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Effects on fertility

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Reference
Endpoint:
toxicity to reproduction
Remarks:
other: OECD 422 Reproductive Screening Study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 April 2012 to 25 June 2012 (in-life phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Study without significant deficiencies
Reason / purpose:
reference to same study
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 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.


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 (95 mg/kg), Group 3 (298 mg/kg) and Group 4 (893 mg/kg) were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).
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.
No. of animals per sex per dose:
10 (see table)
Control animals:
yes, concurrent vehicle
Positive control:
No
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 (see Allocation), 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
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.
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):
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.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see Repeat Dose Toxicity: oral.001
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The spermatogenic staging profiles were normal for all selected Group 1 and 4 males, and for all males suspected to be infertile (Group 1 male no.1, Group 2 male nos.15,17 and 20, Group 3 male nos. 22 and 26 and Group 4 male nos. 31, 33 and 36).
Reproductive performance:
no effects observed
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.

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.

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.
Dose descriptor:
NOAEL
Effect level:
> 893 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
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
Description (incidence and severity):
The nature and incidence of absence of milk in a single surviving pup remained within the range considered normal for pups of this age, and was therefore considered to be of no toxicological relevance.
Histopathological findings:
not examined

Reproductive results:
No reproduction toxicity was observed up to the highest dose level tested (893 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).
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
893 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Reproductive effects observed:
not specified
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 at 298 and 893 mg/kg body weight/day. No reproduction toxicity was observed for treatment up to 893 mg/kg body weight/day.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: 95 mg/kg/day
Reproduction NOAEL: at least 893 mg/kg/day
Developmental NOAEL: 893 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 extremison Day 23 of the post-coitum phase. At 298 mg/kg, one female was sacrificed in extremison 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 phosphataseactivity 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 hyperplasiaofthe 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.

 

Reproductive results:

 

No reproduction toxicity was observed up to the highest dose level tested (893 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 Group 1 and 4 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). Taken together, the evidence for developmental effects at 893 mg/kg/day is weak or equivocal and very likely secondary to maternal

toxicity. As a consequence, for risk assessment purposes the high dose treatment of 893 mg/kg/day is considered the appropriate NOAEL.

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.

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

Parental NOAEL:          95 mg/kg/day

Reproduction NOAEL:   at least 893 mg/kg/day

Developmental NOAEL: 893 mg/kg/day

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
893 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information
Short description of key information:
No reproduction toxicity was observed for treatment up to 893 mg/kg body weight/day.

Justification for selection of Effect on fertility via oral route:
GLP study following current guidelines.

Effects on developmental toxicity

Description of key information
Developmental toxicity in the form of lower pup body weight in female pups was observed at the high dose of 893 mg/kg on day 4 after birth. There were no significant differences on day 1 in female pups, in male pups on day 1 or day 4 or in combined male and female pup weights on either day 1 or day 4 after birth. No developmental toxicity was observed for the lower treatment groups of 95 and 298 mg/kg body weight/day.  Based on the lower female pup weight difference at day 4, the NOAEL was very conservatively set at 298 mg/kg body weight/day. However, clear parental effects were seen at 893 mg/kg bw/d. This included mortality of 4 males and one female and, moreover, some of the clinical signs seen in sacrificed animals were observed in surviving animals. In addtion, most females at 893 mg/kg showed hunched posture and piloerection during the post-coitum and/or lactation phase.Taken together, the evidence for developmental effects at 893 mg/kg/day is weak or equivocal and very likely secondary to maternal toxicity. As a consequence, for risk assessment purposes the high dose treatment of 893 mg/kg/day is considered the appropriate NOAEL.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study without deficiencies
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: OECD 422
Deviations:
yes
Remarks:
Minor deviations occurred. The study integrity was not adversely affected by the deviations.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
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.
Randomization F0: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

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:
unchanged (no vehicle)
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.
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). Female nos. 44 (Group 1), 65, (Group 3) and 77 (Group 4) were not dosed during littering.
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%.
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. 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.
Duration of treatment / exposure:
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, during post-coitum, and during at least 4 days of lactation.
Frequency of treatment:
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.
No. of animals per sex per dose:
10 per sex per dose level including control
Maternal examinations:
Mortality / Viability:
At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).

Clinical signs:
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.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.
Ovaries and uterine content:
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.
Fetal examinations:
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.
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.
Indices:
see table attached.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Most females at 893 mg/kg showed hunched posture and piloerection during the post-coitum and/or lactation phase. The female sacrificed at 893 mg/kg additionally showed laboured respiration, red staining of the snout and diarrhoea. At lower incidence, lethargy and lean appearance were noted among surviving females. At 298 mg/kg, the female sacrificed in extremis during the post-coitum phase (no. 66) showed hunched posture, piloerection, red staining of the snout and diarrhoea predominantly during the week preceding death. A few surviving animals incidentally showed hunched posture.
At 95 mg/kg, no toxicologically relevant clinical signs were noted.

