Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulfonated, sodium salts

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Sodium alkylnaphthalene sulfonate was found to be not toxic to reproduction in the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening study according to OECD 422 at highest dose tested of 1000 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 December 2011 - 20 January 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

no

Principles of method if other than guideline:

In addition, the procedures described in the report essentially conform to the following guidelines:
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

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:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany..
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: mean weight at start of treatment was 297 gr (males) or 201 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
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.

IN-LIFE DATES: From: 04 December 2011 - 20 January 2012

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

- Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for density (1.2 g/mL) and purity (52.95 w/w%) of the test substance.
- Storage conditions of formulations: At room temperature in the dark under nitrogen
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Some animals were not dosed according to the latest body weight. For animals on Day 1 of lactation, the amount of test substance was maximally10% less than intended. For animals on Day 4 of lactation, the amount of test substance wasmaximally 34% more than intended. Given the incidental nature of this deviation, and since inlife data did not indicate an effect of this deviation, interpretation of the study results was considered to have been unaffected.

Details on mating procedure:

- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had 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.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).
Detection of mating was not confirmed for one animal at 100 mg/kg which had total litter loss. One female at 1000 mg/kg was mated with a proven male of the same dose level, since the male with which the female should originally be mated with was sacrificed during the premating period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (22 December 2011), according to a validated method (Project 497219). 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 under nitrogen was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% 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 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 40-47 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Females were not dosed during littering.

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

Frequency of treatment:

Once daily for 7 d/w.

Details on study schedule:

- Age at mating of the mated animals in the study: Approximately 13 weeks

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10, plus 5 males/group in Groups 1 and 4 (Recovery).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of the dose range finding study (Project 497221), and in consultation with the sponsor.
- 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, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.
- Identification of pups: On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.
- Selection of animals for selected measurements: 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.

Positive control:

not required.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: 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). The
circumstance of any death was recorded in detail.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals. 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. Arena observations were not performed when the animals were mating, or housed individually. These clinical observations were at least conducted between 0 and 1 hour after dosing (i.e. based on the peak period of anticipated effects after dosing in the dose range finding study (Project 497221)). The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.

BODY WEIGHT:
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION:
- Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY: yes

WATER CONSUMPTION : No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling).
- Dose groups that were examined: all
- Battery of functions tested: According to test guidelines
Motor activity of males at the end of the recovery period was not determined, although a (potentially) treatment-related effect was noted at the end of treatment. No clinical signs were noted to support the lower motor activity at the end of the treatment period, nor were any clinical signs noted at the end of the recovery period. The mean motor activity did not show a clear dose-related trend over the dose groups. This suggests that an effect (if any) on motor activity at the end of the recovery phase would have been small, and not have any significant impact on the overall conclusion of the study.

GENERAL REPRODUCTION DATA
- Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these 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.

Oestrous cyclicity (parental animals):

Not determined.

Sperm parameters (parental animals):

Slides of the testes were prepared for histopathological staging of spermatogenesis (5 males of the control and high dose group and animals suspected to be infertile).

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.

- Mortality: 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 in all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

GROSS PATHOLOGY:
- All animals were fasted overnight (with a maximum of 20 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Selected 5 animals/sex/group and all animals that died spontaneously or were killed in extremis: According to test guidelines
- All remaining females which failed to deliver and the remaining males: According to test guidelines

ORGAN WEIGHTS
- Selected 5 animals/sex/group: According to test guidelines
- All remaining males: Epididymides and Testes

HISTOPATHOLOGY:
- According to test guidelines

Postmortem examinations (offspring):

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

GROSS NECROPSY
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. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.

HISTOPATHOLOGY / ORGAN WEIGHTS
No.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (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 (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data.
- The following additional methods of statistical analysis were used: Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test 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. No statistical analysis was performed on histopathology findings.

Reproductive indices:

For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

macroscopic and microscopic abnormalities were observed in the 1000 mg/kg group that were considered to be related to treatment in stomach (thickened limiting ridge in 2/5 females and irregular surface in 1/8 males and 1/5 females) and mesenteric lymph nodes (haemorrhagic appearance in 1/8 males and 3/5 females). Such effects were also observed in the animals found dead or sacrificed, but not in animals at the end of the recovery period.

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality only occurred in the high dose group of 1000 mg/kg (2/15 males and 4/10 females) which were attributed to tracheal/pulmonary lesions associated with aspiration of test article (possibly due to the gavage procedure) and/or acute inflammation/ulceration in the stomach.

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

MORTALITY
Two males at 1000 mg/kg were sacrificed in extremis on Days 4 and 5 of the premating period, respectively. Three females at 1000 mg/kg were found dead on Days 20, 39 and 23 of treatment, respectively (i.e. Days 1, 22 and 5 of the post-coitum phase, respectively). One other female at 1000 mg/kg was found dead on Day 25 of treatment (i.e. Day 11 of the mating period). One female at 1000 mg/kg and one female at 100 mg/kg were sacrificed due to total litter loss. The incidence of total litter loss showed no relation to the dose, and the incidence was low. No toxicological relevance was ascribed to these occurrences.

