 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.

Parental results:

The lower motor activity of females at 150 mg/kg was temporary in nature since at commencement

and the end of the measurement interval motor activity appeared similar to control levels. Moreover,

the lower motor activity was not associated with concurrent changes in clinical appearance or changes

during functional observation tests. Therefore, this change in motor activity was considered to be of no

toxicological relevance.

Tubular crystals were noted in the kidneys of one male and one female at 150 mg/kg (up to slight

degree). Crystal deposition can lead to obstructive nephropathy. In this study, this finding was

accompanied in the female by tubular dilatation, epithelial hypertrophy and granular cast(s) (up to

slight degree) in the immediate vicinity as tubular crystals were noted, and is suggestive for findings

secondary to obstruction. Therefore, the presence of tubular crystals is considered to be adverse.

The increased severity of congestion/erythrophagocytosis in the mesenteric lymph nodes of females

treated at 15 mg/kg, 50 mg/kg and 150 mg/kg (up to moderate degree, and corresponding to dark red

foci/reddish discolouration of the mesenteric lymph node in some females at 15 and 150 mg/kg) was

considered to be non-adverse given the absence of any other indicators of cellular damage, e.g.

inflammation or necrosis. The increased incidence and severity of hyperplasia/hypertrophy of the

follicular epithelium of the thyroid gland in females treated at 150 mg/kg was subtle in nature and

occurred in the absence of a dose-related trend, and was therefore considered to be non-adverse.

There were no morphological findings in the reproductive organs of either sex which could be

attributed to the test item.

No toxicologically significant changes were noted in any of the remaining parental parameters

investigated in this study (i.e. clinical appearance, functional observations, body weight, food

consumption, clinical laboratory investigations, macroscopic examination and organ weights).

Reproductive results:

No toxicologically significant changes were noted in any of the reproductive parameters investigated in

this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea

and implantation sites) up to the highest dose level tested (150 mg/kg).

 

No abnormalities were seen in the reproductive organs of the rats who failed to sire or deliver healthy pups, which could account for their infertility. Furthermore, the spermatogenic staging profiles were normal for all selected Group 1 and 4 males, and for all males suspected to be infertile.

 

In conclusion, treatment with Barium bis( di c8-c10, branched, c9 rich, alkylnaphthalene sulphonate)

by oral gavage in male and female Wistar Han rats at dose levels of 15, 50 and 150 mg/kg revealed

parental toxicity at 150 mg/kg, consisting of histopathological findings in the kidneys of the males and

females. No reproduction and developmental toxicity was observed for treatment up to 150 mg/kg.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL: 50 mg/kg (55 mg/kg1)

Reproduction NOAEL: at least 150 mg/kg*

*After completion of the in-life phase indicated that the purity of the test substance was 100% instead of an estimated purity of 90%. Although for 150 mg/kg formulations this would result in an actual dose of 165 mg/kg, formulation analyses showed that accuracy of preparation of was approximately 10% below target.