Dose descriptor:
NOAEL
Effect level:
ca. 893 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
Mean body weight of pups at 893 mg/kg on Day 4 of lactation was lower than those of control pups (achieving statistical significance for male pups only).
No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs and macroscopy) were observed.
The apparent lower gestation index at 893 mg/kg was attributed to the lower number of pregnant females (8) in combination with the higher relative effect on the gestation index of the female (no. 74) that was sacrificed before delivery. Histopathology of this female confirmed presence of a placenta. One female at 893 mg/kg (no. 75) had post-implantation loss (found pregnant based on presence of implantation sites). At 298 mg/kg, of the 10 pregnant females one female (no. 66) was not pregnant and was sacrificed in extremis on Day 23 of the post-coitum phase, and one female (no. 62) had post-implantation loss (found pregnant based on presence of implantation sites). At 95 mg/kg, three females did not become pregnant, and in the control group one female did not become pregnant.
Duration of gestation was similar between the groups.

Parturition/maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Early postnatal pup development
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs and external macroscopy did not reveal treatment-related findings.

Mortality
One pup of the control group, two pups at 95 mg/kg, and two pups at 893 mg/kg were found dead or missing during lactation. Pups missing were most likely cannibalised. 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.

Clinical signs
Incidental clinical symptoms of pups consisted of insufficient milk in the stomach, absence of milk in the stomach and a blue snout or body. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Macroscopy
The nature and incidence of absence of milk in a single surviving pup remained within the range considered normal for pups of this age, and was therefore considered to be of no toxicological relevance.

Dose descriptor:
NOAEL
Effect level:
>= 893 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment related effects
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Developmental toxicity in the form of lower pup body weight in female pups was observed at the high dose of 893 mg/kg on day 4 after birth. There were no significant differences on day 1 in female pups, in male pups on day 1 or day 4 or in combined male and female pup weights on either day 1 or day 4 after birth. No developmental toxicity was observed for the lower treatment groups of 95 and 298 mg/kg body weight/day. Based on the lower female pup weight difference at day 4, the NOAEL was very conservatively set at 298 mg/kg body weight/day. However, clear parental effects were seen at 893 mg/kg bw/d. This included mortality of 4 males and one female and, moreover, some of the clinical signs seen in sacrificed animals were observed in surviving animals. This included mortality of 4 males and one female and, moreover, some of the clinical signs seen in sacrificed animals were observed in surviving animals. Taken together, the evidence for developmental effects at 893 mg/kg/day is weak or equivocal and very likely secondary to maternal toxicity. As a consequence, for risk assessment purposes the high dose treatment of 893 mg/kg/day is considered the appropriate NOAEL.

 
Executive summary:

Di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid(DNNSA) was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 95, 298 and 893 mg/kg/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 31 days). The females were exposed for 2 weeks prior to mating, during mating, duringpost-coitum, and at least 4 days of lactation (for 41-52 days). Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Developmental results

Developmental toxicity in the form of lower pup body weight in female pups was observed at the high dose of 893 mg/kg on day 4 after birth. There were no significant differences on day 1 in female pups, in male pups on day 1 or day 4 or in combined male and female pup weights on either day 1 or day 4 after birth. No developmental toxicity was observed for the lower treatment groups of 95 and 298 mg/kg body weight/day. Based on the lower female pup weight difference at day 4, the NOAEL was very conservatively set at 298 mg/kg body weight/day. However, clear parental effects were seen at 893 mg/kg bw/d. This included mortality of 4 males and one female and, moreover, some of the clinical signs seen in sacrificed animals were observed in surviving animals. In addition, most females at 893 mg/kg showed hunched posture and piloerection during the post-coitum and/or lactation phase.Taken together, the evidence for developmental effects at 893 mg/kg/day is weak or equivocal and very likely secondary to maternal toxicity. As a consequence, for risk assessment purposes the high dose treatment of 893 mg/kg/day is considered the appropriate NOAEL.

 

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 earlypostnatalpup development consisting of mortality, clinical signs and macroscopy).

 

In conclusion, treatment withdi 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, 893 mg/kg body weight/day revealed parental toxicity at 298 and 893 mg/kg body weight/day. Developmental toxicity (lower female pup body weights at one time point - day 4) observed at 893 mg/kg/day was considered equivocal and related to maternal toxicity. No reproduction toxicity was observed for treatment up to 893 mg/kg body weight/day.

 

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

Parental NOAEL:            95 mg/kg/day

Reproduction NOAEL:     893 mg/kg/day

Developmental NOAEL: 893 mg/kg/day

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
893 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information
Justification for selection of Effect on developmental toxicity: via oral route:
GLP guideline study.

Justification for classification or non-classification

In a reproductive toxicity screen combined with repeated dose toxicity according to OECD 422 no fertility effects were seen and developmental effects (reduced female pup body weight at one time point) only occured secondary to maternal toxicity. Therefore, the substance is not classified for reproductive toxicity according to CLP (Regulation EC No 127272008) or DSD (Directive 67/548/EEC).