CLINICAL SIGNS
Hunched posture was shown by all males and most females at 1000 mg/kg (except the two males sacrificed in extremis during the first week of treatment), generally from the first week of treatment onwards. Surviving males and females at 1000 mg/kg showed sporadic incidences of lethargy, breathing difficulties (rales and/or shallow respiration), piloerection and pale appearance. Reduced faeces production was noted for males at 1000 mg/kg during the last week of the mating period. For males, all these findings had resolved at commencement of/during the recovery phase. Clinical signs noted among the four females at 1000 mg/kg found dead included lethargy, flat/hunched posture, uncoordinated movements, ptosis, rales, piloerection, lean or pale appearance, abdominal swelling and/or ptosis. The incidence of these findings was not clearly related to the day of death. Two males at 1000 mg/kg were sacrificed in extremis during the first week of treatment based on their significantly deteriorated health condition (clinical signs were not recorded on the day of sacrifice). Salivation seen among all animals at 1000 mg/kg, and at lower incidence at 100 and 300 mg/kg, was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance. Incidental findings that were noted included rales, a dark left eye, alopecia, scabs, hypersensitivity to touch (single day for a single animal), and chromodacryorrhoea. These findings occurred within the
range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

FUNCTIONAL OBSERVATIONS
Motor activity (total movements and/or ambulations) was reduced for males at 100, 300 and 1000 mg/kg and for females at 100 and 300 mg/kg with statistical significance at the end of the treatment period, but no clear dose-related trend was apparent and control values were considered to be high (motor activity at the end of the recovery period for males was not determined). All groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period. Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.

BODY WEIGHTS
No toxicologically relevant changes in body weights and body weight gain were noted. The slightly lower body weight gain of males at 1000 mg/kg at the end of the premating period did not prevail or became more pronounced as treatment progressed. This change was therefore considered to be of no toxicological significance. One male at 1000 mg/kg and one female at 100 mg/kg showed weight loss of up to 9% over Days 1-8 of the premating period. As these animals again gained weight during the mating period, and similar changes in body weight were absent among other animals of these dose groups, this was considered to be of no toxicological relevance.

FOOD CONSUMPTION
No toxicologically relevant changes in food consumption before or after correction for body weight were noted. Absolute food intake of males and food intake corrected for body weight for both sexes at 1000 mg/kg was lower during the first week of the premating period. Since this change did not prevail or became more pronounced as treatment progressed, it was considered to be of no toxicological significance.

HAEMATOLOGY
The following (statistically significant) changes in haematology parameters distinguished animals at 1000 mg/kg from control animals at the end of the treatment phase:
- Lower red blood cell counts in males and females,
- Higher red cell distribution width (RDW) in two males and one female at 1000 mg/kg
- Lower haemoglobin level in males and females,
- Lower haematocrit level in males and females.
At the end of the recovery phase, males at 1000 mg/kg showed lower red blood cell counts, haemoglobin and haematocrit levels, and a higher mean corpuscular volume (MCV).

CLINICAL BIOCHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher alkaline phosphatase activity (ALP) in two females at 1000 mg/kg (mean not statistically significant).
- Higher glucose levels in females at 1000 mg/kg (mean not statistically significant).
- Lower total protein levels in males at 300 and 1000 mg/kg,
- Lower total bilirubin levels in males at 100, 300 and 1000 mg/kg, and in females at 300 and 1000 mg/kg.
- Higher cholesterol level in females at 1000 mg/kg.
These findings had resolved during the recovery phase in males at 1000 mg/kg.
The lower bile acid level of males at 100, 300 and 1000 mg/kg was considered to be of no toxicological significance, since the opposite effect would be expected in case of target organ toxicity. This change in bile acid level, as well as the higher chloride level of males at 1000 mg/kg and the lower potassium levels in females at 300 mg/kg at the end of treatment showed no dose-related trend. The apparent higher glucose level of females at 1000 mg/kg was due to a high variation in individual values, the range of which was comparable to the variation encountered among control animals. No
toxicological relevance was therefore ascribed to these changes.

MACROSCOPIC EXAMINATION
The following macroscopic abnormalities at the end of the treatment period in surviving animals at 1000 mg/kg were considered to be directly related to treatment with the test substance:
- Thickened limiting ridge of the stomach in 2/5 females.
- Irregular surface of the stomach in 1/8 males, and irregular surface of the forestomach in 1/5 females,
- Reddish/dark red/black discolouration of the mesenteric lymph nodes in 1/8 males, and 3/5 females.
Findings in sacrificed animals (except for the female with total litter loss) or animals found dead at 1000 mg/kg considered to be directly related to treatment included:
- Dark red/black discolouration of the mesenteric lymph nodes in 3/4 females.
- Thickened limiting ridge of the stomach in 3/4 females.
- Irregular surface of the forestomach in 1/4 females,
- Dark red discolouration or dark red foci of the glandular mucosa of the stomach in 2/2 males.
- Dark red discolouration of the gastro-intestinal tract in 1/2 males.
No treatment-related macroscopic abnormalities were noted at the end of the recovery period in males at 1000 mg/kg. Only a background level of hemorrhage/congestion in the mesenteric lymph node was recorded in 1/4 surviving Recovery males at 1000 mg/kg (minimal). Dark red foci/reddish discolouration of the thymus noted in several surviving animals and intercurrent sacrifices/deaths was considered to be related to necropsy procedures (congestion), and the incidence of microscopic correlates did not indicate a relationship to treatment. Other findings among animals sacrificed in extremis or found dead consisted of a reduced size of the thymus and spleen, enlarged liver, an advanced stage of autolysis, incomplete exsanguination, dilation of the gastrointestinal tract and mucous contents of the gastro-intestinal tract. These were considered to be related to the necropsy procedures and/or to be secondary to the autolytic process. One female at 100 mg/kg and one female at 1000 mg/kg showed total litter loss. One female at 1000 mg/kg showed dead foetuses in the uterus (five in each horn). The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, did not show a dose-related incidence trend and/or had no treatment-related microscopic correlates. These necropsy findings were therefore considered to be of no toxicological relevance, and included red foci on the lungs, pelvic dilation of the kidneys, a yellowish or reddish soft or hard nodule on the epididymides, a tan focus on the preputial glands, irregular surface of the liver, a dark red hard nodule on the spleen, uterus containing fluid, tan discolouration of the clitoral glands, and alopecia.

