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

Toxicity to reproduction

Administrative data

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.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 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:

other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.

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. Pups were kept with the dam until termination.
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 oral 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 was maximally 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.

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.

Frequency of treatment:

Once daily, 7 d/w

Remarks:

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

No. of animals per sex per dose:

10, plus an additional 5 males 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.
- 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. Only females with live offspring were selected.

Positive control:

Not required.

Observations and examinations performed and frequency:

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.

Sacrifice and pathology:

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

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.
- 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.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Description (incidence and severity):

Based on subjective appraisal.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

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 (most pronounced in animal nos. 40, 48, and 84) 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.

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'

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.

Critical 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:

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

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting 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:

other: Organisation of Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, March 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The United States Environmental Protection Agency (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:

other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.

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

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.

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%.

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.

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. Main 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.

Duration of test:

Males: 29 days
Females: 40-47 days

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.

Maternal 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.
Clinical signs for one animal at 1000mg/kg on 10 January 2012 were recorded off-line and were not identified with severity grades. Based on clinical signs recorded, an adequate evaluation of the study results could still be made. Clinical signs were entered in the computer system later, and a grade 1 was entered for all symptoms, including those for which a maximum grade 3 or 4 applied (the software requires a grade to be entered).

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.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

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.

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.

Indices:

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 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.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
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 (most pronounced in animal nos. 40, 48, and 84) 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.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

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: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.

Details on embryotoxic / teratogenic effects:
There was no evidence of teratogenic effects based on the absence of relevant clinical signs and external macroscopic findings.

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. 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

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Abnormalities:

not specified

Developmental 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.

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.

Conclusions:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 4

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

800-660-7

Cas Number:

1258274-08-6

Molecular formula:

Not applicable, UVCB substance

IUPAC Name:

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

Constituent 2

Reference substance name:

Sodium alkylnaphthalene sulfonate

IUPAC Name:

Sodium alkylnaphthalene sulfonate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Sodium alkylnaphthalene sulfonate
- Substance type: Amber to greenish liquid
- Physical state: liquid
- Purity: 52.95 w/w%
- Lot/batch No.: Tank 109 2-22-08
- Expiration date of the lot/batch: 22 February 2018
- Storage condition of test material: At room temperature in the dark under nitrogen
- pH: 7.5 – 10 at concentration of 5%
- Conversion factor: The purity of the test substance was 52.95 w/w%. Accordingly, a correction factor of 1.8886 was applied to adjust for the purity of the test substance.
- Volatile: No
- Test substance handling: Flush container with nitrogen after handling
- Specific Gravity / Density: 1.2 g/mL

Test animals

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

Administration / exposure

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

Doses / concentrations

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.

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

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.

Effect levels (P0)

1

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.

2

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'

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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