ORGAN WEIGHTS
No toxicologically relevant changes in organ weights and organ to body weight ratios were noted. The (statistically significant) lower absolute thymus weights and thymus to body weight ratio and higher liver to body weight ratio in males at 1000 mg/kg at the end of the treatment phase were slight
in nature, had no morphological correlates, and these organ weights were similar to control levels at the end of the recovery period. The statistically significant lower absolute thyroid weight of males at 100 and 1000 mg/kg and lower thyroid to body weight ratio of males at 100 mg/kg occurred in the absence of a dose-related trend, and means remained within the range considered normal for rats of this age and strain. The higher thyroid weight, thyroid to body weight ratio, and seminal vesicle weight of males at 1000 mg/kg at the end of the recovery period was absent at the end of the treatment period, and the change was slight. No toxicological relevance was therefore ascribed to these variations.

MICROSCOPIC EXAMINATION
The following treatment-related findings were noted in surviving main study animals at 1000 mg/kg:
- Lymphogranulocytic inflammation of the forestomach in 3/4 males.
- Hyperkeratosis of the stomach in 1/4 males (minimal degree).
- Hyperplasia of the squamous epithelium of the forestomach in 1/4 males (minimal) and 2/4 females (minimal-slight).
- Increased incidence and severity of hemorrhage/congestion in the mesenteric lymph nodes of 4/4 males and 4/4 females.
No treatment-related histopathological findings were noted at the end of the Recovery phase. The major histologic findings for the animals found dead or sacrificed in extremis were present in the respiratory tract (trachea/lungs) and/or stomach, and primarily consisted of tracheal/bronchiolar necrosis with inflammation, ulceration/erosion/inflammation of the glandular stomach and subcapsular necrosis of the liver. The presence of lesions in the trachea/lungs suggests a local reaction to aspiration of test article and the possibility of refluxive inhalation or gavage procedure rather than
systemic toxicity. Stomach lesions were considered to be test substance related. Findings of atrophy and increased apoptosis in the thymus and lymphoid depletion in the spleen of early death animals were considered secondary to stress. Reticuloendothelial hyperplasia in the spleen and histiocytic infiltrates in the mesenteric lymph nodes were possibly treatment related.
Reproductive performance:
No test article related abnormalities were seen in the reproductive organs. Staging of spermatogenesis in testes did not provide any evidence of test article related impairment to the spermatogenetic cycle. Other microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar Han rats of this age and strain.

REPRODUCTIVE DATA
No toxicologically relevant effects on mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were noted. Fertility and conception indices at 1000 mg/kg were lower than controls. This was ascribed to death of three females, and not related to a primary effect of the test substance on reproductive performance. Three females were recorded as non-pregnant, two of which at 1000 mg/kg were found dead early in the post-coitum phase, on respectively Days 1 and 5). Hence, potential pregnancy of these two females could not be confirmed by eg. counting of implantation sites. One female at 1000 mg/kg was found dead on Day 11 of the mating phase, and necropsy of this female showed implantation sites confirming pregnancy. All females which completed the full study duration were found to be pregnant, either based on delivery of pups or based on presence of implantation sites. Two females (one at 100 mg/kg and one at 1000 mg/kg) had total litter loss. Given the low incidence of this finding, this was considered to be of no toxicological relevance. The variation in the number of corpora lutea over the dose groups (achieving a level of statistical significance at 1000 mg/kg) occurred in the absence of a clear dose related trend, and the variation in individual values remained essentially within the range encountered among control animals. Hence, no toxicological relevance was ascribed to these variations.

GESTATION
No toxicologically relevant effects on gestation index and duration were noted. The lower gestation index at 1000 mg/kg was due to total litter loss of one female, and spontaneous death of two females out of the seven pregnant females. This lower gestation index was therefore considered to be of no toxicological relevance.

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.

Key result

Dose descriptor:

NOAEL

Remarks:

parental - systemic effects

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: An increased incidence and severity of hemorrhage/congestion was noted in the mesenteric lymph nodes of all surviving selected females at 1000 mg/kg.

Key result

Dose descriptor:

NOAEL

Remarks:

parental - local effects

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

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

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, body weight and external macroscopy did not reveal treatment-related findings. The higher sex ratio at 100 mg/kg occurred in the absence of a dose-related trend, and was therefore considered to be of no toxicological relevance.

MORTALITY PUPS
Three pups of the control group, four pups at 100 mg/kg, and eight pups at 1000 mg/kg (due to total litter loss of a single female (no. 90) were found dead or missing during lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing 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 PUPS
Clinical symptoms of pups were confined to a scab on the nose of one pup. The nature and incidence of this clinical sign remained within the range considered normal for pups of this age, and was therefore considered to be of no toxicological relevance.
Clinical signs of several pups were not recorded on 10 January 2012. Sufficient pup data were available for adequate interpretation of the study results.

BODY WEIGHT PUPS
Body weights of pups were considered to have been unaffected by treatment.

MACROSCOPY PUPS
Incidental macroscopic findings of pups that were found dead included absence of milk in the stomach, a stage of beginning autolysis and partial cannibalism. A hard nodule on the lower lip was noted for one of the surviving pups. The nature and incidence of this finding remained within the range
considered normal for pups of this age, and was therefore considered to be of no toxicological relevance.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects.

Reproductive effects observed:

not specified

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). 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 under nitrogen at room temperature under normal laboratory light conditions for at least 6 hours.

Conclusions:

Sodium alkylnaphthalene sulfonate was found to be not toxic to reproduction in the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening study according to OECD 422.

Executive summary:

In an OECD 422 study, oral treatment with Sodium alkylnaphthalene sulfonate resulted in deaths at 1000 mg/kg which were attributed to tracheal/pulmonary lesions associated with aspiration of test article (possibly due to the gavage procedure) and/or acute inflammation/ulceration in the stomach. The primary effects at 1000 mg/kg resulting from treatment in surviving animals and early deaths/sacrifices were confined to local effects in the stomach (being most pronounced in those animals sacrificed of found dead during the study), and an increased incidence and/or severity of hemorrhage in the mesenteric lymph nodes.

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

- Parental NOAEL: 300 mg/kg/day (local and systemic)

- Reproduction NOAEL: at least 1000 mg/kg/day

- Developmental NOAEL: at least 1000 mg/kg/day

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Valid and GLP compliant study.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In an OECD 422 study, oral treatment with Sodium alkylnaphthalene sulfonate resulted in deaths at 1000 mg/kg which were attributed to tracheal/pulmonary lesions associated with aspiration of test article (possibly due to the gavage procedure) and/or acute inflammation/ulceration in the stomach. The primary effects at 1000 mg/kg resulting from treatment in surviving animals and early deaths/sacrifices were confined to local effects in the stomach (being most pronounced in those animals sacrificed of found dead during the study), and an increased incidence and/or severity of hemorrhage in the mesenteric lymph nodes. Clinical signs, clinical biochemistry, haematology parameters and motor activity were unremarkable.

Specifically, histopathological examinations showed no remarkable findings in reproductive organs: cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles,testes, uterus and vagina). No toxicologically relevant effects on mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were noted.

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

-        Parental NOAEL: 300 mg/kg/day (local and systemic)

-        Reproduction NOAEL: at least 1000 mg/kg/day

-        Developmental NOAEL: at least 1000 mg/kg/day

Effects on developmental toxicity

Description of key information

Results from a prenatal developmental toxicity study (OECD 414) in rat, resulted to a developmental NOAEL of at least 600 mg/kg bw/day, being the highest dose tested.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 2016 - March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Januari 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: SR123887B
- Expiration date of the batch: 21 February 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature container flushed with nitrogen
- Stability under test conditions: yes
- Solubility and stability of the test substance in the vehicle: Stability for at least 6 hours at room temperature under normal laboratory light conditions and under nitrogen is confirmed over the concentration range 20 - 200 mg/mL

Species:

rat

Strain:

other: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: approximately 10-14 weeks
- Weight at study initiation: 169 to 237 grams
- Housing: Females were individually housed in Macrolon plastic cages; sterilized sawdust as bedding material and paper as cage-enrichment/nesting material were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water.
- Acclimation period: At least 5 days prior to treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 – 20.3
- Humidity (%): 45.3 – 48.8
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 February 2016 To: 17 March 2016

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test
item, vehicle, and/or formulation. No correction was made for the purity/composition of the test item. Formulations were stored at room temperature flushed with nitrogen.
Dose volume was 5 mL/kg bw. Actual dose volumes were calculated according to the latest body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

Details on mating procedure:

Untreated females were mated at the Supplier, and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum was the day of successful mating; confirmed by vaginal plug).

Duration of treatment / exposure:

From Days 6 to 20 post-coitum, inclusive.

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.

Duration of test:

until Day 21 post-coitum

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

22 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were selected by the sponsor based on the results of a combined 28-day/repro
screening study, in which dose levels of 0, 100, 300 and 1000 mg/kg bw/day were tested. At 1000 mg/kg bw/day, mortality was observed in 2/10 males after 4 to 5 days and in 4/10 females after 20 to 39 days. This was attributed to tracheal/pulmonary lesions associated with aspiration of test article and acute inflammation/ulceration in the stomach. Moreover, hemorrhage in the mesenteric lymph nodes was noted at 1000 mg/kg bw/day. At 300 mg/kg bw/day, only minimally increased incidence and severity in hemorrhage was observed which was considered not adverse. No reproduction or developmental toxicity was observed by treatment up to 1000 mg/kg bw/day.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from Day 2 post-coitum onwards up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION: Yes
Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative assessment was introduced as no treatment related effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 21 post-coitum
- Organs examined: according to guidelines

OTHER: the stomach of all animals was dissected and examined

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

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 (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-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. No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

Pre-implantation loss (%) = ((number of corpora lutea - number of implantation sites) / number of corpora lutea) x 100
Post-implantation loss (%) = ((number of implantation sites – number if live fetuses) / number of implantation sites) x 100
Viable fetuses affected/litter (%) = ((number of viable fetuses affected/litter) /( number of viable fetuses/litter)) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant clinical signs were noted by treatment up to and including 600 mg/kg bw/day. Rales was noted for three females at 100 mg/kg bw/day and five females at 600 mg/kg bw/day for 1-3 consecutive days. Piloerection was observed for one control female, one female at 300 mg/kg bw/day and nine females at 600 mg/kg bw/day for 1-3 consecutive days. As these findings were not consisted over time, they are considered not toxicologically relevant.
Salivation was noted for two females at 100 mg/kg bw/day, eight females at 300 mg/kg bw/day and almost all females at 600 mg/kg bw/day. This is considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test item.
Alopecia was noted for one control female, two females at 100 mg/kg bw/day and two females at 300 mg/kg bw/day. As this occurred within the range of background findings observed in rats of this age and strain under the conditions in this study and no dose response relationship is observed, this is not considered to be treatment related.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Weight gain corrected for uterus weight was significantly lower at 300 and 600 mg/kg bw/day (23.9 g and 23.1 g, respectively), compared to controls (30.7 g). As no dose response relationship is noted and the control value is relatively high compared to our historical control data (mean corrected body weight gain: 27 g), this was considered not toxicologically relevant.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

One female at 600 mg/kg bw/day showed an irregular surface of the glandular stomach, which was probably related to treatment with the test item, but there was no microscopic correlate in this part of the stomach. However, an erosion/ulceration with a lymphogranulocytic inflammation was recorded in the forestomach for this animal.
Alopecia noted for some females confirmed the clinical sign observed in the in-life phase of the animals and was considered not to be treatment related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the stomach at 600 mg/kg bw/day. A slightly increased incidence of minimal inflammation of the glandular stomach was recorded at 600 mg/kg bw/day (5/10 females), compared to 1/10 females of the control group. In one female at 600 mg/kg bw/day a moderate erosion/ulceration with a moderate lymphogranulocytic inflammation of the forestomach was recorded, which was considered to be related to the treatment with the test item.
The remainder of the recorded microscopic findings including the minimal inflammation in the forestomach in single animals were within the normal range of background pathology encountered in rats of this age and strain.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

A relatively high mean value of pre-implantation loss (14.0% per litter) was noted in the 100 mg/kg bw/day group, compared to the other groups. This was not considered to be treatment related as treatment started from implantation onwards (i.e. Day 6 post-coitum). As this value was within normal limits and no dose response relationship was observed, this was considered not treatment related.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

One female at 100 mg/kg bw/day and one female at 300 mg/kg bw/day were not pregnant. This observation is considered not treatment-related.

Other effects:

no effects observed

Details on maternal toxic effects:

Test item-related morphological alterations were present in the stomach at 600 mg/kg bw/day. A slightly increased incidence of minimal inflammation of the glandular stomach was recorded at 600 mg/kg bw/day (5/10 females), compared to 1/10 females of the control group. In one female at this dose a moderate erosion/ulceration with a moderate lymphogranulocytic inflammation of the forestomach was recorded, which was considered to be related to the treatment with the test item.
No maternal toxicity was observed in the 100 and 300 mg/kg bw/day groups.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

>= 600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: no systemic effects observed up to and including the highest dose tested.

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

no systemic effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: morphological alteration in stomach: slightly increased incidence in minimal inflammation of the glandular stomach; one high dose female with moderate erosion/ulceration with moderate lymphogranulocytic inflammation of the forestomach

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically relevant effects on fetal body weights (per sex and combined for both sexes) noted by treatment up to and including 600 mg/kg bw/day.
Female fetal weights were significantly lower at 600 mg/kg bw/day (4.9 g) when compared to the control group (5.2 g). As this decrease was minimal, the value was only just outside the historical control range (5.0 g - 5.2 g) and fetal skeletal development was unaffected, this is considered to be a result of a slightly high control value (mean historical control value: 5.1 g) and not toxicologically relevant. Mean combined (male and female) fetal body weights were 5.3, 5.3, 5.2 and 5.1 gram for the
control, 100, 300 and 600 mg/kg bw/day groups, respectively.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There were no toxicologically relevant effects on fetal body weights (per sex and combined for both sexes) noted by treatment up to and including 600 mg/kg bw/day.
Female fetal weights were significantly lower at 600 mg/kg bw/day (4.9 g) when compared to the control group (5.2 g). As this decrease was minimal, the value was only just outside the historical control range (5.0 g - 5.2 g) and fetal skeletal development was unaffected, this is considered to be a result of a slightly high control value (mean historical control value: 5.1 g) and not toxicologically relevant. Mean combined (male and female) fetal body weights were 5.3, 5.3, 5.2 and 5.1 gram for the
control, 100, 300 and 600 mg/kg bw/day groups, respectively.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Mean litter sizes were 9.6, 9.0, 10.5 and 10.0 fetuses/litter for the control, 100, 300 and 600 mg/kg bw/day groups, respectively.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to and including 600 mg/kg bw/day.
Mean sex ratios (males:females) were 48:52, 52:48, 49:51 and 48:52 for the control, 100, 300 and 600 mg/kg bw/day groups, respectively.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on external morphology following treatment up to and including 600 mg/kg bw/day. Malformations were noted in two fetuses at the high dose level and in one control fetus. At the high dose, these were general anasarca and small lower jaw, astomia (absent mouth) and absent eye bulges (all together in one fetus), and in the control group a combination of exencephaly and open eyelids was observed in one fetus. The occurrence of two dissimilar malformed fetuses at the high dose does not indicate a relation to treatment.
External variations were not seen in any group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on skeletal morphology following treatment up to and including 600 mg/kg bw/day.
Of the malformations, bent limb bones (scapulas and/or humeri) were most frequently observed. These occurred in one control fetus, two fetuses from dose group 100 mg/kg bw/day and three fetuses from dose group 600 mg/kg bw/day. The mean litter incidence at the high dose level was above the maximum historical control value (1.8% versus 0.9% per litter). However, because no dose response was observed for this malformation and it is the most frequently observed skeletal abnormality among historical control fetuses, the finding was not considered to be treatment related. The other skeletal malformations that were noted (rib anomaly in one high dose fetus and fused skull bones in one low dose fetus) occurred singly and therefore were considered chance findings.
Skeletal variations occurred at an incidence of 81.0%, 75.3%, 83.2% and 93.3% per litter in dose groups 0, 100, 300 and 600 mg/kg bw/day, respectively. All the ones noted, were not considered treatment related as they occurred in the absence of a dose-dependent relationship, infrequently, in control fetuses only and/or at frequencies that were within the range of available historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on visceral morphology following treatment up to and including 600 mg/kg bw/day.
Three fetuses in the high dose group had visceral malformations. The one with multiple malformations observed externally also appeared to have a narrow pulmonary trunk and atrial septum defect and two other fetuses had a small eye observed at soft tissue cephalic examination. The latter finding was also observed in one low dose fetus. The externally noted absent eye bulges in the high dose fetus should be mentioned here as well, but these occurred in a fetus with multiple external and visceral
malformations. As such, this grossly abnormal fetus was considered to have fortuitously occurred. The two remaining small eyes at the high dose level were not considered treatment related as these pertains two cases only without presence of a dose relationship. Moreover, this malformation was previously noted in historical control fetuses.
Besides the occurrence of situs inversus in one control fetus, there were no other viscerally malformed fetuses in this study.
The variations that were noted in this study were small supernumerary lobe(s) and appendix of the liver, convoluted and dilated ureter(s), small renal papilla and partially undescended thymus horn(s). These variations occurred at low incidences and/or in the absence of a doserelated incidence trend and therefore were not considered to be treatment related.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

No developmental toxicity was observed in the 100, 300 and 600 mg/kg bw/day groups.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of adverse effects up to and including the highest dose tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

The concentrations analysed in the formulations of 100, 300 and 600 mg/kg bw/day groups were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%), with mean accuracies of 103%, 96% and 103% in these groups, respectively

The formulations of 100 mg/kg bw/day and 600 mg/kg bw/day were homogeneous (i.e. coefficient of variation ≤ 10%), with a homogeneity (coefficient of varaition) of 1.6 and 3.8% in these groups, respectively.

Table 1: overview developmental toxicity data

Dose level (mg/kg bw/day)	0	100	300	600
Pregnant/total dams	22/22	21/22	21/22	22/22
-early resorptions -late resorptions (% per litter)	8.7 0.4	7.7 0.0	4.7 0.0	6.6 0.3
Dams with abortion, early deliveries, stillbirths, resorptions only and/or dead fetuses	0	0	0	0
Pre-implantation loss (number and percent)	16 (7.4%)	30 (14%)	9 (3.5%)	15 (5.8%)
Post-implantation loss (number and percent)	22 (9%)	11 (7.7%)	11 (4.7%)	17 (6.9%)
Body weight on day 21	327	314	322	315
Body weight gain day 6-21 (%)	44	41	43	41
Gravid uterine weight (g)	69.1	63.8	72.5	68.3
Mean live offspring (number)	9.6	9.0	10.5	10.0
Live offspring (percent)	91.0	92.3	95.3	93.1
Mean fetal/pup body weight males (g)	5.4	5.5	5.3	5.2
Mean fetal body weight females	5.2	5.1	5.1	4.9**
Mean fetal body weight (sexes combined)	5.3	5.3	5.2	5.1*
Malformations (including runts) number and percent of fetuses per litter	3 (2.3%)	4 (7.6%)	0 (0%)	8 (6.6%)
Variations (% per litter) -external -soft tissue -skeletal	0 7.7 81.0	0 18.3 75.3	0 11.0 83.2	0 8.6 93.3

Table 2: Historical Control Data Rat: Crl:WI(Han) (outbred, SPF-Quality)

Gestation Day 21

Study Date Range: 2014 – 2015

Mean of Study Means

Endpoint	Total	Mean	SD	Median	Min	Max	P5	P95
No of Studies	13
Total No. of Animals in the Control Group	304
No. of Animals that Died	0
No. of Animals that were Euthanized	0
No. of Animals that Aborted or Delivered	3
Percent Pregnant		98.8	2.73	100.0	90.9	100.0	97.1	100.0
No. of Animals Examined at Laparohysterectomy	301
No. Nongravid	4
No. Gravid	297
No. with Only Resorptions	2
No. of Dams with Live Fetuses	295
Mean No. Viable Fetuses/Dam		10.7	0.71	10.6	9.1	11.6	10.3	11.2
Total No. Viable Fetuses	3194
Viable Fetuses (%/Litter)		95.2	2.63	95.9	88.9	98.4	93.6	96.8
Mean No. Postimplantation Loss/Dam		0.5	0.15	0.4	0.2	0.7	0.4	0.6
Total No. Postimplantation Losses	134
Postimplantation Loss (%/Litter)		4.8	2.63	4.1	1.6	11.1	3.2	6.4
Dead Fetuses (%/Litter)		0.0	0.11	0.0	0.0	0.4	0.0	0.1
Early Resorptions (%/Litter)		4.7	2.62	4.1	1.6	11.1	3.2	6.3
Late Resorptions (%/Litter)		0.0	0.11	0.0	0.0	0.4	0.0	0.1
Mean No. Implantations/Dam		11.2	0.69	11.1	9.6	12.0	10.8	11.6
Mean No. Corpora Lutea/Dam		11.9	0.71	11.7	10.9	13.2	11.5	12.3
Mean No. Preimplantation Loss/Dam		0.7	0.32	0.6	0.2	1.3	0.5	0.9
Total No. Preimplantation Losses	207
Preimplantation Loss (%/Litter)		6.2	3.43	5.8	2.0	14.5	4.2	8.3
Total No. Male Fetuses	1617
Total No. Female Fetuses	1577
% Males/Litter		50.8	2.12	50.7	46.6	53.7	49.5	52.0
% Female/Litter		49.2	2.12	49.3	46.3	53.4	48.0	50.5
Mean Fetal Body Weight (g)		5.2	0.08	5.2	5.1	5.3	5.1	5.2
Mean Male Body Weight (g)		5.4	0.10	5.4	5.2	5.5	5.3	5.4
Mean Female Body Weight (g)		5.1	0.07	5.1	5.0	5.2	5.0	5.1
Mean Male Placenta Weight (g) 1		0.46	0.01	0.47	0.44	0.47	0.4	0.5
Mean Female Placenta Weight (g) 1		0.44	0.01	0.44	0.42	0.45	0.4	0.5

Historical Control Data Rat: Crl:WI(Han) (outbred, SPF-Quality) 

Gestation Day 21 

Study Date Range: 2014 -2015 

 

No. of Studies             13      

 Total No. Fetuses/Litters Examined Externally  3194 /295 

 Total No. Fetuses/Litters Examined Viscerally  2061 /295 

 Total No. Fetuses/Litters Examined Skeletally  2059 /295

Mean of Study Means                            Summary Incidence

(%Per Litter Basis)                                (Total No. Affected)

MALFORMATIONS	Mean	SD	Median	Min	Max	P5	P95	Fetuses	Litters
Total External Malformations								1	1
Total Visceral Malformations								7	7
Total Skeletal Malformations								15	15
Total Malformations								22	22
EXTERNAL
Exencephaly	0.0	0.14	0.0	0.0	0.5	0.0	0.1	1	1
Eye(s)-Open	0.0	0.14	0.0	0.0	0.5	0.0	0.1	1	1
VISCERAL
Diaphragmatic Hernia	0.0	0.08	0.0	0.0	0.3	0.0	0.1	1	1
Eye(s)-Absent and/or Small	0.1	0.26	0.0	0.0	0.9	0.0	0.2	3	3
Hydrocephaly-External	0.0	0.12	0.0	0.0	0.5	0.0	0.1	1	1
Situs Inversus	0.2	0.34	0.0	0.0	1.0	0.0	0.4	3	3
SKELETAL
Jaw-Upper Jaw Small	0.1	0.22	0.0	0.0	0.8	0.0	0.2	1	1
Jaw-Lower Jaw Absent or Small	0.1	0.22	0.0	0.0	0.8	0.0	0.2	1	1
Limb Bone(s)-Bent	0.3	0.44	0.0	0.0	1.1	0.0	0.5	4	4
Rib Anomaly	0.1	0.31	0.0	0.0	1.1	0.0	0.3	1	1
Skull Anomaly	0.1	0.34	0.0	0.0	1.2	0.0	0.3	2	2
Sternebra(e)-Fused	0.1	0.29	0.0	0.0	1.0	0.0	0.3	2	2
Sternebra(e) Malaligned (Severe)	0.0	0.08	0.0	0.0	0.3	0.0	0.1	1	1
Sternoschisis	0.1	0.22	0.0	0.0	0.8	0.0	0.2	1	1
Vertebral Anomaly With or Without Associated Rib Anomaly	0.2	0.53	0.0	0.0	1.9	0.0	0.5	3	3
Vertebral Centra Anomaly	0.1	0.22	0.0	0.0	0.8	0.0	0.2	1	1

Mean of Study Means                         Summary incidence

(%Per Litter Basis)                             (Total No. Affected)

VARIATIONS	Mean	SD	Median	Min	Max	P5	P95	Fetuses	Litters
EXTERNAL
None Observed
VISCERAL
Kidney(s)-Renal Papilla(e) Absent and/or Small	0.1	0.25	0.0	0.0	0.9	0.0	0.2	2	2
Liver-Appendix	1.2	0.56	1.3	0.3	2.3	0.9	1.6	23	21
Liver-Discolored	0.1	0.30	0.0	0.0	1.0	0.0	0.3	3	3
Liver-Small Supernumerary Lobe(s)	4.0	1.96	4.0	1.3	7.7	2.8	5.2	69	58
Spleen-Supernumerary	0.0	0.14	0.0	0.0	0.5	0.0	0.1	1	1
Thymus-Partially Undescended Horn(s)	1.3	1.55	0.8	0.0	4.3	0.3	2.2	34	23
Thyroid-Discolored	0.1	0.36	0.0	0.0	1.3	0.0	0.3	1	1
Ureter(s)-Convoluted	1.0	2.39	0.0	0.0	8.7	0.0	2.5	43	28
Ureter(s)-Dilated	0.9	2.33	0.0	0.0	8.5	0.0	2.3	44	19
SKELETAL
7th Cervical Rudimentary Rib(s)	1.7	1.34	1.2	0.0	4.4	0.9	2.5	30	26
7th Cervical Full Rib(s)	0.1	0.36	0.0	0.0	1.1	0.0	0.4	2	2
14th Full Rib(s)	5.7	4.65	5.2	0.0	13.1	2.9	8.5	88	64
14th Rudimentary Rib(s)	44.1	19.84	54.4	19.0	72.0	32.1	56.1	798	250
Metacarpal(s) and/or Metatarsal(s) Unossified	2.2	1.97	1.0	0.0	6.3	1.0	3.4	41	24
Pelvic Girdle-Caudal Shift	6.6	3.77	7.1	1.7	12.8	4.3	8.9	127	71
Rib(s)-Bent	10.6	7.78	10.2	0.8	22.3	5.9	15.3	162	85
Rib(s)-Short	0.0	0.06	0.0	0.0	0.2	0.0	0.0	1	1
Skull-Reduced Ossification	2.7	2.55	1.8	0.0	7.0	1.2	4.3	81	46
Skull-Supernumerary Site	0.0	0.14	0.0	0.0	0.5	0.0	0.1	1	1
Sternebra(e) #1, #2, #3 and/or #4 Unossified	0.2	0.31	0.0	0.0	0.8	0.0	0.3	3	3
Sternebra(e) #5 and/or #6 Unossified	0.9	1.33	0.0	0.0	4.1	0.1	1.7	37	23
Sternebrae-Malaligned (Slight or Moderate)	11.1	5.72	8.9	4.4	21.3	7.6	14.5	188	131
Sternum-Supernumerary Ossification Site	0.1	0.31	0.0	0.0	1.1	0.0	0.3	1	1
Vertebral Centra-Reduced Ossification	0.6	0.88	0.4	0.0	3.0	0.1	1.2	12	12

Conclusions:

Based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for Sodium alkylnaphthalene sulfonate was established as being 300 mg/kg bw/day for local toxicity based on morphological alterations of the stomach. The maternal NOAEL for systemic toxicity was at least 600 mg/kg bw/day.
The developmental NOAEL was established as being at least 600 mg/kg bw/day.

Executive summary:

Developmental toxicity/teratogenicity for Sodium alkylnaphthalene sulfonate was studied in rats according to OECD 414 guideline. Eighty-eight mated female Wistar Han rats were assigned to four dose groups. Sodium alkylnaphthalene sulfonate was administered once daily by oral gavage from Days 6 to 20 post-coitum at doses of 100, 300 and 600 mg/kg bw/day. The rats of the control group received the vehicle, water, alone. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on one day during treatment were analyzed for accuracy and homogeneity.

All animals surviving to Day 21 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. The stomach was collected and fixed from all animals at necropsy. Histopathological examination was performed on the stomach from 10 selected females per group. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placenta and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative; these fetuses were dissected and examined for visceral anomalies. The other one-half of the fetuses were processed and stained with Alizarin Red S for skeletal examinations.

Accuracy and homogeneity of formulations were demonstrated by analyses.

Test item-related morphological alterations were present in the stomach at 600 mg/kg bw/day. A slightly increased incidence of minimal inflammation of the glandular stomach was recorded at 600 mg/kg bw/day (5/10 females), compared to 1/10 females of the control group. In one female at this dose a moderate erosion/ulceration with a moderate lymphogranulocytic inflammation of the forestomach was recorded, which was considered to be related to the treatment with the test item. No maternal toxicity was observed in the 100 and 300 mg/kg bw/day groups. No developmental toxicity was observed in the 100, 300 and 600 mg/kg bw/day groups.

In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for Sodium alkylnaphthalene sulfonate was established as being 300 mg/kg bw/day for local toxicity based on morphological alterations of the stomach. The maternal NOAEL for systemic toxicity was at least 600 mg/kg bw/day. The developmental NOAEL was established as being at least 600 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Valid and GLP compliant study.

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

Developmental toxicity/teratogenicity for Sodium alkylnaphthalene sulfonate was studied in rats according to OECD 414 guideline. Eighty-eight mated female Wistar Han rats were assigned to four dose groups. Sodium alkylnaphthalene sulfonate was administered once daily by oral gavage from Days 6 to 20 post-coitum at doses of 100, 300 and 600 mg/kg bw/day. The rats of the control group received the vehicle, water, alone. All animals surviving to Day 21 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. The stomach was collected and fixed from all animals at necropsy. Histopathological examination was performed on the stomach from 10 selected females per group. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placenta and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative; these fetuses were dissected and examined for visceral anomalies. The other one-half of the fetuses were processed and stained with Alizarin Red S for skeletal examinations.

Results: Test item-related morphological alterations were present in the stomach at 600 mg/kg bw/day. A slightly increased incidence of minimal inflammation of the glandular stomach was recorded at 600 mg/kg bw/day (5/10 females), compared to 1/10 females of the control group. In one female at this dose a moderate erosion/ulceration with a moderate lymphogranulocytic inflammation of the forestomach was recorded, which was considered to be related to the treatment with the test item. No maternal toxicity was observed in the 100 and 300 mg/kg bw/day groups. No developmental toxicity was observed in the 100, 300 and 600 mg/kg bw/day groups.

In conclusion, based on the results in this prenatal developmental toxicity study the following NOAELs were established:

- NOAEL maternal, local = 300 mg/kg bw/day based on morphological alterations of the stomach.

- NOAEL maternal, systemic toxicity = 600 mg/kg bw/day.

- NOAEL developmental = 600 mg/kg bw/day.

 

Supportive information comes from an available OECD 422 study, showing a relative low toxicity for sodium alkylnaphthalene with no impact to developmental parameters at all up to the highest tested dose of 1000 mg/kg bw/day. Specifically, # of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings. The study concluded that there was no evidence of teratogenic effects based on the absence of relevant clinical signs and external macroscopic findings.

Mode of Action Analysis / Human Relevance Framework

Based on its surfactant properties, the structure is not expected to easily pass membrane structures, but cytotoxicity through disruption of cell membrane is expected. This is supported by study results: All available data indicates that the NOAEL is driven by local effects of the substance on skin and gastro-intestinal tract. Consequently, Sodium alkylnaphthalene sulfonate is not likely to reach reproductive organs or exert effects on the developing foetus.

Justification for classification or non-classification

Available data from a prenatal developmental toxicity study (OECD 414) of Sodium alkylnaphthalene sulfonate in rats showed no developmental toxicity at the highest dose test of 6000 mg/kg bw.

Additionally, Sodium alkylnaphthalene sulfonate was found to be not toxic to reproduction in the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening study according to OECD 422 at highest dose tested of 1000 mg/kg bw/day.

Based on mechanistic considerations,Sodium alkylnaphthalene sulfonate is not likely to reach reproductive organs or exert effects on the developing foetus.

For the same reasons, also effects via lactation are not to be expected.

 

Consequently, no classification for reproduction toxicity is needed.